JP2577114C - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION   The present invention relates to a cell growth medium.   Most currently used for the persistence of normal mammalian cells, i.
In any medium or system, the proteins necessary to support such growth and growth are Incorporate indeterminate proteins into the system or use feeder cells to supply
I have.   The presence of such uncertain proteins interferes with the desired end use of the cultured cells
Cells under conditions that minimize the presence of indeterminate proteins.
It is desirable to culture. For example, living skin used as a skin graft for burn patients
Uncertain protein in cultured epidermal cells used to prepare skin equivalents
When such a protein is used, it is possible that such a protein may cause an immune response.
It is also anticipated that equivalents will not be suitable for the desired application. Provide such biological skin equivalents
When using mouse 3T3 foster cells to culture epidermal cells, they are captured by the epidermis.
Residual 3T3 cells or a part of the cells are used for cross-species immunereactio
n) is very likely to occur. Bovine serum is used for the production of such biological skin equivalents.
When used, a similar immune reaction is likely to occur.   Thus, for the sustained growth and proliferation of cells, uncertain proteins at very low concentrations
Chemically defined cell culture media and systems containing or more preferably without any
is necessary. The term “determined” means that a component contains trace amounts of contaminants.
Media or supplements that do not contain any intentionally added uncharacterized supplements
Means a component.   Numerous chemically defined cell culture media for cell growth that meet these needs
It has been developed. Examples of such a medium include a modified Eagle medium (hereinafter referred to as MEM), Ham's
F12 medium, Dulbecco's modified Eagle medium (hereinafter DMEM), MCDB 153 and medium
There is ground 199. However, there are many details in existing chemically defined media.
For successful cell culture, supplemental proteins such as serum or serum-derived factors
It was found that the addition of or the use of cultured cells was necessary. As a result of this,
Uncertain components will be introduced to the ground. Supplementary complex proteins in cell culture
Addition not only introduces uncertain proteins but also increases cell culture costs
.   Problems encountered during culture and sometimes maturation of epithelial cells are now available
Identify the drawbacks of the culture medium and system. Epithelial cells such as epidermal cells are used for living skin, etc.
Production of Valuables and Production of Cultured Epidermal Sheets for Use in Test Systems and Skin Grafts Used for Living skin and other tissue equivalents, and the
Methods of manufacture and use are described in U.S. Pat.Nos. 4,485,096, 4,835,102, 4,837,379, 1
U.S.S.N. 07 / 505,678 filed on April 6, 990, U.S.S.N. 07/40 filed on September 15, 1989
No. 8,052. The contents of these documents are included in this specification
The text will be referred to hereinafter as "patents". These patents and patent applications are
Requires large growth of skin cells, but currently available media meets this requirement
Can not.   Mammalian epidermis is primarily derived from a single cell type, keratinocytes, at various stages of differentiation.
It is configured. The basal layer isolated from the underlying dermal fibroblasts is mitotic keratin
Including cells. These keratinocytes produce progenitor cells, some of which are
It no longer divides, moves outward from the basal layer, and finally differentiates to form the stratum corneum,
From the surface. Some applications, such as those used for testing percutaneous absorption
In experimental systems, biological skin equivalents must contain a well-developed keratinocyte layer
Therefore, differentiation and maturation of cultured epidermal cells are required.   Form keratinocyte sheets that closely resemble keratinocytes observed in vivo
This creates additional requirements that existing cell growth media and systems must satisfy. Or
The formation of such a sheet involves differentiation of epidermal cells while maintaining a proliferative subpopulation of basal cells.
Sufficient calcium concentration is needed to stratify older and more mature keratinocytes.
It is important. The culture and maturation of keratinocytes are tested in various ways and supplemented serum or
Even when protein was added, the degree of success was not uniform. For example
Keratinocyte proliferation when supplemented with conventional serum in chemically defined media
It was found that the requirements were not met. Achieve keratinocyte proliferation and maturation
To do this, add mouse serum simultaneously with little or no added serum or other uncertain factors.
