Different Components of Animal Cell Culture Media
Animal-cell culture media: History, characteristics, and current issues
Abstract
Background Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal‐cell culture media. Methods A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. Results At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical‐based synthetic media because naturally derived ingredients have their disadvantages such as large batch‐to‐batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum‐containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Conclusions Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.
Discover the world's research
- 20+ million members
- 135+ million publications
- 700k+ research projects
Join for free
Content may be subject to copyright.
Reprod Med Biol. 2017;1–19.
|
1
wileyonlinelibrary.com/journal/rmb
|
DOI: 10.1002/rmb2.12024
REVIEW ARTICLE
Animal- cell culture media: History, characteristics, and current
issues
Tatsuma Yao1,2 | Yuta Asayama1
1Research and Development Center, Fuso
2Faculty of Biology-Oriented Science and
Technology, Kindai University, Wakayama,
Correspondence
Tatsuma Yao, Research and Development
Center, Fuso Pharmaceutical Industries, Ltd.,
Email: tat-yao@fuso-pharm.co.jp
Abstract
Background: Cell culture technology has spread prolifically within a century, a variety
of culture media has been designed. This review goes through the history, character-
istics and current issues of animal- cell culture media.
Methods:
Results:
naturally derived products such as serum. The field then gradually shifted to the use of
chemical- based synthetic media because naturally derived ingredients have their dis-
advantages such as large batch- to- batch variation. Today, industrially important cells
can be cultured in synthetic media. Nevertheless, the combinations and concentra-
tions of the components in these media remain to be optimized. In addition, serum-
containing media are still in general use in the field of basic research. In the fields of
assisted reproductive technologies and regenerative medicine, some of the medium
components are naturally derived in nearly all instances.
Conclusions: Further improvements of culture media are desirable, which will certainly
contribute to a reduction in the experimental variation, enhance productivity among
biopharmaceuticals, improve treatment outcomes of assisted reproductive technolo-
gies, and facilitate implementation and popularization of regenerative medicine.
KEYWORDS
cell culture technique, cell proliferation, culture media, cultured cells, serum
1| INTRODUCTION
The influence of cell culture technology on human society has been
immeasurable. Progress in biology in recent years, for example,
has depended heavily on cell culture technology.1 In addition, cell
culture- based practical technologies have been developed in vari-
ous areas, including the assessment of the efficacy and toxicity of
new drugs, manufacture of vaccines and biopharmaceuticals, and
-
matic cells became technically feasible recently, researchers around
the world are fiercely competing for leadership in the advances
of regenerative medicine. In this area likewise, cell culture tech-
nology is regarded as a foundation for further development and
popularization.
No one probably would argue against the claim that a culture
medium supports cell survival and proliferation, as well as cellular
functions, meaning that the quality of the medium directly affects the
research results, the biopharmaceutical production rate, and treat-
ment outcomes of assisted reproductive technology. It is essential,
therefore, for investigators who are working with cell cultures to se-
lect an appropriate medium that is suitable for their aims. In some
medium, provided the original work is properly cited and is not used for commercial purposes.
2
|
YAO And ASAYAMA
cases, researchers should modify a medium themselves. In addition,
when facing problems, researchers have to know the properties of
the medium in order to identify the cause of any problem with their
experiments.
groups, based on the type of supplements added; for example,
serum- containing media, serum- free media, protein- free media, and
chemically defined media (Tables 1 and 2). Serum- containing media
naturally contain various serum- derived substances, which make the
medium composition unclear and whose concentrations can fluctu-
ate from batch to batch. This situation makes the culture results less
reproducible and poses a risk of microbial contamination. Serum-
containing media, however, can be designed easily and be used ef-
fectively for a variety of cell types because serum includes a lot of
active substances that are necessary for the survival and growth of
animal cells.2 Serum- free media, in contrast, have a defined composi-
tion, resulting in a high reproducibility of results, and the cultivation
process can be validated. In addition, target cells can be grown selec-
tively in an intermingled cell population if the culture conditions are
of protein- free media (which do not contain any protein at all) and
chemically defined media (which do not contain any undefined in-
gredient) provide additional stability and reproducibility for culture
systems, facilitating the identification of the cellular secretions and
reducing the risk of microbial contamination. However, the serum-
free media are difficult to design: only specific cell types have been
cultivated this way to date.3
available commercially. Thus, there are investigators who are using
culture media without understanding their details and background,
particularly regarding the rationale for their development, the
exact ingredients, as well as the cell types that these media are
suitable for. This review article briefly describes the history of the
development of animal- cell culture media, with comments on the
types of media in general use today regarding their characteristics,
roles of the medium components, and pitfalls or problems with
their use.
2| HISTORY OF CELL CULTURE MEDIA
2.1| Dawn of cultivation experiments (1882–1907)
In 1882, Sydney Ringer developed Ringer's solution, a balanced salt
solution of a composition that is close to that of bodily fluids, and suc-
cessfully kept frog hearts beating after dissection and removal from
the body.4,5 This is said to be the first instance of in vitro cultivation
of animal tissue. Balanced salt solutions were developed one after
another in the wake of Ringer's report, including Locke's solution,6
Tyrode's solution,7 the Krebs–Ringer bicarbonate solution,8
solution,9 Earle's solution,10 and Hanks' solution.11 The composition
of these balanced salt solutions is simple and includes only inorganic
salts, sometimes with glucose added as a nutrient. Nonetheless, their
pH, osmotic pressure, and inorganic salt concentrations were cali-
brated to physiological conditions and these solutions can be used
successfully to keep tissues and cells outside the body alive for short
periods, generally up to a few days.
attention to cells in culture devices and tried to maintain the cells.
Nonetheless, the cells usually did not survive and rarely showed mitotic
figures.12–14
an apparent outgrowth of nerve fibers of a frog for several weeks in
lymph fluid that had been freshly drawn from the lymph sacs of an adult
frog.15 This experiment is considered to be the beginning of animal cell
cultivation.
TABLE1Categories of animal- cell culture media
Category Definition Type Example
Natural media Consisting of natural
biological substances, such
as plasma, serum, and
embryo extract
Coagulant or clots Plasma separated from heparinized blood, serum, and
fibrinogen
Tissue extracts Extracts of chicken embryos, liver, and spleen and bone
marrow extract
Biological fluids Plasma, serum, lymph, amniotic fluid, and pleural fluid
Synthetic media Composed of a basal medium
and supplements, such as
serum, growth factors, and
hormones
Serum- containing media Human, bovine, equine, or other serum is used as a
supplement
Serum- free media Crude protein fractions, such as bovine serum albumin or
α- or β- globulin, are used as supplements
Xeno- free media Human- source components, such as human serum albumin,
are used as supplements but animal components are not
allowed as supplements
Protein- free media Undefined components, such as peptide fractions (protein
hydrolysates) are used as supplements
Chemically defined media Undefined components, such as crude protein fractions,
hydrolysates, and tissue extracts, are not appropriate as
supplements, but highly purified components, such as
recombinant proteins are appropriate supplements
|
3
YAO And ASAYAMA
TABLE2Types and characteristics of basal media
Category Name (author, year) Features
Research
Laboratories
1950)
Developed in order to cultivate chicken embryonic cells under protein- free conditions, it is prepared by the sequential addition of amino acids,
vitamins (including fat- soluble vitamins), and nucleic- acid precursors. Its composition is extremely complex because the components that are
thought to be necessary on theoretical grounds, including inactive components, are added to the medium. Often used for organ culture
of reducing substances (cysteine, glutathione, and ascorbic acid), the elimination of fat- soluble vitamins, changes in the types of nucleic- acid
precursors, and the addition of coenzymes
Eagle media
1955)
Supplemented with the minimal components that are necessary for mouse L cells and human HeLa cells to reach the index of proliferative
capacity and including 13 amino acids and eight vitamins, it is unsuitable for cells whose cultures require many components because of its
simple composition
can add non- essential amino acids to reduce the biosynthetic load
Freeman 1959)
ability of the polyoma virus in mouse embryonic cells. Various modifications have been made since, with supplementation, for example, of
the non- essential amino acids, glycine and serine, iron, and pyruvate. The glucose concentration also can be increased to 25 mmol L in
order to accommodate cells with high nutritional requirements. In the event that pH changes are suspected due to metabolites, sodium
bicarbonate is doubled in concentration, equilibrated, and then used at 10% CO2
α
vitamins (ascorbic acid, biotin, and cyanocobalamin), pyruvate, lipoic acid, and nucleosides
selenite, pyruvate, and HEPES. Transferrin, bovine serum albumin, and soybean lipids are added as serum substitutes. With its high
Tissue Culture
Section of the
National Cancer
Institute (NCTC)
media
Developed to culture L cells under protein- free conditions, its amino acid composition is based on the results of a componential analysis of the
compounds that have been ultrafiltered from horse serum and chicken embryonic- tissue extract. Its composition is quite complex, with not
vitamins). Cysteine was included in the original composition, but after it was found to negatively affect cells, a version was developed from
which cysteine was removed (NCTC135)
Ham media Ham's F- 10 (Ham 1963) Enables colony formation by a single Chinese hamster ovary (CHO) cell under serum- free conditions, developed by adding two kinds of
purified serum proteins (serum albumin and fetuin), instead of serum, and by examining in detail the types and concentrations of amino acids
and trace elements This medium was the first to contain the trace elements, copper and zinc (iron having been included in other media
already). The CHO cells have an inferior proliferative capacity in this medium alone, compared to the one with serum added. Furthermore,
culture of the cell lines other than CHO necessitates the addition of serum
Ham's F- 12 (Ham 1965) The serum albumin and fetuin (used in Ham's F- 10) are replaced by two compounds with definite chemical composition: linoleic acid and
putrescine, which enables colony formation by a single CHO cell under protein- free conditions. Often cited as the world's first chemically
defined medium, the levels of several amino acids are higher than in Ham's F- 10, while those of the vitamins (except choline and inositol) and
potassium phosphate are reduced. Its composition must be modified (eg, by reducing the zinc concentration) for a protein- free culture of
elements contaminating water or raw materials are necessary for a protein- free culture of CHO cells in Ham's F- 12.
Kaighn's modified Ham's F- 12
(Ham's F- 12K) (Kaighn 1974)
The concentrations of the amino acids, pyruvate, biotin, calcium, magnesium, putrescine, and phenol red are increased with respect to those in
Ham's F- 12, among other compositional modifications, in order to support the proliferation and differentiation of primary cultured cells
(Continues)
4
|
YAO And ASAYAMA
Category Name (author, year) Features
Roswell Park
lymphocytes, and hybridomas
and Development
media
1977)
1984)
serum- free culture of human vascular endothelial cells
1979)
-
ments of a variety of cell types, it is most often used as a basal medium for serum- free culture
serum- free medium with added insulin, transferrin, ethanolamine, and selenite
Other media
(Waymouth 1959)
Developed with as simple a composition as feasible, so that mouse L929 cells could be cultured without the addition of serum and other
proteins, it is composed of a total of 40 components, including glucose, inorganic salts, amino acids, vitamins, purine bases, and hypoxan-
thine. It is characterized by high concentrations of glucose, histidine, lysine, glutamine, choline, and thiamine
Trowell's T- 8 (Trowell 1959) Designed for the long- term culture of adult rat liver epithelial cells. With its comparatively simple composition, it contains no non- essential
amino acid and hardly any vitamins, but is characterized by high glucose and insulin concentrations. It is used for short- term organ culture
Leibovitz's L- 15 (Leibovitz 1963) The buffering capacity is mediated by phosphates and free basic amino acids instead of sodium bicarbonate, so that a culture's pH is
maintained in ambient air without the use of a CO2 incubator. Instead of glucose, pyruvate (and galactose) is added at a high concentration in
order to control pH drops due to the lactic acid that is produced during glucose metabolism and to promote the release of CO2 from the
concentration. Once used for cell and tissue transport and primary cultures, its popularity has declined as researchers began to use HEPES as
a buffering agent and as they realized, moreover, that a certain amount of sodium bicarbonate is necessary for optimal cell proliferation
Sartorelli 1964)
Contains a high concentration of folate because this medium was developed by using folate- dependent L5178Y lymphoma cells
HEPES, 4- (2- hydroxyethyl)- 1- piperazineethanesulfonic acid.
TABLE2(Continued)
|
5
YAO And ASAYAMA
2.2| Use of natural media (1907- )
the vascular suture and transplantation of blood vessels and organs.
He contributed greatly to tissue culture technology by devising a pro-
totype of the cell culture flask that is used widely today and by estab-
lishing the aseptic manipulation technique.16
The first success of animal cell culture by Harrison inspired Carrel
in 1909. There, Burrows found that lymph is unsuitable for the culti-
vation of cells from warm- blooded animals and used plasma instead.
Thereafter, blood plasma had become a major culture medium for a
variety of animal cells. He successfully cultivated chicken embryonic
cells by using chicken blood plasma, which is readily available,17 and
later successfully cultivated mammalian cells as well.18 In 1912, Carrel
demonstrated that the long- term cultivation of the cells that have been
obtained from the connective tissues of chick fetuses is possible (for
several months) with a periodic exchange of the medium.19 In 1913,
he discovered that adding embryonic extract to blood plasma can dra-
matically increase cellular proliferation and extend the culture period
of fibroblasts from the chick embryo heart.20,21
the composition of the lymph, plasma, and embryonic extract was un-
known, it became a new scientific inquiry regarding which of their com-
ponents affected the survival and growth of animal tissues and cells.
This situation led to a period when researchers attempted to identify
the growth- promoting substances within these ingredients of natural
origin and to replace them with ingredients of definite composition.
Carrel is widely believed to be the first person in the world to
successfully culture mammalian somatic cells, but the Biographical
-
22
2.3| Endeavors toward synthetic media (1911- )
the Locke–Lewis solution—which is modified Locke's solution that ad-
ditionally contains amino acids, bouillon, and glucose (or maltose)—is
more effective for chick embryo cell cultivation than simple balanced
salt solutions.23,24 They reported that glucose is especially important:
if the concentration of glucose is not sufficient in the medium, the
chick embryo cells show vacuolar degeneration and die within a few
days.25 In contrast, in their search for the active ingredients in em-
bryonic extract, it was ascertained that the active substance is in the
protein fraction26 and that the partially hydrolyzed proteins effec-
tively promote the cell growth of chick embryo fibroblasts.27 Those
researchers also confirmed the growth- promoting activity of amino
acids28 and glutathione for chick embryo fibroblasts.29 They hypoth-
esized that glutathione is required for the control of the redox envi-
ronment during cell cultivation. Carrel's medium was supplemented
with several natural products, such as casein digests (thymus- derived)
nucleic acids, liver ash, and hemoglobin. Thus, their research did not
lead to the molecular identification of any substance that is essential
contrast, confirmed the effectiveness of two hormones (insulin and
thyroxine) by successfully cultivating human fibroblasts for >3 months
in a medium that consisted of Ringer's solution with these hormones,
along with glucose, cysteine, hemin, peptone, and blood plasma.30
Researchers continued the efforts to specify the composition of media
thereafter, including the development of Baker's medium, which con-
1 , vitamin B2, glutathione, and
blood plasma.31 Nevertheless, this approach did not lead to culture
media that did not include natural products, such as blood serum or
plasma.
2.4| Birth of established cell lines (1940- )
It is rare for healthy somatic cells that are derived from animals to
acquire unlimited proliferative capacity during cultivation. They typi-
cally stop growing after a certain number of divisions (ie, the Hayflick
limit).32
that had been sampled from animals in each experiment. In 1940,
Wilton R. Earle et al. used carcinogens to successfully create immortal
mouse fibroblasts (L cells),10 revealing that proliferation from a single
cell is possible.33
an infinitely proliferating human cell line from a tissue of a patient with
uterine cervical cancer (HeLa cells).34 Due to the emergence of these
established cell lines, the sampling of cells from the tissues of animals
in each experiment became unnecessary, enabling researchers world-
wide to perform assays by using the same homogenous population of
cells. This state of affairs made it easier to examine and to precisely
quantify the subtle differences in the effects of culture media on cells.
Thus, the development of culture media advanced rapidly as a result.
2.5| Establishment of basal media and research into
protein- free media (1946- )
Baker's medium and the other media that had been developed up to
this point contained naturally derived components of unknown compo-
sition, including plasma, serum, bouillon, peptone, and tissue extracts.
In order to find the crucial components in those natural materials and
to develop defined media that are comparably efficient in the cultiva-
tion of cells, relative to the media containing natural ingredients, two
main strategies were undertaken. The first strategy was to use dialyzed
serum for the support of cells at minimum levels and to add defined
components to maximize the proliferation of cells. The second strategy
was not dependent on serum, or even proteins at all, and involved the
formulation of media exclusively from definitive components.
Fischer was a pioneer of the first strategy. He dialyzed blood plasma
to remove the low- molecular- weight fraction. Culture media that were
supplemented with dialyzed blood plasma could sustain cells only for
a short period, indicating that the low- molecular- weight fraction was
essential for the survival of cells. Then, he discovered that the amino
acids are the key substance in the low- molecular- weight fraction.35,36
This creation of dialyzed media, along with the established cell lines,
6
|
YAO And ASAYAMA
made it possible to determine, in a systemic way, whether the cells
require low- molecular- weight substances under culture conditions.
For example, in 1955, Harry Eagle studied the minimum necessary
amounts of low- molecular- weight components that are required by
mouse L cells and HeLa cells by using a balanced salt solution with the
addition of dialyzed serum, on the basis of Fischer's method.37,38 He
found that 13 amino acids and eight vitamins are necessary.39
on to study the amino acid requirements of a variety of cells, Eagle
-
posed of the minimal essential components that he identified (glucose,
six inorganic salts, 13 amino acids, eight water- soluble vitamins, and
dialyzed serum).40 41 Clifford
P. Stanners et al.,42 43 made
purpose (Table 2). Later, using dialyzed serum that had been prepared
-
firmed that carcinosarcoma cells require pyruvate.44
45 in terms of its calcium and magne-
medium for use in lymphocyte cultivation.46,47
The other strategy—the total omission of serum and proteins—
is thought to have begun in 1946, with full- scale research by Philip R.
White. He developed a chemically defined medium that was composed
of glucose, inorganic salts, amino acids, iron, vitamins, and glutathione,
with no protein at all. Using this medium, he successfully cultivated chick
embryo- derived fibroblasts and cardiac muscle cells for ~2 months.48
Other researchers were unable to repeat his findings and argued that
49
vitamins, cholesterol, and nucleic acid precursors, in addition to the ingre-
Parker's team used the strategy of adding as many low- molecular- weight
substances as possible, according to what was considered theoretically
necessary for cell culture.50 Using this medium, they cultured chick
embryo- derived cells for 3- 4 weeks. Serum and embryonic extract were
still necessary for the primary culture. Other researchers also reported
-
eration.37
consisting of 58 components, was developed through several modifi-
cations, such as increasing the levels of reducing substances (cysteine,
glutathione, and ascorbic acid), removing fat- soluble vitamins, chang-
ing the nucleic acid precursors, and adding coenzymes (Table 2).51,52 In
contrast, during the same period, the National Cancer Institute's Tissue
Culture Section (NCTC), led by Wilton R. Earle, developed a chemically
defined medium that was composed of 68 ingredients, named NCTC109
(Table 2).53 These two media were difficult to prepare because of their
752/1 medium (Table 2), which is composed of 40 ingredients (glucose,
inorganic salts, amino acids, vitamins, purine bases, hypoxanthine, and
glutathione). This was the simplest- possible chemically defined medium at
the time.54 -
signed for the best growth of mouse L cells. Therefore, they were not nec-
essarily suitable for the serum- free culture of other cell types. In addition,
cell cloning was not possible in those protein- free media. Waymouth hy-
pothesized that the proteins that are produced by cells are necessary for
proliferation under protein- free conditions: the so- called "auto/paracrine
medium in 1963 (Table 2). With two kinds of serum protein fractions (al-
bumin and fetuin) instead of serum, he successfully made a single Chinese
hamster ovary (CHO) cell form a colony under serum- free conditions.53
The composition of this medium, however, was undefined because it con-
tained serum protein fractions and the proliferative capacity it conferred
fell short of that induced by serum- containing media. There was also a
challenge: serum supplementation remained necessary for the cultivation
of cell types other than CHO cells. In order to solve these issues, Ham
replaced the albumin and fetuin of biological origin with low- molecular-
weight substances—linoleic acid and putrescine55,56—and developed
Ham's F- 12, a completely synthetic medium of definite composition.
During this refinement of the medium, the concentration of each ingre-
dient in Ham's F- 10 was reviewed and modified (Table 2).57 With the re-
duced amount of zinc, this medium supported colony formation by mouse
L cells, but protein- free cultivation of other cells remained difficult.58,59
It became clear in later studies that trace elements contaminating the
water or raw materials they used are necessary for the protein- free culti-
vation of CHO cells in Ham's F- 12 medium, leading to the development
(Table 2).60 Toshiko Takaoka and Hajim Katsuta successfully cultured var-
ious kinds of cells long- term in a simple medium that did not contain pro-
teins or lipids;61 however, only a few cell types can adapt to protein- and
lipid- free conditions and the fatty- acid composition of the cells thus cul-
tured differs greatly from the cells cultured in serum- containing media.62
In the field of life sciences, the "reductive approach" and the "syn-
thetic approach" are often used for elucidating a complicated biological
process. The reductive approach is used to analyze and identify the es-
sential parts in the complex biological process. The synthetic approach
is used to reconstruct the biological process by putting the known parts
together. Both approaches were instrumental in the development of
synthetic culture media because these approaches had disadvantages
relative to each other. First, the reductive approach was undertaken
strenuously by Carrel and others because the synthetic approach was
almost impossible without any information about the active substances
for the cultivation of cells; accordingly, the substances that were im-
portant for cell culture in natural media were lined up. In contrast, the
reductive approach could not be entirely successful at elucidating the
complexities of the natural media because of the quantitative and qual-
itative limitations of the analytical method, complicated components of
the candidate substances based on the knowledge of the reductive
approach were tested by means of the synthetic approach by Eagle
and others and the substances that are essential for cell culture were
eventually identified. It seems to be important when and how these
approaches are used for the successful development of culture media.
|
7
YAO And ASAYAMA
2.6| Identification of serum substitutes and the
development of serum- free media tailored to a cell
type (1970- )
Insulin was discovered earlier by Frederick Banting and Charles Best
(1921), but full- scale research into this peptide as a supplement for
culture media began in the 1960s.63,64 Initially, the effectiveness of in-
sulin alone was found to be inferior to that of serum,65,66 but the use of
insulin in combination with low- concentration serum yielded a higher
level of efficacy of baby hamster kidney (BHK)21 cell growth. This
finding led researchers to conclude that insulin acts in a coordinated
one after another during this era: nerve growth factor,67 epidermal
growth factor,68,69 insulin- like growth factor,70–73 fibroblast growth
74,75 platelet- derived growth factor,76–78 and transform-
79,80 The addition of these growth factors to
a culture medium increased cellular proliferation. Nevertheless, their
effect on cell proliferation, as with insulin, was found to be almost
always inferior to the effect of serum.81–85
Under these circumstances, in 1976, three key reports were pub-
lished that accelerated the development of serum- free media. Ham's
group discovered that a trace element of selenite is necessary for the
serum- free cultivation of human diploid cells86
and Iscove showed that, besides selenite, a combination of transferrin
and albumin is a good serum substitute.87
H. Sato discovered that a combination of several hormones and
growth factors is an effective serum substitute.58 Prompted by their
discoveries, attempts at serum- free culture by using serum substitutes
(eg, several hormones and growth factors, transferrin, and selenite;
Table 3) grew in number and a variety of serum- free media was devel-
oped, with each medium tailored to researchers' cell type of interest.