A chemically defined culture that does not use cultured cells such as embryonic fibroblasts (3T3 cells)
The use of nutrient groups is preferred.   Considering the current state of keratinocyte culture technology, some problems emerge
(Breidahl et al.,Aust. N. Z. J. Surg. 59 (1989) 485-497).   Rheinwald and Green describe a 3T3 foster cell layer that produces clonal growth and multiple passages
Keratinocyte growth method using Rheinwald & Green,Cell 6 (1975) 331-344). In this method, keratinocytes are additionally cloned during passage.
Stratification and separation maintaining a proliferative or relatively undifferentiated basal cell population that can proliferate
It was considered to be a very advanced method because a transformed culture could be formed. Rhei
Despite such advantages of the method of nwald and Green, cultured cells such as mouse 3T3
The use of cells is not preferred. The reason for this is that the 3T3 cells
This is because there is a possibility that the defined protein is present in the cultured cells. 3T3 cells and
And cell fragments can be removed with 0.02% EDTA (Sun & G
reen,Cell 9 (1976) 512-521), complete removal is often difficult and
Removal and consequent effects of stress on cultured epidermal cells are undesirable
Conceivable. The 3T3 rearing system requires a large amount of serum (about 5%),
The defined protein will be added further.   Rheinwald and Green (Above) Even after the presentation, researchers, etc.
Development of a method for culturing epidermal cells to mature keratinocytes in the absence of a cell support
Departure continued. Significant advances in this area have been made by Boyce and HamJ. Invest. Der
matol.81 (1983) reported to 33S-40S. In this method, first, a small amount of dialysis FBS (2%)
F-12 (Peehl & Ham,In Vitro 16 (1980) 526-538)
Used. The keratinocytes were then cultured in a serum-free medium containing a small amount of calcium.
Developed the optimal formulation of this medium to grow by changing the concentration of various components
The medium was named MCDB 153 (Boyce & Ham 1983,Aboveas well asJ. Tiss.Cult.Meth.
9 (1985) 83-93).   MCDB 153 is used in the fields of keratinocyte biology, toxicology, pathology and epidermal cell transfer.
It was reported to be useful in the field of transplant cell transplantation (Boyce & Ham 1985,Above). MC
DB153 is a serum-free medium that does not require cultured cells or has some disadvantages. For example
MCDB153 is extremely flexible in the presence of, for example, calcium and / or serum.
Its use is limited because it is not a medium.   The keratinocyte culture capacity in MCDB 153 is strictly dependent on the calcium concentration of the medium.
As a result, the flexibility of this medium is further limited. In MCDB 153
Growth is maintained by keeping the calcium concentration below about 1.0 mM for optimal
The calcium concentration is about 0.3 mM (Boyce & Ham 1983, Fig. 1, p. 35S,Above). Adding calcium at a concentration of about 1.0 mM or more in MCDB 153 to cause stratification
Then, cell division stops, and terminal differentiation occurs, and subsequent culturing becomes impossible. That is, the cells
Proliferative basal cell population is lost due to not being resistant to high calcium concentrations in the medium (B
oyce & Ham 1983,Above). Approximately 1.for proper layering and differentiation of adhesive multilayer skin sheets.
Because a physiological concentration of 8 mM calcium is required, MCDB 153
It is not a suitable medium for forming a rat cell layer. Pittelkow and Scott in MCDB 153
Reported formation of implantable epidermal sheets using cultured cells (Mayo Cl
in. Proc. 61 (1986) 771-777) to achieve stratification while maintaining adequate viability.
It was found that it was necessary to return to high serum (10%) DMEM medium in order to achieve this.   The limitations of MCDB 153 also indicate that cells grown in MCDB 153 will grow
Dramatic loss of ability (Boyce & Ham 1983,Above). This finding is
Number of cells show continued differentiation even when the calcium concentration is less than 1.0 mM
This suggests that one of the reasons why the MCDB 153 line is extremely calcium intolerant.