When developing serum- free culture media, researchers typically
insulin, transferrin, and selenite,88 ITS, a supplement that contains a mix-
ture of these three substances, has become commercially available. In
the serum- free cultivation of hybridomas and developed the supplement,
ITES, which consists of ITS plus ethanolamine.89 Various other supple-
ments were designed for different cell types and cultivation purposes,
with the aim of the addition of various trace elements to protein- free
culture media. These include Synthetic Serum Replacement, in which
various trace elements are stabilized by ethylenediaminetetraacetic acid,
citric acid, and aurintricarboxylic acid;90 the B- 27 supplement that was
created for use with nerve cells (it contains progesterone, putrescine,
glutathione, in addition to ITS);91,92 and Knockout Serum Replacement
amino acids, antioxidants, ITS, and trace elements).93
2.7| Improvements to basal media (1970- )
In addition to being a source of hormones, growth factors, carrier proteins,
and lipids, serum increases the levels of various low- molecular- weight
which serum is excluded were sometimes unable to adequately support
cell growth.94
-
ter performance occasionally when used for certain types of cells.95,96
The reason seems to be the large number of constituents in Ham's F- 12
mixing the two allows each to complement the weaknesses of the other.
97
media, however, do not always show a level of performance that is bet-
ter than that of a single medium. For example, the ferrous sulfate that is
contained in Ham's F- 12 is toxic to nerve cells and nerve cells proliferate
-
91 Naturally, the composition of a
basal medium that is used for serum- free culture should be optimized for
each cell type. In addition, it seems that the optimization also depends on
the scale of the culture and its method.98
2.8| Medical and industrial applications of animal-
cell culture technology (1978- )
2.8.1| Culture media for the production of
recombinant pharmaceuticals
Inspired by the 1982 clinical application of recombinant human insulin
expressed in Escherichia coli, researchers actively proceeded to pro-
duce growth hormones, interferon α, and other substances by using
E. coli or yeast as a host. With E. coli and yeast, however, it was impos-
to be used for the production of recombinant proteins, like tissue
plasminogen activator, erythropoietin, interferon β, and monoclonal
antibodies. The host cells that have been used in the manufacture of
biopharmaceutical products include CHO cells, mouse myeloma NS0
cells, BHK cells, human embryonic kidney 293 cells, and human retinal
popular in the field of biopharmaceutical manufacturing for the fol-
lowing reasons: (1) technological advances in mass- culture methods
for these two cell lines; (2) sufficient knowledge about the safety of
viruses that these two cell lines contain; and (3) remarkable advances in
high- expression sublines that were derived from these two cell lines.99
In order to enhance the efficiency of the production of biophar-
maceuticals, one must increase the production rate of the target pro-
tein in a culture medium that contains none or a minimal amount of
ingredients of biological origin, like serum, because they significantly
hamper the process of product purification. Research in this direction
has been conducted to efficiently optimize the medium's composition,
for example, by means of approaches that are based on the monitoring
of changes in the concentration of the medium components and by-
products in the culture,100 as well as genomics- and proteomics- based
8
|
YAO And ASAYAMA
TABLE3Characteristics and limitations of serum substitutes
Category Name Characteristic Limitations
Serum, tissue
extracts
For example, fetal bovine
serum protein,bovine
pituitary extract
Contain various components, including proteins
and lipids of serum or tissue origin and
contribute to improved cellular proliferation
and survival
Composition is undefined and therefore
there is large lot- to- lot variation and a high
risk of contamination; for example, by
viruses
Hydrolysates For instance, animal-
derived (animal tissues,
milk), microorganism-
derived (yeast),
plant- derived (soy,
wheat, rice)
Supply cells with vitamins, lipids, inorganic salts,
low- molecular- weight peptides, and amino
acids. Confirmed efficacy for culturing Chinese
hamster ovary, hybridoma, baby hamster
kidney, Vero, and lymph cells. Purification of
antibodies and recombinant proteins is
simplified because the components in question
contain only low- molecular- weight substances,
owing to ultrafiltration. Very low- cost, as
compared with serum
Composition is undefined; thus, there is a
-
nation, for example, by viruses when the
origin is from animals. The risk is non- zero
even when the origin is plants: for instance,
when in contact with animals or animal-
source products during cultivation or the
manufacturing process. Caution is necessary
because the raw materials could have been
exposed to high concentrations of
pesticides or herbicides
induce proliferation, differentiation, migration,
supplementation of the medium with growth
present on the surface of the target cells;
therefore, heparin (or synthetic dextran as a
substitute) is added to the medium in some
situations
contamination; for example, by viruses. The
use of recombinant proteins reduces the
risk of contamination; however, the risk is
non- zero because the proteins could have
been produced by means of animal- derived
enzymes in the manufacturing process and
for other reasons. Released from platelets,
β acts as a growth inhibitor on many
epithelial cells
Hormones For example, growth
hormone, insulin,
hydrocortisone,
triiodothyronine,
estrogen, androgens,
progesterone, prolactin,
follicle- stimulating
hormone, gastrin-
releasing peptide
proliferation of a variety of cells.
Hydrocortisone improves the cloning efficiency
of the glial cells and fibroblasts and is
necessary for the maintenance of the
epidermal keratinocytes and several other
endothelial cell types. Triiodothyronine is
hydrocortisone and prolactin, various
combinations of estrogen, androgens, and
progesterone are necessary for the mainte-
nance of the mammary epithelium
Insulin is unstable at 37°C (especially in the
presence of a high concentration of
cysteine) and therefore must be added to a
medium at a comparatively high concentra-
tion. In addition, zinc is necessary for insulin
to exert its biological action and researchers
ideally should use a zinc- supplemented
medium. The hydrocortisone that is present
in the fetal bovine serum acts as a growth
inhibitor in high- density cultures (many cells
that are closely packed; eg, glial cells,
pulmonary epithelial cells). Conversely, it
sometimes promotes growth in low- density
cultures
Carrier proteins
lactoferrin, and others
substances, including lipids (eg, fatty acids,
cholesterol), trace elements (eg, copper, nickel),
amino acids (cysteine, tryptophan), and
vitamins (pyridoxal phosphate: ie, the active
form of vitamin B6
an aqueous solution alone, they are more
effectively supplied to cells after the formation
of complexes with albumin. In addition,
albumin has toxin- neutralizing, antioxidant, and
shear stress- reducing effects. Transferrin is
used as a carrier of iron. Lactoferrin can serve
as a substitute for transferrin
If these agents are serum- derived, there is a
risk of contamination; for example, by
in distribution today is purified from corn by
using the cold ethanol fractionation method:
the products that are prepared this way
contain lower proportions of other proteins.
elements that are bound to albumin vary
from lot to lot. Sometimes, differences
between lots are observed as a result:
researchers should perform batch screening
before using these products. Serum- derived
transferrins include compounds of porcine,
transferrin typically has low activity,
researchers must in some cases work
around this issue; for example, by raising the
concentration
(Continues)
|
9
YAO And ASAYAMA
Category Name Characteristic Limitations
Lipids and related
components
Cholesterol, steroids, fatty
acids (eg, palmitate,
stearate, oleate,
linoleate), ethanolamine,
choline, inositol, and
others
Serve various roles: as membrane components,
in nutrient storage and transport, and in signal
biosynthesize the lipids that are necessary for
adding the lipids to the medium lessens the
biosynthetic load. In addition, some cells lack
the enzymes that are necessary for the
cholesterol- biosynthetic pathway: a source of
sterols must be added to the medium in order
to culture such cells. The use of lipoproteins
and albumin is the physiologically closest and
most effective way to solubilize proteins and
deliver them to the cells
When building a culture medium under
protein- free conditions, researchers must
use ethanol, surfactants (eg, Pluronic F- 68,
Tween 80), or cyclodextrin to solubilize the
lipids. If ethanol is used for solubilization,
one must typically add a quantity of
on cells. Caution is advised when using
surfactants and cyclodextrin: they are toxic
to cells at high concentrations, result in poor
lipid solubility at low concentrations, and
commercially available lipids are animal-
derived and their performance varies
Transition metals For example, Fe, Zn, Cu,
These are transition elements because they
readily undergo electron transfer, so they
function in the active centers of enzymes and
physiologically active substances inside the
cell. Se, Fe, Cu, and Zn, in particular, generally
are used in cell culture. Se has an antioxidant
activity in the form of selenoproteins, such as
glutathione peroxidase and thioredoxin
reductase
Chelating agents can serve as a substitute for
the Fe carrier, transferring, in cases where it
must be removed from the medium. Caution
is still necessary: depending on the species
and concentration of the chelating agent,
not only could it be ineffective as a carrier,
it might also promote the production of
reactive oxygen species
Vitamins
E, K), water- soluble
vitamins (eg, B1 , B2, B6,
B12 , C, folate)
Necessary for cell division and growth as
precursors of various cofactors. Vitamins C and
E additionally have antioxidant effects.
Vitamins are present in most of the basal
media, but their types and amounts (especially
of the fat- soluble vitamins) are limited in some
situations; therefore, they are added according
to the needs of the cell type
by air oxidation. Vitamin C, moreover, reacts
with trace elements and oxidatively
decomposes, sometimes generating reactive
1 , B2, B12, C,
and K are readily degraded by light; vitamins
B1 and B5 are easily degraded by heat.
Folate has poor solubility and is partially
removed during filtration sterilization in
some cases. Hydroxocobalamin and vitamin
C interact, promoting mutual degradation
Polyamines Putrescine, spermidine,
spermine
Low- molecular- weight, basic, physiologically
active amines that exist ubiquitously in cells
and promote protein or nucleic- acid synthesis.