Conceivable. Because calcium triggers the final process of terminal differentiation in related cells
(Rice & Green,Cell, Vol. 18, 681-694, November 1979). Change
In addition, 30% colony formation efficiency and about 24 hours
A population doubling time between is achieved. These properties decline after three generations (Boyce
 & Ham1983,Above).   The quality of cells grown in MCDB 153 is sufficient to use in the production of epidermal cell sheets.
High quality, can maintain the consolidation layer for some time in the current MCDB 153 system
It is considered that a stratified culture cannot be obtained.   Others use sub-optimal media and use growth substrates in their systems.
And attempts to obtain an optimal system by, for example, Karasek and Charlton (J.In
vest.Dermal. 56 (1971) 205-210) and Gilchrest et al. (J. Cell Physiol. 112 (1982
197-206) reported the benefits of a protein substrate in the culture of epidermal keratinocytes
But with only marginal success, mainly due to shortcomings in the media formulation
won. Karasek and Charlton (Above) And other researchers have high serum concentrations
(Usually about 10%) was found to be necessary. Gilchrest etc. (CellBiol
. Intl. Rpt. 4 (1980) 1009-1016) Lack of supplemented M199 medium used The point is that basic nutrients are deficient, such as extremely low inositol concentration
Met.   Eisinger's work (Methods in Skin ReseachSkerrow & Skerrow, Chap. 7, J
. Wiley & Sons Ltd. 1985) and Eisenger et al. (Proc. Natl. Aca. Sci.76 (19
79) 5340-5344) report a method for expanding epidermal cells. According to the author's description
The advantage of their method is that cells can be grown without the use of cultured cells and epidermal components.
It is to let. However, Eisinger (AboveIn the method (),
High cell density is required. Furthermore, the growth rate is slow, and the doubling rate of the total number of cells is
Low (partly because of poor plate efficiency). Therefore, this method
It is not suitable for commercialization of large-scale growth or continuous culture from a single source.
No.   Supplementation of epidermal growth factor and hydrocortisone to basal medium increases keratinocytes
It is well known to those skilled in the art that propagation and spread are enhanced (Barrandon & Green,Cell50
(1987) 1131-1137; Bertolero et al.,Exp. Cell Res. 155 (1984) 64-80). Cyclic
AMP level enhancer (U.S. Pat.No. 4,456,687) and bovine neutral extract (Gilchrest
other,J. Cell Phys.120 (1984) 377-383) or placenta extract (O'Keefe & Chiu,Soc
. Invest. Dermatol. 90 (1988) 2-7) is also advantageous in many cell culture systems.   Looking at the literature, many researchers have found that collagen substrates (Liu et al.,In Vitro 15 (197
9) 813-822) or fibronectin substrate (Kubo et al.,Soc.Invest.Dermatol.88 (19
87) Use 594-601) or try to grow epithelial cells using various
I have seen that most studies only achieve short-term cultures at relatively high seeding densities.
It turns out that it was. Among these modified media containing MCDB 153, the 3T3 cultured cell line
Nothing has approached the level of success observed using.   However, 3T3 rearing systems require cAMP level enhancers to achieve optimal growth.
Typeically requires the use of cholera toxin (Green,Cell 15 (1978) 801-811). this
Artificial enhancement of cAMP levels can complicate studies of the effects and mechanisms of actual growth factors.
Mess up.   Provides prolonged growth and proliferation of cells and, in some cases, There is a need for a culture medium and system that matures predetermined cells while actively dividing the bottom cells.
You.Summary of the Invention   Cell growth in the cell media and systems of the present invention has a number of advantages, including:
. However, it is not limited to these. (1) No need for supporting or growing cells. (2) The growth rate is high even in the absence of serum and / or bovine hypothalamus extract. (3) Cell proliferation is rapid even in the absence of cAMP level enhancers. (4) The plate efficiency and colony formation rate can be changed by freely changing the calcium concentration.