Intracellular concentrations of polyamines are
regulated and maintained both by biosynthesis
or decomposition inside the cell and by
transport from outside the cell
Cell growth halts if the intracellular
polyamine concentrations drop too low due
to a disrupted balance among polyamine
biosynthesis, decomposition, and transport.
polyamine concentration rises too much
Reductants 2- mercaptoethanol,
α- thioglycerol, reduced
glutathione
Import of cystine or cysteine into cells is
necessary to maintain the intracellular redox
environment and the addition of reducing
agents to the culture medium of cells that lack
cystine transporters (eg, lymphocytes and
embryonic stem cells) converts cystine into
cysteine, which the cells then are able to
import
Caution is necessary when adding reducing
agents in the absence of albumin: this
approach can damage cells
Protective
additives,
detergents
Carboxymethyl cellulose,
polyvinyl pyrrolidone,
Pluronic F- 68, Tween
80, and others
Reduce the shear stress generated in stirred
cultures and by pipette manipulation. Pluronic
F- 68 and Tween 80 also are used as solubiliz-
ers of lipophilic substances (eg, lipids,
fat- soluble vitamins)
Surfactants sometimes show cytotoxicity,
depending on their concentration
For example, fibronectin,
laminin
Promote the adhesion of anchorage- dependent
cells to vessels
Pose a risk of viral contamination if
components of biological origin are used
-
TABLE3(Continued)
10
|
YAO And ASAYAMA
approaches.101 Through such efforts, as well as host cell modifica-
tions,102 the per- cell production yield has increased nearly 10- fold
from 1986 to 2004.103 The composition of the various culture media
that are used in biopharmaceutical manufacturing today has not been
disclosed for commercial reasons, but the composition of a previously
reported serum- free culture medium that is used for CHO cells is de-
tailed in Table 4 for reference.
2.8.2| Culture media for use with pluripotent
stem cells
in 1998 and human iPS cells by Shinya Yamanaka et al. in 2007, the
demand for these cells has increased rapidly due to their usefulness in
basic and clinical studies for regenerative medicine, as well as in a vari-
ety of possible applications, such as disease modeling, drug discovery,
and cytotoxicity studies. Thus, culturing these cells in a simple, low-
cost, and highly productive way became an important issue. These
cells initially were cultivated on a layer of feeder cells in a serum- or
KSR- supplemented medium,104 but in anticipation of clinical applica-
tions, the efforts shifted to the development of culture conditions
that are feeder- free and xeno- free (Table 5).105–111
with insulin, sodium selenite, transferrin, ascorbic acid (stable form),
β1 (or Nodal), and sodium bicarbonate—this medium al-
lows for the long- term growth of human iPS cells.109 -
cent years, developments have continued in the field of culture media
that contain low- molecular- weight compounds instead of expensive
β1; Table 5).112
2.8.3| Culture media for use in assisted
reproductive technology
The development of in vitro fertilization, embryo culture, and embryo
transfer technologies has progressed on the basis of animal experi-
ments, primarily on rabbits and mice.113–116 In 1978, the first clinical
application was successful, as implemented by Patrick C. Steptoe and
117 The medium they preferred to use for human
zygote cultures was Ham's F- 10, supplemented with serum.118 It was
later revealed, however, that hypoxanthine and the trace elements
in Ham's F- 10 negatively affected the embryos via the production of
reactive oxygen species.119–122 The metabolism of a pre- implantation
embryo differs greatly from that of somatic cells; therefore, the em-
bryo can be cultured up to the blastocyst stage by using a simple me-
dium that consists of a balanced salt solution that is supplemented
with glucose, pyruvate, lactate, and albumin.123 The concentration of
glucose that was used usually for somatic cells in those days, which
was as high as 5.5 mmol L, turned out to be unfavorable to zygote
cleavage. Therefore, glucose is often added to the culture medium for
use with zygotes at the low concentration of 0.2- 0.5 mmol L .124 The
detailed composition of the embryo culture media that is used clini-
cally has not been published to date for commercial reasons; however,
125 it resembles either the
126 or the
medium that was developed by Biggers et al.127 -
mation on the culture media for assisted reproductive technology is
available elsewhere.124
3| SELECTION OF A BASAL MEDIUM
Each basal medium has been designed in each case on the basis of
the cell type, the origin (animal species), and the purpose of the cul-
turing. In fact, the medium composition can differ greatly depending
on such background factors. Whether supplementation with natu-
ral products is allowed is another important presupposition for the
was designed under the assumption of serum supplementation and
accordingly includes only the minimum necessary components (inor-
ganic salts, sugar, essential amino acids, and water- soluble vitamins).
culture, contain various other components. Regarding the selection
of basal media, readers can be referred to the literature and suppliers'
TABLE4Serum- free culture media for Chinese hamster ovary cells
Name (author[s], year) Basal media Supplements Remarks
(Hamilton and Ham 1977)
Ham's F- 12
are not present in Ham's F- 12
3
Insulin, transferrin, and selenite Developed because Chinese hamster
ovary cells could not be cultured in the
(Keen and Rapson 1995)
ferric citrate, insulin, ethanolamine, putrescine,
Pluronic F- 68, and soy peptone
Lacking high- molecular- weight proteins,
it was developed for use with
citrate is used instead of transferrin
Name unspecified
(Sung and Lee 2009)
Zn), ferric citrate, selenite, insulin, ethanolamine,
phosphatidylcholine, hydrocortisone, putrescine,
pyruvate, ascorbate, Pluronic F- 68, dextran sulfate,
and a hydrolysate mixture (yeast, soy, and wheat)
The combination and concentrations of
the added hydrolysates were
determined by using an experimental
design method. It was developed to
increase antibody productivity
|
11
YAO And ASAYAMA
information; for instance, cell banks' sites (eg, www.atcc.org, www.
phe-culturecollections.org.uk, cellbank.nibiohn.go.jp, and cell.brc.
riken.jp). Table 2 shows the basic characteristics of each medium and
researchers can select several candidates after comparing them ex-
perimentally. α
-
3.1| Selection of a basal medium: the roles of the
medium components
3.1.1| Serum
Serum serves as a source of amino acids, proteins, vitamins, carbohy-
drates, lipids, hormones, growth factors, inorganic salts, trace elements,
and other compounds. It also improves the pH- buffering capacity of
the medium and helps to reduce shear stress (ie, physical damage that
is caused by pipette manipulation and stirring). Furthermore, serum
alters the conditions at a culture substratum, allowing the adherent
cells to readily proliferate there. Fetal bovine serum (FBS) is the most
popular and widely applicable serum today. Other kinds of serum are
used in certain situations, including calf serum (CS) and horse serum.
Researchers can select an appropriate serum type on the basis of its
characteristics. Fetal bovine serum generally is rich in growth fac-
tors and contains low levels of γ- globulins (which have a cell growth-
inhibitory activity). Thus, it is suitable for cells that are difficult to
coax to proliferate in culture and for the cloning of cells. In contrast,
because CS has a weak growth- promoting activity, it has been used
effectively for studies of contact inhibition on the 3T3 cell line. It is
also suitable for cellular differentiation studies, in which growth fac-
tors can interfere with the results.128 Lipid levels in serum rise with
increasing calf age (ie, days after birth)129 and therefore CS or adult
bovine serum is sometimes selected, instead of FBS, when cells with
high lipid requirements are cultured. Horse serum that is harvested
from adult horses via a closed system of collection has a compara-
tively high homogeneity between lots. Its characteristics include a low
concentration of polyamine oxidase,130 which makes polyamines; the
latter have a cell- proliferative effect and are metabolically degraded
less readily.
3.1.2| Alternatives to serum
When serum supplementation is inappropriate or undesirable, re-
searchers can choose several substitutes for serum. They include
serum extracts, tissue extracts or hydrolysates, growth factors, hor-
mones, carrier proteins like albumin and transferrin, lipids, metals,
vitamins, polyamines, and reductants (Table 3). The number of com-
binations of these supplements is nearly infinite and they often inter-
act with each other. Their selection thus incurs an enormous effort,
time, and cost: one cannot design an optimal medium by simply try-
ing them in arbitrary combinations at random. Thorough studies on
past records of successful combinations, if available, are helpful. The
TABLE5Serum- free culture media for embryonic stem/induced pluripotent stem cells
Name (author[s], year) Basal media Supplements Remarks
Knockout Serum Replacement (KSR)
containing animal- source components (KSR),
it is for use with mouse embryonic stem cells.
The cultures require feeder cells
TeSR
(Ludwig et al. 2006)
selenite, LiCl, insulin, transferrin, human serum
β1, γ- aminobutyric acid, pipecolic
acid, glutathione, 2- mercaptoethanol, lipids (fatty
acids, cholesterol), Pluronic F- 68, and Tween 80
feeder cells
E8
(Chen et al. 2011)
β
NaHCO3
large between- lot variation) and
2- mercaptoethanol (which negatively affects
cells) were removed and supplements were
refined down to the necessary minimum
(Name undefined)
(Hasegawa et al.
2012)
transferrin, Wnt3a, and indole derivative (ID)- 8
(DYRK inhibitor)
β are replaced
with Wnt3a and the low- molecular- weight
conventional media
(Name undefined)
(Hasegawa et al.
2015)
β inhibitor (eg, 1- azakenpaullone), and
β inhibitor and
compounds), it can be manufactured cheaply,
and quality management is simple
factor of activated T cells.
12
|
YAO And ASAYAMA
following is an example of a protocol when researchers try to imple-
ment a serum- free culture by themselves:131
1.
2. If using glutamine, set its concentration to 2–4 mmol L and
consider the stable form: L-alanyl-L-glutamine (see the next
section).
3.
according to the requirements of the cell type under study.
4. Pay attention to the osmotic pressure.
5. Use antibiotics as little as possible.
6. When culturing adherent cells, consider using substrates, such as
fibronectin and laminin, for cell attachment.
7. For a stirred culture, consider supplementing the medium with pro-
tective agents like Pluronic F-68 to minimize the shear force.
8.
9. Verify that the cell performance has not changed in the new
medium.
The serum- free and protein- free media that are sold by a vari-
ety of manufacturers largely show good performance and research-
ers can use them to culture their cells of interest. Nonetheless, their
composition almost has never been disclosed for commercial reasons,
and therefore, technically, such media cannot be termed "chemically
defined."
4.| POINTS OF CAUTION WHEN USING
CULTURE MEDIA
4.1| Setting up a serum- free culture
There are two points to note when working with a serum- free cul-
ture system. First, unintended clones of cells in a culture vessel can be
selected during subculturing because serum- free media can promote
the proliferation of a particular cell clone or subtype of cell more often
than can serum- containing media. Second, impurities in a serum- free
medium, both avoidable and inevitable, might have stronger effects
on the cultured cells than can serum- containing media because of a
lack of the toxin- neutralizing activity that serum contains. In addition,
there are other precautions for serum- free cultures: that is, the mini-
mization of the concentration of trypsin and the careful selection of
adhesion factors.57
4.2| pH changes
The pH of the culture medium that is used for animal- cell cultures
generally is maintained by the equilibrium relationship between so-
dium bicarbonate (NaHCO3) in the culture medium and CO2 in the
2 concentration in
the incubator to 5%- 6% by convention, but technically, it should
be adjusted according to the concentration of the NaHCO3 in the
culture medium. For example, 5% CO2 is appropriate for a medium
with 26 mmol L NaHCO3 2 for
a medium with 14 mmol L added (eg, Ham's F- 12 medium), and
10% CO2 for a medium with 44 mmol L NaHCO3
medium). These numbers are theoretical values that are calculated
by using the Henderson–Hasselbalch equation (Fig. 1): in practice,
checking the pH of a culture medium after it reaches equilibrium
and then making minute adjustments to the CO2 concentration are
preferable.