30% or more. This is roughly 10% for 3T3, and MCDB 153-
Equivalent to 30% for low calcium. (This property is not
Extremely desirable. ) (5) The cells can be easily used for the production of biological skin equivalents, and the other methods described above.
Can form a multilayered epidermis that is as good as cells grown in MCDB 153 (MCDB 153).
Proliferating cells do not survive well). (6) Epidermal cells grown on collagen-coated surface are used for transplantation if desired
In order to be able to exfoliate as sheet-like epidermal cells using collagenase
You. (7) Ca⁺⁺The flexibility of the system in the concentration range and density dependence is
It offers a number of advantages that are compatible with high volume microparticle carrier culture. (8) When cells reach about 60% confluence due to rapid growth rate
Approximately synchronized cell populations are generated. Many rounded dividing cells and the present invention
Therefore, the high plating efficiency of the expanded cells means that the transition from beads to beads is
This suggests that it may be possible in carrier culture.   The cell culture medium (culture medium) of the present invention comprises a) about 0.5 to about 50 μg / ml of insulin or
Is about 10^-Ten~Ten^-8M insulin-like growth factor; b) about 0.05 to about 50 μg / ml
Transferrin, c) about 2-200 pM triiodothyronine, d) about 10^-6~ About 10
^-2At least one of M ethanolamine or O-phosphoryl-ethanolamine
And e) about 0.005 to about 2.0 mM calcium, and f) DMEM, IDMEM, MEM, M199, RPMI 1640, Ham's F12, Ham's F10, NCTC 109 and at least NCTC 135
A nutrient source selected from one and contain no undefined mammalian hormones.
And a chemically defined cell culture medium.   Other components that may be included in the medium of the present invention include hydrolysates of about 0.04 to about 4.0 μg / ml.
Cortisone; about 1 to about 50 ng / ml epidermal growth factor; about 0.02 to 2 mM adenine; about 2 × 1
0^-Ten~ 2 × 10^-8M progesterone; about 10^-9~ About 10^-7M selenium; about 10^-Four~ About 2 x
Ten^-3M strontium; cholera toxin, foreskolin, isop
Roterenol, methyl isobutyl xanthine, dibutyryl c-AMP, theophylline,
About 10 selected from at least one of fein and pertussis toxin.^-9~Ten^-3M1
One or more cAMP level enhancers; about 1 to 50 ng / ml of EGF. Ferrous iron
The ions are from about 0.05 to about 50 μg / ml, ie about 5.6 × 10^-Five~ About 5.6 × 10^-2M Transfer
Supplied by phosphorus.   It is expected that the media of the present invention may be effective for culturing various cell types. Departure
The cells that are preferably cultured according to the light commercialization are epithelial cells. Preferred epithelial cells
The vesicles can include epidermal cells or epidermal keratinocytes.   The cell culture system includes the above-described cell culture medium, plastic; glass, and collagen.
Fibronectin; laminin; heparan sulfate proteoglycan;
Collagen, fibronectin, laminin or heparan sulfate proteoglycan
A fine particle carrier coated with; or (a) Collagen solution is contractile agent and contractile agent is dispersed in it
Mixing under conditions to form a gel mixture, (b) the gel mixture prepared in step (a) is shrunk to a tissue equivalent
Maintain conditions that can form Support for cells comprising tissue equivalents produced by a method comprising:
trate, also referred to herein as a substrate).   A preferred type of collagen is natural type I collagen from bovine tendon.   One method of culturing epithelial cells is to inoculate the growth support with epithelial cells as described above.
And maintaining the cell culture system under conditions that promote cell growth. This
Cell proliferation obtained by such a method should have a population doubling time of about 16 to about 33 hours. Features. Further cultivate cells continuously (passage) and achieve about 20 to about 50 doublings
can do. In yet another embodiment, the method further comprises:
At a concentration of about 1.0 mM or more, while maintaining a colony formation rate of about 20 to about 60%.
This enables stratification and differentiation of cells. this
Is important in the formation of living skin equivalents for transplantation and sheet-like epidermis. Change
An embodiment of the invention provides for clonal expansion of cells, i.e., at a cell density of about 30 to about 1000 cells.
Provide seeding.   With the realization of the present invention, a primary (primary) having a colony formation rate of about 20 to about 100%
Provides rapid cell expansion and more than 50 population doublings
. In addition, calcium can be added to 1.0 mM or more, and colony formation of about 20 to about 60%
Cell stratification and differentiation can be achieved while maintaining the rate.   Further, epithelial cells were placed on a collagen-coated fine particle carrier in the cell culture system.
Plate culture at a plate efficiency of about 20 to about 40% to promote cell growth of the microparticle carrier and the plated cells.
A method for culturing epithelial cells in a particulate carrier, including maintaining them under advancing conditions
Provide the law. With the implementation of this method, even when cell growth reaches confluence,
A colony formation rate of ~ 100% is maintained. In addition, calcium concentration,
During periods of rapid congestion and rapid growth, increase from about 0.08 mM to about 1.8 mM,
Cell adhesion due to the shearing force applied to the fine particle carrier
Loss can be reduced.   In addition, fully epithelialized living tissue equivalents can be produced. This way
Is (a) inoculating the tissue equivalent with epithelial cells, (b) maintaining the obtained cell culture system under conditions that promote cell growth; and (c) Calcium is added to physiological concentration and the system is fully keratinized
Maintaining the conditions that allow the formation of layers And   The media of the present invention is regulated by rapidly growing epithelial cells, thus
Conditioned media is effective as a growth aid and in therapeutic applications such as wound healing
It is. Details of the Invention   The chemically defined medium of the present invention provides a support that can provide undefined proteins to the medium.
Provide a cell culture in the absence of cells, serum or other components.   Some advantages of the culture medium of the present invention include easy production and flexibility in use
I.e., it can be used at different calcium concentrations and can be added with different growth factors.
And the ability to alter extracellular matrix components. Medium and system of the present invention
Of the epithelial cellsin vitroBiologists and other people involved in manufacturing and / or research
Provide people with a system that satisfies most of their needs.   One medium of the present invention comprises insulin or an insulin-like growth factor, tiger.
Sferrin, triiodothyronine, ethanolamine and / or O-phosphoryl
Contains ethanolamine, calcium and nutrients. For example, a specific cell
Depending on the circumstances, such as, but not limited to, epidermal growth factor (EGF),
Locortisone, strontium, progesterone, selenium and cAMP level enhancers
Other components can also be added to the medium. Hydrocortisone is normal in humans.
Keratinocytes (Rheinwald and Green,supra) Is important for long-term culture
And is therefore a preferred component of intact mSBM. However, most
Hydrocortisone is not necessary if a few auxiliaries are desired, and without it
Would be preferable.   Insulin is present at a concentration of about 0.05 to about 500 μg / ml, preferably about 0.5 to about 50 μg / ml.
Although present, a particularly preferred concentration is 5.0 μg / ml. Insulin is now
Although preferred for economic reasons, proinsulin IGF-1 (10^-Ten~Ten^-8M)
Alternatively, other insulin-like growth factors can be substituted for insulin. Honcho
Insulin-like growth factors in the textbook are structurally similar to insulin.
Compositions that stimulate insulin-like growth factor receptors, such as insulin
Mean growth factors I and II.   Transferrin is provided at about 0.05 to about 50 μg / ml, preferably at about 5 μg / ml.
More preferably, it is supplied in a concentration.   Triiodothyronine has a stronger effect in the cell culture system of the present invention
Therefore, it is preferable to thyroxine. Triiodothyronine is preferred Alternatively, it is present at a concentration of about 2 to about 200 pM, more preferably about 20 pM.   Ethanolamine and / or O-phosphoryl-ethanolamine are also examples of the present invention.
It can be used in embodiments. However, these components are combined
It is preferable to use them. Ethanolamine is about 10^-6~ About 10^-2M, more preferred
About 10^-FourM concentration, O-phosphoryl-ethanolamine is about 10^-6~about
Ten^-2M, more preferably 10^-FourIt is present at a concentration of M.   Compared to currently available media, the calcium concentration of the media of the present invention is increased.