The 4- (2- hydroxyethyl)- 1- piperazineethanesulfonic acid (HEPES),
with its strong buffering capacity, might have buffering effects in a
medium. Especially when cultured cells are handled outside the CO2
incubator, the pH of the medium with HEPES is more stable than with
only bicarbonate. It also might be beneficial for cultures of high cell
density; for instance, when the pH can drop rapidly due to an ac-
cumulation of metabolites, such as lactate. With serum- free media,
which lack the buffering capacity of serum, the pH- buffering role of
HEPES could be more relevant. Nevertheless, HEPES might have neg-
ative effects on certain types of cells, such as chick embryo epiphy-
seal chondrocytes and ES cells.107,132 It also has been demonstrated
that HEPES- containing media produce an increased level of cytotoxic
products, mostly hydrogen peroxide, during exposure of the medium
to visible light.133 Researchers should be aware of such drawbacks of
FIGURE1The pH control mechanism of culture media, based
on the bicarbonate buffer system and the Henderson–Hasselbalch
equation. When dissolved in water, sodium bicarbonate (NaHCO3 )
dissociates to form a sodium ion (Na+) and a bicarbonate ion
(HCO3
). The latter reacts with H+ in solution to form carbonic acid
(H2 CO3 ), which dissociates into CO2 and H2O. These two reactions
attain their respective equilibria. The CO2 in solution also reaches
equilibrium with CO2
concentration of gas phase CO2 increases the amount of CO2
that is dissolved in the culture medium, in turn raising the H2 CO3
concentration and lowering the pH. In contrast, if the concentration
of the gas phase CO2 is lowered, then the pH rises due to the
reverse reaction. The relationship between the culture medium
pH and the concentrations of CO2 and NaHCO3 can be expressed
by the Henderson–Hasselbalch equation: pH=p Ka+log[HCO3
[CO2 Liquid phase , where: p Ka is the negative log of the acid
dissociation constant.
|
13
YAO And ASAYAMA
HEPES and confirm that the addition of HEPES is harmless to the cells
that they use.
Phenol red, added to a medium as a pH indicator, sometimes af-
fects the proliferation of cells and shows estrogenic activity.134,135
Therefore, investigators should not forget to test whether such char-
acteristics of phenol red affect their cells and empirical results, es-
pecially when estrogen- sensitive cells, like mammary gland cells, are
involved.
4.3| Oxidative stress
Culture experiments in general and especially when performed under
a higher oxygen concentration, such as organ culture experiments,
can expose cells to oxidative stress, which negatively affects the
cells and tissues in culture. Supplementation with substances with
β-
mercaptoethanol, dithiothreitol, or lipoic acid) is effective, especially
when serum (it contains antioxidants) is not added to the medium.
In addition, iron and copper ions in a free state promote the produc-
tion of reactive oxygen species. It is therefore best for these ions to
be complexed with appropriate carriers (eg, transferrin, albumin, or
chelating agents) and to be supplied to the cells in this state, while
they are isolated from reactive systems. Caution is also necessary with
respect to free iron ions, which are readily hydroxylated and precipi-
tate in an aqueous solution.
4.4| Nutrient requirements
Sufficient amounts of nutrients in the medium are a prerequisite for
cells to behave properly. Some cell types require higher levels of nutri-
ents than others do, depending on their metabolic activity and prolif-
eration rate. Such characteristics of cells should be taken into account
-
nally to contain glucose at 5.6 mmol L. Now, a modified version of
, which can be
used for cells requiring greater amounts of glucose, is available from
various suppliers. One caveat when using this high- glucose medium
for actively proliferating cells is the accumulation of metabolites, like
lactate, and a plunge in the pH. Researchers are advised to replace the
medium at proper intervals or to use HEPES to confer a stronger pH-
buffering capacity onto the medium.
for mammalian cells in culture, in addition to being a biosynthetic
material for nucleic acids and proteins. The glutamine requirements
for cells in culture are ~3- 40- fold greater than those of other amino
acids.40 During the cultivation of cells with high nutrient requirements,
the addition of glutamine could be helpful. In contrast, glutamine
readily decomposes in culture media and generates cytotoxic ammo-
nia. Consequently, researchers may consider adding glutamine to the
medium immediately before use or to consider using glutamine deriv-
atives, such as L- alanyl- L- glutamine or glycyl- L- glutamine, which are
resistant to degradation.
medium, therefore, could ensure more favorable culture conditions.
alleviates the biosynthetic load of the cells.
isoleucine, require special attention. They all belong to the group of es-
sential amino acids and several cell types, including human fibroblasts
proper intervals can be considered in order to obtain the best results
of an experiment.
5| CURRENT ISSUES WITH
CULTURE MEDIA
5.1| Design of experiments for the optimization of
the medium components
a basal medium and several supplements. Researchers pay no atten-
tion to the possible interactions among the components of the basal
medium and these supplements. The fact is that each component of
a culture medium does not always act in isolation. The components
can interact and their effects on the cells should be expected to be
a medium component and its effect on the cells might not follow a
linear dependence. Cells' response to each component could be cur-
vilinear, as shown in Figure 2. Therefore, the best way to find the op-
timal concentration and interactions of each component in a culture
medium might be a statistical experimental strategy called a "design
of experiments" (DoE), rather than the classical one- factor- at- a- time
experiments. The latter approach might miss the optimal point of con-
centration because there are some unexamined areas in the range
design points, namely the concentrations of the components to be
examined, evenly throughout the area of settings. Then, the obtained
empirical data will be applied to the appropriate mathematical models,
such as the general linear model or the logistic regression model. In
general, when the data corresponding to the response variable (also
called the "objective variable") are continuous, such as the production
volume of a biopharmaceutical, the general linear model is used. With
binominal data, such as the survival rate of cells, the logistic regres-
sion model is preferable. By means of these models, an optimal con-
centration of each component (which is expected to induce the best
response of the cultured cells) will be estimated (Fig. 3B). The DoE
screening design is used first when one is trying to extract the most
important components from many, say more than five, candidates of
14
|
YAO And ASAYAMA
the initial components. The strength of the screening designs, such
as fractional factorial or the Plackett–Burman design, is that they can
reduce the number of experimental runs significantly, in comparison
with the number that otherwise has to be done with the full factorial
design, which involves every possible combination of components. For
consists of 60 components, only 64 experiments are needed with the
Plackett–Burman design, whereas 260 experiments are required with
the full factorial design. Once the key factors are identified, prefer-
ably eight or fewer, a response surface design, such as the central
composite design or Box–Behnken design, will be used to estimate
a non- linear response of the cells and to identify the optimized con-
centrations and interactions of each component. These approaches
have become a powerful tool for the improvement of productivity in
the field of cell culture.136
a "definitive screening design," recently was reported, with the claim
that it has the advantages of both a screening design and a response
surface design.137 This design enables the estimation of not only the
main effects (the effect of a certain component alone) but also the in-
teractions of the components and the factors with non- linear effects,
only 121 experiments are needed. This approach could be especially
useful for studies where the sample size is limited, such as a primary
culture, organ culture, and embryo culture, although there are some
restrictions in this design. The establishment of effective develop-
ment systems for culture media that combine in vitro and in silico
approaches and the optimization of the combinations of all the com-
ponents in the culture medium are crucial for further improvements in
cell culture performance.
5.2| Undefined medium supplements
Supplements of biogenic origin, like serum, can be a cause of varia-
tion in the experimental results from batch to batch. They also carry
a risk of microbial contamination of the culture medium. Thus, the
replacement of those supplements with defined ones has been pur-
sued in the history of culture media, as described above.43,87
dawn of the technology, a human embryo was cultured in a medium
containing serum. In the mid- 1980s, the serum could be replaced by
serum albumin.138
and is multifunctional. It binds to various water- insoluble substances
like lipids. The lipids that are carried by albumin become an energy
source and biosynthetic substances for an embryo.139–141 In addition,
serum albumin serves as an antioxidant, osmotic regulator, and neu-
tralizer of toxins. These functions are key benefits that serum usu-
ally provides to the media. Nonetheless, the use of serum albumin
in place of serum has not contributed much to the development of
chemically defined media. First, most, if not all, commercial serum
albumin versions contain >100 serum proteins, although these ad-
mixtures have very low concentrations.142,143 Second, albumin can
bind to potential toxins, like phthalates144 (common plasticizers) or
endotoxins. Thus, those toxins can be present in the commercial ver-
sions of serum albumin. These impurities in serum albumin products
even were reported to vary in concentration from batch to batch,
thus affecting the results of the embryo culture.145–150 Therefore,
the development of the culture media without undefined supple-
ments is desirable, especially for human embryos. Recently, highly
purified recombinant human albumin became commercially avail-
able. It is worth trying it as a substitute for serum- derived albumin.
Summarized in Table 3 are the points of caution when researchers
use undefined supplements.
5.3| Contamination of a medium
Foreign substances from unidentified sources can contaminate a cul-
ture medium, thus possibly affecting the empirical results. Typically,
those contaminants include viruses, bacteria, mycoplasma, and endo-
toxins. There are, however, other types of contaminants, like plasticiz-
ers that might be eluted from plastic instruments144 or trace elements,
even in water. These substances also can affect the cells in culture.60
It also was reported that some toxic substances are eluted from the
microfilters that are used for sterilization.151 Some of this contamina-
tion seems to be even inevitable, but care must be taken to minimize it
in order to make culture experiments reliable and highly reproducible.
Thus, researchers may consider practicing the sterile technique strictly
and selecting culture instruments carefully. Washing the instruments
FIGURE2
When Component B's concentration is low (eg, 0mg/L), antibody
when Component B's concentration is high (eg, 20mg/L), antibody
phenomenon—one component influencing the response of another
component—is called a "two- factor interaction." The relationship
between the concentration and response is not necessarily linear,
by using at least a three- level screening design is necessary to
understand such responses
|
15
YAO And ASAYAMA
with the culture medium immediately before use is recommended in
some special cases.151–153
6| CONCLUSION
Ever since Harrison's successful cultivation of animal cells, cell culture
technology has developed in leaps and bounds, with many break-
throughs. With the consumables (eg, culture media) and cell culture
equipment now supplied on a commercial basis, it became possible for
fewer opportunities lately to appreciate the research value of culture
media, as well as their shortcomings and limitations. With progress in
regenerative medicine and biopharmaceuticals, the creation of culture
systems that do not require a human intervention is expected to con-
tinue: this trend will probably intensify in the future. Even with this trend,
the culture medium is of paramount importance for the best quality of
cell culture experiment, as well as biopharmaceutical work. Here again,
it should be noted that the current culture media and their formulations
have been established through the timeless efforts of innumerable re-
searchers. From now on, investigators should drive the further evolution
of culture media, with the aim of improving culture performance.
ACKNOWLEDGEMENTS
-
script. We are especially thankful to Takehiko Ogawa for his advice
and expertise.
FIGURE3Concepts of the one- factor- at- a- time experiment and design of experiment (DoE). These figures show the difference in strategies
has been used, has a big disadvantage of missing the optimal point because there are some unexamined areas in the range of parameters. B, In
contrast, the DoE is a model- based statistical method that can clarify the relationship between the response of the cells and the concentrations
of the tested components in the range of settings. The process of the DoE is mainly composed of four steps. First, allocate the design points
evenly throughout the area. Second, record the response of the cells for each run. Third, fit the collected data to an appropriate model (eg, a
logistic regression model for a binomial response) and validate the relevance of the model to decide whether it is available for the next step.