Range and still maintain a viable cell culture. Cells
It can grow in medium with 5% serum concentration and 1.8 mM physiological calcium concentration,
Loss of proliferation and terminal differentiation (ie, proliferating basaloid ce
ll population)) reduces serum to 0% and reduces calcium concentration.
A calcium concentration of about 0.005 to about 2.0 mM, more preferably about 0.08 to about 1.0 mM
And optimized by   Minimal layer formation is seen under the calcium conditions used for optimal growth rates
It is. If these cultures are kept confluent, remove larger cells from the culture dish.
Remove and leave the dish with the population formed by the tightly packed layer of small cells. Cal
The addition of calcium allows for more mature cells while maintaining a confluent basal cell layer.
The cells can be stratified. 1.8mM calcium before confluence
The colony appearance and cell distribution will continue to grow until the cells are confluent.
Comparable to those seen with feeder cells. If calcium is below 1.0mM
If maintained, involucrin or transgluta
Few transglutaminase (a marker for epidermal cell differentiation) is found. Mosquito
Lucium is present at about 0.005 to about 2.0 mM, depending on the purpose of culturing the cells. If
Depending on the other components in the medium
provide.   Epidermal growth factor (EGF) is from about 1 to about 50 ng / ml, preferably about 10 ng / ml (mouse EGF) and
Present at about 1 ng / ml (human EGF). EGF is preferred, but human or mouse tumor cells
Vesicle growth factor-α is preferably replaced by EGF at about 10 ng / ml (mouse) and about 1 ng / ml (human).
Can be EGF is an optional component in the medium of the present invention, It may be preferred for certain applications, such as batch cultivation.   Preferably, hydrocortisone is at a concentration of about 0.04 to about 4.0 μg / ml, but especially
The preferred concentration is 0.4 μg / ml.   In yet another embodiment of the present invention, the medium of the present invention is supplemented with additional components.
it can.   In some cases, components that inhibit fibroblast growth, such as cholera toxin,
It would be desirable to include it.   Belonging to fibroblast growth factor, such as acidic FGF, epidermal keratin growth factor and
And basic FGF (slightly higher concentration) may be used.^-Teng / ml ~ about 10^-6g / ml
, Preferably about 10^-7The effect is obtained at g / ml. Usually, 1 × 10 cells^ThreeCells / cm^Twothat's all
When arranged on a flat plate, the use of BHE has little or no effect. Said
The media contains another component, about 0.02 mM to about 2.0 mM adenine, about 2 × 10^-Ten~ 2 × 10
^-8M, preferably 2 × 10^-9M progesterone, about 1 × 10^-9~ About 1 × 10^-7M selenium also
May be added. Progesterone, selenium, in some cases to optimize growth
Can be added.   The system of the present invention establishes a primary cell culture and passages human epithelial cells.
In addition, cyclic AMP enhancers (also referred to as leveling agents), serum and
No bovine hypothalamus extract is required. Prior art studies have shown the addition of cAMP enhancers and
Presence of bovine pituitary extract is not essential for establishing primary cultures of epithelial cells
(Green, 1978, supra, and Boyce and Ham, supra).
, 1985). Although the addition of BHE may have some effect, the effect of
The data show that primary and subculture of epithelial cells using the medium of the invention
Does not require BHE or cAMP enhancer. However, if desired, cAMP enhancer
The present invention may be implemented using   Cyclic AMP enhancers that can be used in the practice of the present invention include cholera toxin,
Β-adrenergic substances such as foreskolin, adrenaline, te
Ofillin, dibutyryl cyclic AMP, methyl isobutyl xanthine, iso
Proterenol, caffeine and pertussis toxin. The preferred substance is
Latoxin and forescoline. Cholera toxin is about 10^-8~ About 10^-FiveUse with M Preferably, forescoline is about 10^-9~ About 10^-3M, preferably about 10^-7~ About 10^-FiveM
, More preferably 10^-6Use with M.   The use of cAMP enhancers in the systems and media of the present invention may
Effective in establishing culture or reestablishing frozen primary cultures or other cells
May also bring. However, in the present invention, a cAMP enhancer or BHE is added.
Cells can be easily established without it. The cells then receive the cAMP enhancer
Any number of subcultures can be performed using the medium of the present invention that does not contain the medium.   It can be used in place of calcium's constitutive need.
Thus, the addition of cationic substitutes has been studied. All cells
It requires some calcium, but calcium promotes differentiation in epidermal keratinocytes.
It also shows the effect. Induction of differentiation (Praeger et al.,J.Cell.Physiol., 132 (1987) 81-89, F
urakawa et al.J.Invest.Dermatol., 90 (1988) 690-696)
It has already been demonstrated that other divalent cations can be used in place of the required components (Ru
bin,J.Cell.Physiol., 91 (1977) 449-458). In the system of the present invention, the stronch
The effect of um and magnesium was investigated. Magnesium is sometimes effective
It turned out to be. When strontium is added, the percentage of proliferating cell population
(9-14 μm distribution) does not change much, but the final cell yield is significantly increased and the population
The doubling time was reduced by about 4 hours. For this, reference was made to Reference Example 7 described below.
No.   Nutrient sources useful in the practice of the present invention are known nutrients essential for cultured cells,
An energy source such as glucose, fructose or galactose;
Essential and non-essential amino acids; water-soluble vitamins (Bs, biotin, folic acid,
(Cotinamide, pantothenic acid, pyroxydine, riboflavin and thiamine)
Fat-soluble vitamins (A, D, E, K and ubiquinone); bicarbonate, calcium, salt
Major inorganic ions such as chloride, magnesium, phosphate, potassium and sodium
Trace elements such as As, Co, Cr, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Sn, V and Zn
Element; lipid; CO_Two/ HCO_ThreeAnd buffers such as HEPES; gases (oxygen and carbon dioxide);
Supply nucleic acid precursors such as adenine, cytidine, hypoxanthine and thymidine
Dulbecco's Modified Eagle Medium (DMEM) (MEM), M199, RPMI 1640 (all commercially available from Flow Laboratories), and Iscove
(EDMEM, commercially available from Gibco Labs). Minimum required culture
Ground and M199 also need to add phospholipid precursors and non-essential amino acids. Yet another
Vitamin that supplies amino acids, nucleic acids, enzyme cofactors, phospholipid precursors and inorganic salts
Rich commercial mixtures include Ham's F12, Ham's F10 and NCTC109 (all from Flow Laborat
ories) and NCTC 135 (Irvine Scientific).   The components of each of the above nutrient sources are shown in the table below. * NCTC 109 and 135 list the same components (Ref. 2) with the exception of NCTC 135
Excludes L-cysteine HCl. Ref. 1, page 466, shows that the 135
One of the reasons for removing cysteine is cited by the toxicity of cysteine. * NCTC 109/135 base medium (9172 & 9502) does not contain components marked with (*). Be
See the Media Introduction for details of the culture medium. A. In Ref. 2, L-cysteine 10.49 mg / l, L-cysteine HCl 250 mg / l, L-tyrosine 1
6.44mg / l. B. Not included in powdered media. C. Plaque Assay (2x) medium does not contain phenol red and glutamine.