Finally, use the model to optimize the concentrations of the components or to predict a response of the cells
16
|
YAO And ASAYAMA
DISCLOSURES
Conflict of interest: The authors declare no conflict of interest. Human
and Animal Rights: This article does not contain any study on human or
animal participants that was performed by any of the authors.
REFERENCES
In Vitro Cell Dev Biol.
1990;26:9–23.
Serum- free cell culture: the serum- free media interactive online da-
tabase. ALTEX. 2010;27:53–62.
3. Freshney RI. Serum-free media. In: Freshney RI, ed. Culture of Animal
Cells
4. Ringer S. Concerning the influence exerted by each of the constit-
uents of the blood on the contraction of the ventricle. J Physiol .
1882;3:380–393.
-
ferent constituents of the blood on the contraction of the heart. J
Physiol. 1883;4:29–42.
6. Locke FS, Rosenheim O. Contributions to the physiology of the
isolated heart: the consumption of dextrose by mammalian cardiac
muscle. J Physiol. 1907;36:205–220.
Arch Int
Pharmacodyn. 1910;20:205–223.
8. Krebs H, Henseleit K. Untersuchungen über die harnstoffbildung im
tierkörper. Klin Wochenschr. 1932;11:757–759.
tumor cells in continuous culture: I. Preliminary report: cultivation
of mesoblastic tumors and normal tissue and notes on methods of
cultivation. Am J Cancer. 1936;27:45–76.
of malignancy in vitro; IV: the mouse fibroblast cultures and changes
seen in the living cells. J Natl Cancer Inst. 1943;4:165–212.
preservation of tissues by refrigeration. Proc Soc Exp Biol Med.
1949;71:196–200.
12. Roux W. Beitriige zur entwicklungsmechanik des embryo. Z Biol .
1885;21:411.
13. Loeb L. Über die enstehung von bindegewebe, leucocyten und roten
blutkörperchen aus epithel und über eine methode. In: Loeb L, ed.
Isolierte Gewebsteile zu Züchten
Company; 1897:1–56.
-
les animales en dehors de l'organisme. Compt Rend Soc Biol Paris.
1903;55:1266–1268.
the living developing nerve fiber. Anat Rec. 1907;1:116–128.
J
Exp Med. 1923;38:407–418.
the body. JAMA. 1910;55:2057–2058.
-
nique. J Exp Med. 1911;13:387–396.
J
Exp Med. 1912;15:516–528.
-
sue. J Exp Med. 1913;17:14–19.
J Exp Med.
1922;35:755–759.
Biograph. Memoirs Natl Acad. Sci.
1967;39:323–358.
-
bryos in solutions of NaCl, CaCl2 , KCl and NaHCO3 . Anat Rec.
1911;5:277–293.
known chemical constitution. Anat Rec. 1912;6:207–211.
-
tures. J Exp Med. 1922;35:317–322.
embryonic tissue extract. J Exp Med. 1926;44:387–395.
cell multiplication. J Exp Med. 1926;44:503–521.
-
uents of embryonic tissue juice on the growth of fibroblasts. J Exp
Med. 1926;44:397–407.
29. Baker LE. The chemical nature of the substances required for cell
on the growth of fibroblasts. J Exp Med. 1929;49:163–182.
fibroblasts. Am J Cancer. 1933;18:28–38.
cells and monocytes. Science. 1936;83:605–606.
strains. Exp Cell Res. 1961;25:585–621.
-
lated tissue cells. J Natl Cancer Inst. 1948;9:229–246.
-
erative capacity of cervical carcinoma and normal epithelium. Cancer
Res. 1952;12:264–265.
tissue cells in artificial media. Proc Soc Exp Biol Med. 1948;67:40–46.
Biochem J.
1948;43:491–497.
37. Eagle H. The specific amino acid requirements of a mammalian cell
(strain L) in tissue culture. J Biol Chem. 1955;214:839–852.
38. Eagle H. The specific amino acid requirements of a human carcinoma
cell (strain HeLa) in tissue culture. J Exp Med. 1955;102:37–48.
39. Eagle H. Nutrition needs of mammalian cells in tissue culture.
Science. 1955;122:501–514.
Science.
1959;130:432–437.
Virology. 1959;8:396–397.
hamster hybrid cells. Nature New Biol. 1971;230:52–54.
-
min, transferrin, and soybean lipid in cultures of lipopolysaccharide-
reactive b lymphocytes. J Exp Med. 1978;147:923–933.
oxaloacetate, and α- ketoglutarate for isolated Walker carcinosar-
coma 256 cells. Exp Biol Med. 1958;98:303–306.
Cancer Res. 1959;19:591–595.
cells. Cancer. 1966;19:713–723.
-
cytes. JAMA. 1967;199:519–524.
48. White PR. Cultivation of animal tissues in vitro in nutrients of pre-
cisely known constitution. Growth. 1946;10:231–289.
Nature. 1948;161:768–769.
-
sue culture. I. Initial studies on a synthetic medium. Exp Biol Med .
1950;73:1–8.
-
Spec Publ NY Acad Sci. 1957;5:303–313.
|
17
YAO And ASAYAMA
52. Parker RC. Chemically defined media. In: Parker RC, ed. Methods of
Tissue Culture
-
fined medium for long- term cultivation of strain L- 929 cells. Cancer
Res. 1956;16:87–94.
54. Waymouth C. Rapid proliferation of sublines of nctc clone 929
752/1). J Natl Cancer Inst. 1959;22:1003–1017.
mammalian cells. Science. 1963;140:802–803.
mammalian cell line. Biochem Biophys Res Commun. 1963;14:34–38.
synthetic medium. Proc Natl Acad Sci USA. 1965;53:288–293.
growth of cells in a defined medium. Nature. 1976;259:132–134.
Adv Biotechnol Processes. 1989;11:107–141.
in protein- free media. In Vitro Cell Dev Biol. 1977;13:537–547.
61. Takaoka T, Katsuta H. Long- term cultivation of mammalian cell
strains in protein- and lipid- free chemically defined synthetic media.
Exp Cell Res. 1971;67:295–304.
fatty acids in mammalian cell strains cultured for more than 10 years
in serum- free synthetic media. Jpn J Exp Med. 1987;57:351–354.
J
Biol Chem. 1959;234:2754–2758.
64. Higuchi K. Studies on the nutrition and metabolism of animal cells
in serum- free media: I. Serum- free monolayer cultures. J Infect Dis.
1963;112:213–220.
-
sion and virus transformation. Nature. 1970;227:798–801.
-
Ciba
Foundation Symposium – Growth Control in Cell Cultures. Chichester:
nervous system. Ann NY Acad Sci. 1952;55:330–344.
68. Cohen S. Isolation of a mouse submaxillary gland protein accelerat-
ing incisor eruption and eyelid opening in the new- born animal. J Biol
Chem. 1962;237:1555–1562.
and chemical properties. J Biol Chem. 1972;247:5928–5934.
-
tor which stimulates sulfate incorporation by cartilage in vitro. J Lab
Clin Med. 1957;49:825–836.
suppressible and nonsuppressible insulin- like activities in human serum
increased precision and specificity. J Clin Invest. 1963;42:1816–1834.
factor. Nature. 1972;235:107.
73. Rinderknecht E, Humbel RE. Polypeptides with nonsuppressible
insulin- like and cell- growth promoting activities in human serum:
Isolation, chemical characterization, and some biological properties
of forms I and II. Proc Natl Acad Sci USA. 1976;73:2365–2369.
effect alone and with hydrocortisone on 3t3 cell growth. Nature .
1974;249:123–127.
-
vine pituitary. J Biol Chem. 1975;250:2515–2520.
promoting activity. Exp Cell Res. 1974;87:297–301.
factor that stimulates the proliferation of arterial smooth muscle
cells in vitro. Proc Natl Acad Sci USA. 1974;71:1207–1210.
derived growth factor. Proc Natl Acad Sci USA. 1979;76:1809–1813.
factor- like peptide from cells transformed by a temperature- sensitive
sarcoma virus. J Cell Physiol. 1981;109:143–152.
-
gistic interaction of two classes of transforming growth factors from
murine sarcoma cells. Cancer Res. 1982;42:4776–4778.
in human fibroblasts and modulation of action by cholera toxin. Proc
Natl Acad Sci USA. 1973;70:2964–2968.
confluent 3t3 cell populations by a fibroblast growth factor, dexa-
methasone, and insulin. Proc Natl Acad Sci USA. 1974;71:4584–
4588.
3t3 cells: serum factors. Proc Natl Acad Sci USA. 1974;71:2908–2911.
from rat liver cell conditioned medium: comparison of biological
activities with calf serum, insulin, and somatomedin. J Cell Physiol .
1974;84:181–192.
control in balb/c 3t3 fibroblasts. Exp Cell Res. 1975;90:279–284.
trace nutrient for growth of wi- 38 diploid human fibroblasts. Proc
Natl Acad Sci USA. 1976;73:2023–2027.
transferrin, albumin and lecithin in haemopoitec cell cultures. Nature .
1976;263:594–595.
Cell.
1980;22:649–655.
essential component. Proc Natl Acad Sci USA. 1982;79:1158–1162.
-
dium. Cytotechnology. 1993;11:219–231.
hippocampal neurons in b27- supplemented neurobasal™ , a new
serum- free medium combination. J Neurosci Res. 1993;35:567–576.
-
tiated growth of neurons from the striatum, substantia nigra, sep-
tum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res.
1995;42:674–683.
for the large scale production of recombinant protein from a Chinese
hamster ovary cell line. Cytotechnology. 1995;17:153–163.
serum- free medium. Nature . 1979;281:388–389.
-
eloma mpc- 11 cells in serum- free growth factor supplemented me-
dium. Agric Biol Chem. 1982;46:1831–1837.
-
Methods for Serum-Free
Culture of Neuronal and Lymphoid Cells
1984:197–206.
antibodies with human–human hybridomas and their serum-free,
Modern
18
|
YAO And ASAYAMA
Approaches to Animal Cell Technology. London: Butterworths;
1987:52–76.
-
Handbook of Industrial Chemistry and Biotechnology. Boston: Springer;
2012:1229–1248.
and media optimization for high productivity cell culture process de-
velopment. Biotechnol Bioeng. 2013;110:191–205.
and proteomic approaches for the development of cell culture me-
of recombinant proteins: current state and further potential. Appl
Microbiol Biotechnol. 2012;93:917–930.
-
vated mammalian cells. Nat Biotech. 2004;22:1393–1398.
embryonic stem cell lines maintain pluripotency and proliferative po-
tential for prolonged periods of culture. Dev Biol. 2000;227:271–278.
embryonic stem cells in defined conditions. Nat Biotechnol .
2006;24:185–187.
differentiation of human embryonic stem cells in chemically defined
conditions. Proc Natl Acad Sci USA. 2006;103:6907–6912.
human embryonic stem cells in a defined serum- free medium. Proc
Natl Acad Sci USA. 2008;105:13409–13414.
culture method enabling the establishment of clinical- grade human
embryonic, induced pluripotent and adipose stem cells. PLoS ONE.
2010;5:e10246.
-
ditions for human iPSC derivation and culture. Nat Methods.
2011;8:424–429.
small molecule DYRK inhibitor provides long- term xeno- free human
pluripotent cell expansion. Stem Cells Transl Med. 2012;1:18–28.
free culture system for the derivation of human induced pluripotent
stem cells. Sci Rep. 2014;4:3594.
112. Hasegawa K, Yasuda S, Shahsavarani H, Yoshida N. Culture medium
113. Thibault C, Dauzier L, Wintenberger S. Cytological study of
fecundation in vitro of rabbit ovum. CR Seances Soc Biol Fil .
1954;148:789–790.
Nature.
1959;184:466–467.
implantation stages of the mouse in a simple chemically defined me-
dium. J Reprod Fertil. 1968;17:399–401.
Nature. 1963;200:281–282.
embryo. Lancet. 1978;2:366.
pregnancies using cleaving embryos grown in vitro. Br J Obstet
Gynaecol. 1980;87:737–756.
119. Noda Y. Embryo development in vitro. Assist Reprod Rev. 1992;2:
9–15.
-
tive oxygen species in embryos cultured in vitro. Free Radic Biol Med.
1993;15:69–75.
effect of hypoxanthine in Ham's nutrient mixture F- 10 culture me-
dium on human oocyte fertilization and early embryonic develop-
ment. Fertil Steril. 1993;60:876–880.
human zygote culture in Ham's F- 10 without hypoxanthine. J Assist
Reprod Genet. 1993;10:271–275.
vitro fertilization with the use of a medium based on the composition
of human tubal fluid. Fertil Steril. 1985;44:493–498.
past, present, and future. J Mamm Ova Res. 2016;33:17–34.
T. Composition of commercial media used for human embryo cul-
ture. Fertil Steril. 2014;102:759–766.
Handbook of In Vitro Fertilization. New York: CRC
Press; 2000:205–264.
of mammalian preimplantation embryos: historical perspective and
current issues. Hum Reprod Update. 2003;9:557–582.
Cell Culture
Models of Biological Barriers In-Vitro Test Systems for Drug Absorption
and Delivery
calves and adult steer: a study of developmental changes. Lipids .
1981;16:240–245.
130. Freshney RI. Defined media and supplements. In: Freshney RI, ed. Culture
of Animal Cells
-
ically defined cell culture media – replacing fetal bovine serum in
mammalian in vitro methods. Toxicol In Vitro. 2010;24:1053–1063.
BES zwitterion buffers on the ultrastructure of cultured chick embryo
epiphyseal chondrocytes. In Vitro Cell Dev Biol. 1982;18:755–765.
-
toxic effects of light- exposed HEPES- containing culture medium. In
Vitro Cell Dev Biol. 1985;21:282–287.
breast cancer cells. Endocrinology. 1991;129:3321–3330.
-
ithelial cells. Am J Physiol Renal Physiol. 1992;262:F687–F691.
experiments methodology. Biotechnol Prog. 2008;24:1191–1203.
screening in the presence of second- order effects. J Qual Technol .
2011;43:1–15.
in vitro fertilization, early embryo culture, and transfer. Fertil Steril.
1984;42:750–755.
and development of mouse embryos in vitro. J Androl. 1982;3:
440–444.
acids on mouse and rat embryo development. J Mamm Ova Res.
1995;12:113–118.
human preimplantation embryos. Hum Reprod. 2006;21:766–773.
Proteomics.
2010;10:172–181.
143. Dyrlund TF, Kirkegaard K, Poulsen ET, et al. Unconditioned commer-
cial embryo culture media contain a large variety of non- declared
|
19
YAO And ASAYAMA
proteins: a comprehensive proteomics analysis. Hum Reprod. 2014;29:
2421–2430.
ethylhexyl)phthalate and mono(2- ethylhexyl)phthalate in media for
in vitro fertilization. Chemosphere . 2012;86:454–459.
albumin affects cell multiplication and hatching of rabbit blastocysts
in culture. J Reprod Fertil. 1983;69:555–558.
mouse embryo development in vitro. J In Vitro Fert Embryo Transf.
1984;1:183–187.
-
tein source affect the development of preimplantation goat embryos
in vitro. Reprod Fertil Dev. 1991;3:601–607.
human serum albumin on mouse embryo culture. J Assist Reprod
Genet. 1992;9:45–52.
inhibit or stimulate in vitro development of hamster embryos. In Vitro
Cell Dev Biol
bovine embryos as affected by different lots of bovine serum albu-
min and citrate. Theriogenology. 1994;42:397–403.
Embryotoxicity of micropore filters used in liquid sterilization. J In
Vitro Fert Embryo Transf. 1990;7:347–350.
the success of human in vitro fertilization and embryo transfer. Fertil
Steril. 1984;41:202–209.
-
portation of mouse embryos using microtubes and a warm box. PLoS
ONE. 2015;10:e0138854.
How to cite this article:
media: History, characteristics, and current issues. Reprod Med
Biol. 2017;00:1–19. https://doi.org/10.1002/rmb2.12024
... However, this solution condition might not be friendly to general biological cells. The biochemical activities occurring within a living cell are well recognized to normally require the participation of various chemicals, biomolecules, or ions (Yao and Asayama, 2017), which could accordingly lead to a high conductivity environment (e.g., conductivity of commercially-available RPMI cell culture medium: 11,770 μS cm −1 ). As a result, the background solution with low conductivity required for ODEP-based cell manipulation could affect the properties (e.g., cell viability) of the manipulated cells. ...
... Moreover, the viability of cells in sucrose solution significantly (p < 0.05) declined during 4 h (95.3 ± 0.7%, 86.3 ± 0.7%, and 56.8 ± 1.5% after 0, 2, and 4 h of incubation, respectively), within which ODEP-based cell manipulation was generally performed. This phenomenon could be due to the aforementioned noncellfriendly environment of low conductivity (e.g., 6.9 μS cm −1 ), which could limit the biological activity within a cell (Yao and Asayama, 2017). This result could also be due to the sucrose solution-induced cell autophagy (Higuchi et al., 2015), which leads to cell death (Shimizu et al., 2014). ...
... Similarly, dextrose [e.g., 0.3% (w/v)] and BSA are commonly used as supplements in the background solution for DEP-or ODEP-based cell manipulation (Fatima H Labeed et al., 2003;Srinivasu Valagerahally Puttaswamy et al., 2010;Gupta et al., 2012;Song et al., 2015;Chiu et al., 2016). The former can serve as the energy source for biological cells (Yao and Asayama, 2017), and the latter is reported to prevent cell adsorption on the substrate surface, facilitating subsequent ODEP-based cell manipulation (Gupta et al., 2012;Song et al., 2015;Chiu et al., 2016), to reduce ROS (reactive oxygen species) production (Liu et al., 2012) and to prevent cell death (Liu et al., 2012). In addition, RPMI cell culture medium is commonly used for cell culture practice and can provide various basic nutrients (e.g., glucose or amino acids) for manipulated cells. ...
Optically induced dielectrophoresis (ODEP) is effective for cell manipulation. However, its utilization has been limited by the requirement of solution with low conductivity. This issue has been ignored in ODEP-relevant studies. To address this issue, this study aims to investigate to what extent the cell viability and performance of ODEP-based cell manipulation are affected by low conductivity conditions. Additionally, this study aims to modify sucrose solutions to reduce the impacts caused by low-conductivity solutions. Results revealed the use of sucrose solution in ODEP operation could significantly reduce the viability of the manipulated cells by 9.1 and 38.5% after 2- and 4-h incubation, respectively. Prolonged operation time (e.g., 4 h) in sucrose solution could lead to significantly inferior performance of cell manipulation, including 47.2% reduction of ODEP manipulation velocity and 44.4% loss of the cells manipulatable by ODEP. The key finding of this study is that the use of bovine serum albumin (BSA)-supplemented sucrose solution (conductivity: 25–50 μS cm−1) might significantly increase the cell viability by 10.9–14.8% compared with that in sucrose solution after 4 h incubation. Moreover, the ODEP manipulation velocity of cells in the BSA-supplemented sucrose solution (conductivity: 25 μS cm−1) was comparable to that in sucrose solution during 4-h incubation. More importantly, compared with sucrose solution, the use of BSA-supplemented sucrose solution (conductivity: 25–50 μS cm−1) contributed high percentage (80.4–93.5%) of the cells manipulatable by ODEP during 4-h incubation. Overall, this study has provided some fundamental information relevant to the improvement of background solutions for ODEP-based cell manipulation.
... Culture media generally consists of glucose, inorganic salts, water-soluble vitamins, and amino acids formulated to optimize growth for the cell type selected. Additional inputs, such as insulin, transferrin, serum proteins, growth factors, immortalization agents, antibiotics, antimycotics, and antioxidants may be used to support proliferation, differentiation, protein synthesis and degradation, and glucose uptake (Burton et al., 2000;Datar & Betti, 2010;Yao & Asayama, 2017). Some substances may be added to impart organoleptic properties (e.g., proteins or pigments that impart a meat-like color) or add nutritional value (e.g., vitamins) (Simsa et al., 2019). ...
... Characterizing the components of the media to identify potential hazards is therefore an important part of safety assessment. This endeavor is complicated by the different types of media required for different species, cell types, and for different stages of manufacture (Burton et al., 2000;Yao & Asayama, 2017). There are hazards associated with the intentional components and from impurities or contaminants. ...
Cell-cultured meat and seafood offer a sustainable opportunity to meet the world's increasing demand for protein in a climate-changed world. A responsible , data-driven approach to assess and demonstrate safety of cell-cultured meat and seafood can support consumer acceptance and help fully realize the potential of these products. As an initial step toward a thorough demonstration of safety, this review identifies hazards that could be introduced during manufacturing, evaluates applicability of existing safety assessment approaches, and highlights research priorities that could support safe commercialization. Input was gathered from members of the cultured meat and seafood industry, researchers, regulators , and food safety experts. A series of workshops were held with 87 industry representatives and researchers to create a modular manufacturing process diagram , which served as a framework to identify potential chemical and biological hazards along the steps of the manufacturing process that could affect the safety of a final food product. Interviews and feedback on draft documents validated the process diagram and supported hazard identification and evaluation of applicable safety methods. Most hazards are not expected to be novel; therefore, safety assessment methods from a range of fields, such as conventional and novel foods, foods produced from biotechnology, pharmaceuticals, and so forth, are likely to be applicable. However, additional assessment of novel inputs or products with significant differences from existing foods may be necessary. Further research on the safety of the inputs and associated residues, potential for contamination, and development of standardized safety assessment approaches (particularly animal-free methods) is recommended. K E Y W O R D S cultured meat and seafood, hazard, methods, risk assessment, safety.
... Cell Culture Media is composed of nutrient substances necessary for the growth and proliferation of cultured cells. The components of the media depend on the cell type (Yao & Asayama, 2017). In the study, the culture media used for the breast cancer cells was mainly composed of Dulbecco's Modified Eagle Media and Fetal ...
-
![Athena Vega Oliva]()
- Louis Matthew Flores
- Maureen Mae Tolfo
As cancer is the second leading cause of mortality in the world, efficient treatment for it remains under extensive research. Kantutay (Lantana camara), an invasive plant species in the Philippines, was sought to be an alternative treatment for breast cancer. Several studies show that L. camara contains bioactive compounds which were proven to exhibit a highly selective mechanism of inducing p53 proteins, S cycle arrest, and DNA damaging of signaling pathways, signifying its plausible antineoplastic activity. Phytochemical screening showed that the flower contains phenols, tannins, and cardiac glycosides, with a total phenolics content of 0.1±0.2mg GA/g extract, all of which are reported to be cytotoxic and proapoptotic. The antineoplastic activity of the ethanolic flower extract was tested on MCF-7 then compared to Doxorubicin through the MTT Assay protocol.
... This requirement is usually fulfilled by the addition of fetal calf serum (FCS). FCS consists of an undefined cocktail of growth factors, vitamins, hormones and, in contrast to adult serum, low immunoglobulin levels, which may otherwise inhibit cell growth [2]. FCS is harvested from bovine fetuses at the abattoir and as the level of fetal awareness is unknown, it is also not clear whether or not the unborn calves experience distress or even suffer from the procedure [3,4]. ...
Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS.
... Optimising culture media is a costly and time-consuming endeavour due to the complexity of potential formulations. However, a well-defined medium may be invaluable when it comes to reducing the usage of expensive growth factors and serum [80][81][82]. Another factor is the choice of scale-up cell culture platforms, the costs of which depend on the medium consumption over CDM yield and whether the technology requires extra downstream processing steps (such as the removal of microcarriers). ...
Cell-derived matrices (CDM) are the decellularised extracellular matrices (ECM) of tissues obtained by the laboratory culture process. CDM is developed to mimic, to a certain extent, the properties of the needed natural tissue and thus to obviate the use of animals. The composition of CDM can be tailored for intended applications by carefully optimising the cell sources, culturing conditions and decellularising methods. This unique advantage has inspired the increasing use of CDM for biomedical research, ranging from stem cell niches to disease modelling and regenerative medicine. However, while much effort is spent on extracting different types of CDM and exploring their utilisation, little is spent on the scale-up aspect of CDM production. The ability to scale up CDM production is essential, as the materials are due for clinical trials and regulatory approval, and in fact, this ability to scale up should be an important factor from the early stages. In this review, we first introduce the current CDM production and characterisation methods. We then describe the existing scale-up technologies for cell culture and highlight the key considerations in scaling-up CDM manufacturing. Finally, we discuss the considerations and challenges faced while converting a laboratory protocol into a full industrial process. Scaling-up CDM manufacturing is a challenging task since it may be hindered by technologies that are not yet available. The early identification of these gaps will not only quicken CDM based product development but also help drive the advancement in scale-up cell culture and ECM extraction.
... This was the original method to obtain cell lines beginning with the first immortal cell line derived in the 1940s from mouse fibroblasts, as well as the HeLa cell line isolated from the cervical cancer of Henrietta Lacks. Immortality of the HeLa cells could be attributed to the cells' infection with human papillomavirus 18, which may have either degraded the tumor-suppressor protein p53 or caused chromothripsis, a chromosome-shattering and rearrangement associated with 2-3% of all cancers and which changes the expressions of thousands of protein-coding genes [14,15]. ...
-
![Emily Soice]()
- Jeremiah Johnston
The need to produce immortal, food-relevant cell lines is one of the most pressing challenges of cellular agriculture, the field which seeks to produce meat and other animal products via tissue engineering and synthetic biology. Immortal cell lines have a long and complicated story, from the first recognized immortal human cell lines taken from Henrietta Lacks, to today, where they are used to assay toxicity and produce therapeutics, to the future, where they could be used to create meat without harming an animal. Although work in immortal cell lines began more than 50 years ago, there are few existing cell lines made of species and cell types appropriate for cultured meat. Cells in cultured meat will be eaten by consumers; therefore, cultured meat cell lines will also require unique attributes not selected for in other cell line applications. Specifically, cultured meat cell lines will need to be approved as safe for consumption as food, proliferate and differentiate efficiently at industrial scales, and have desirable taste, texture, and nutrition characteristics for consumers. This paper defines what cell lines are needed, the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines used in cultured meat.
... Beginning with the initial hanging drop method of tissue or cell culture with semi-coagulated serum or lymph [24], cell culture methods have been modified throughout the years to allow for aseptic and precise spatial and temporal control of nutrient availability in culture. A comprehensive review of the development of cell culture methods was presented by Millet and Gillette [25] and Yao and Asayama [26]. A neuroblastoma is an embryonic malignancy of the sympathetic nervous system, which shares features of plasticity with developing neural crest stem cells. ...
- Xin Yi Yeo
- Grace Cunliffe
- Tang Jiong
-
![Sangyong Jung]()
Preclinical neurodegenerative disease models have been the cornerstone of neurodegenerative research in the past century. Although these models are inherently flawed, it is undeniable that they have provided rare access to the complexities of the nervous system and linkages between mechanistic and behavioral changes in the study of neuropathology. In this chapter, we discussed the development of models used in Alzheimer's disease (AD) research. We have also looked at the insights obtained about AD pathology and the possible limitations of using these models.
-
![Christine Poon]()
Culture medium is frequently modelled as water in computational fluid dynamics (CFD) analysis of in vitro culture systems involving flow, such as bioreactors and organ-on-chips. However, culture medium can be expected to have different properties to water due to its higher solute content. Furthermore, cellular activities such as metabolism and secretion of ECM proteins alter the composition of culture medium and therefore its properties during culture. As these properties directly determine the hydromechanical stimuli exerted on cells in vitro, these, along with any changes during culture must be known for CFD modelling accuracy and meaningful interpretation of cellular responses. In this study, the density and dynamic viscosity of DMEM and RPMI-1640 media supplemented with typical concentrations of foetal bovine serum (0, 5, 10 and 20% v/v) were measured to serve as a reference for computational design analysis. Any changes in the properties of medium during culture were also investigated with NCI–H460 and HN6 cell lines. The density and dynamic viscosity of the media increased proportional to the % volume of added foetal bovine serum (FBS). Importantly, the viscosity of 5% FBS-supplemented RPMI-1640 was found to increase significantly after 3 days of culture of NCI–H460 and HN6 cell lines, with distinct differences between magnitude of change for each cell line. Finally, these experimentally-derived values were applied in CFD analysis of a simple microfluidic device, which demonstrated clear differences in maximum wall shear stress and pressure between fluid models. Overall, these results highlight the importance of characterizing model-specific properties for CFD design analysis of cell culture systems.
Cell culture method is a method developed in a biotechnological context and refers to the cultivation of cells outside of their natural environment under laboratory conditions and special conditions. It first came into our lives with the enlargement of adult frog nerve fibers in lymph fluids. Studies have shown that the needs of cell lines are different from each other. Considering these differences, tissue, media and environment conditions have been changed and different cell lines and culture methods that are in use today have been found. Various cell lines are currently used in pharmaceutical, medical and biological research. Cell culture studies are carried out to understand the biological structure and tissue morphology of the cell, thus improving tissue engineering. Although two-dimensional cell culture models are generally used, three-dimensional cell culture models are also preferred recently. Although the media serve for different needs, it has been found that some common components are extremely important for cell culture media in general. When these common components are examined, especially carbohydrate sources and serum are in the first place. Since carbohydrates are the main energy sources and serum contains different hormones and components as well as necessary growth factors, it is added to the medium. In cell culture practices, the environmental factors of the environment where the cells are located and the chemi-cal components of the environment are important for cell growth and continuity. In addition, cell culture methods should be sensitive to hygiene. Because when the culture environment, chemical components and environmental factors are taken together, if the aseptic conditions are not studied, there is a risk of contamination. In this review, an overview of cell culture methods and applications will be presented.
The clonogenic assay measures the capacity of single cells to form colonies in vitro. It is widely used to identify and quantify self-renewing mammalian cells derived from in vitro cultures as well as from ex vivo tissue preparations of different origins. Varying research questions and the heterogeneous growth requirements of individual cell model systems led to the development of several assay principles and formats that differ with regard to their conceptual setup, 2D or 3D culture conditions, optional cytotoxic treatments and subsequent mathematical analysis. The protocol presented here is based on the initial clonogenic assay protocol as developed by Puck and Marcus more than 60 years ago. It updates and extends the 2006 Nature Protocols article by Franken et al. It discusses different strategies and principles to analyze clonogenic growth in vitro and presents the clonogenic assay in a modular protocol framework enabling a diversity of formats and measures to optimize determination of clonogenic growth parameters. We put particular focus on the phenomenon of cellular cooperation and consideration of how this can affect the mathematical analysis of survival data. This protocol is applicable to any mammalian cell model system from which single-cell suspensions can be prepared and which contains at least a small fraction of cells with self-renewing capacity in vitro. Depending on the cell system used, the entire procedure takes ~2–10 weeks, with a total hands-on time of <20 h per biological replicate. This Protocol Extension discusses several approaches to analyzing clonogenic growth of mammalian cells in vitro, using a modular framework to facilitate the use of various formats to fully optimize clonogenic growth.
Screening designs are attractive for assessing the relative impact of a large number of factors on a response of interest. Experimenters often prefer quantitative factors with three levels over two-level factors because having three levels allows for some assessment of curvature in the factor—response relationship. Yet, the most familiar screening designs limit each factor to only two levels. We propose a new class of designs that have three levels, provide estimates of main effects that are unbiased by any second-order effect, require only one more than twice as many runs as there are factors, and avoid confounding of any pair of second-order effects. Moreover, for designs having six factors or more, our designs allow for the efficient estimation of the full quadratic model in any three factors. In this respect, our designs may render follow-up experiments unnecessary in many situations, thereby increasing the efficiency of the entire experimentation process. We also provide an algorithm for design construction.
- Tatsuma Yao
-
![Yuta Asayama]()
Systematic studies of mouse embryo culture beginning in 1949 led to an understanding of essential medium components for early mammalian embryos, and embryo culture from the zygote to the blastocyst stage was achieved in 1968. Since then, medium components that are either beneficial or detrimental for embryo culture have been identified. A variety of culture media that mimic the female reproductive tract, such as human tubal fluid medium and sequential media, were developed from the 1970s to the 1990s, and a single medium in which the concentrations of components were determined by a simplex optimization method was introduced for clinical use in 2002. While either sequential media or a single medium is currently used in most cases, no conclusion has yet been reached as to which of the two approaches is the best. That we are now easily able to culture embryos is the result of the work of pioneers. This review presents a chronological overview of media development from initial attempts at mouse embryo culture using synthetic media to the human embryo culture media used today. It also presents the characteristics of sequential media and a single medium. Finally, problems observed with current embryo culture media are discussed, along with future development in this area.
-
![Patrick Quinn]()
- John F. Kerin
- G M Warnes
Significantly more mouse zygotes developed to blastocysts in culture in a medium formulated on the composition of human tubal fluid (HTF) than in modified Tyrode's medium (T6). In a randomized 2×2 factorial trial of human in vitro fertilization that compared the two media and culture under oil versus culture in loosely capped tubes, significantly more clinical pregnancies (30% of 60 transfers) were obtained with HTF medium than with T6 medium (11% of 53 transfers). Decreasing the K + content of HTF medium to that present in T6 medium significantly decreased the number of mouse zygotes that developed in culture. Modifying Ca + + levels had no effect. It is therefore likely that the higher K + content in HTF medium is primarily responsible for the superiority of HTF medium over T6 medium, but other differences in the composition of the two media could contribute to the results observed.
Different Components of Animal Cell Culture Media
Source: https://www.researchgate.net/publication/315504318_Animal-cell_culture_media_History_characteristics_and_current_issues
0 Response to "Different Components of Animal Cell Culture Media"
Post a Comment