Are twice as large. * A trademark of ICI America's Inc.   In addition, the above-mentioned nutrient sources are commercially available with slightly changed ingredients, and some of them are
Constitutes a nutrient source that can be used in the present invention.   One of the preferred nutrient sources used in the present invention is from about 90 to about 10% Ca⁺⁺Free DMEM and
About 10% to about 90% of Ham's F12. Particularly preferred nutrients are Ca⁺⁺Free DMEM
A nutrient source that contains about 75% Ham and about 25% F12. The medium of the present invention is usually
It contains at least about 90%, preferably more than 95%, of the nutrients.   The following shows one specific example of a preferred medium composition of the present invention: Insulin 5μg / ml Transferrin 5μg / ml Triiodothyronine 20pM Ethanolamine 1 × 10^-FourM O-phosphoryl-ethanolamine 1 × 10^-FourM Adenine 0.18mM Selenium 3 × 10^-8M Strontium 1mM Calcium 0.08mM Nutrition source Ca⁺⁺Free DMEM: about 90 to about 10%, Ham                                   F12: about 10 to about 90%.   If desired, to further increase the growth rate or extend the lifespan,
About 1.1 mM hydrocortisone, 10 ng / ml EGF, 2 × 10^-9With M progesterone
, About 9 ng / ml of cholera toxin. The pH of this medium is usually near neutral,
For example, it is about 6.8 to 7.4.   One important criterion for measuring the effectiveness of culture media is population fold per day.
The growth rate of the cell type as indicated by the addend. Using the culture system of the present invention
Then, epithelial cells can be cultured without using serum and feeder cells.
Population doubling at a growth rate comparable to that of the primary cell culture
Proliferate more than once. For example, neoplastic foreskin epithelial keratinocytes are
The doubling rarely drops below 0.5 / day on land and in the system.
Shows an average doubling of 1 / day, with a doubling of 1.3-2.0 / day between passages 2 and 5. (See Table 2). Such a growth rate is that the population doubles every 12-18 hours.
Means   Using the media and systems of the present invention, calcium can be maintained while maintaining the cell substratum.
Can be added to physiological concentrations. Such results have previously been
It could only be obtained with the stem alone. In the culture system and medium of the present invention,
This is because calcium can be added at 1.0 mM or more without destroying the cell population.
Continuous cells of epithelial cells suitable for the production or transplantation of living cells equivalent to such cells
(See Reference Example 5). It also has a negative effect on cell growth
Can be used to culture epithelial cells in microparticle carrier cultures.
It is also possible to grow to an amount (see Reference Example 6). For example, in MCDB 153, cells
Calcium concentration must be low to grow,
If it is low, the cells will not adhere sufficiently to the surface of the fine particle carrier. It shears the particulate carrier
This is a disadvantageous condition when it comes to force. Rheinwald and Green's 3T3 system
In the case of, first coat the microparticle carrier with 3T3 cells and then epithelial cells with 3T3
It may need to be grown on cells.   The medium of the present invention is a calcium-free nutrient that may be
Manufactured using readily available, commercially available ingredients, including sources. In addition, the composition of the medium of the present invention
The fractions are made and combined by methods known to those skilled in the art. In contrast, the city
According to the results of culture studies performed using commercial MCDB 153 equivalents, MCDB 153 equivalents
It seems that there is a limit to the production of.   The system of the present invention is one of the most suitable systems for epithelial cell culture to date.
Not only is it comparable to the 3T3 system, which has been known as
Clearly surpassed. The present invention relates to serum or parenteral
Provided is a cell growth medium for appropriately growing cultured cells independently of vesicles. Of the present invention
Using culture media and systems, primary cultures can be enriched for about 0.5-2.0 / 24 hours (PDL
) Can be established.   The table below compares the currently available media described above with the media and systems of the present invention.
You.   In the present invention, various substrates such as those described in various patents, such as collagen
, Fibronectin, laminin, heparan sulfate proteoglycan and
Tissue equivalents can be used. Natural collagen I derived from bovine tendon is a preferred collagen
It is one of the gen substrates. Under certain conditions, the system of the present invention
Proliferation can be promoted using substrates coated with tin or collagen (see
See Example 3.) When establishing a primary culture or using a serum-containing medium,
For example, use collagen in an amount of about 1% or more, or remove cells from frozen stocks.
Standing may be advantageous. However, if the serum concentration is reduced to about 0.3% or less,
Reduces the serum effect of promoting differentiation, and the matrix components provide the benefits described above.
No longer. If a substrate is used, the substrate must be of high quality. why
However, poor quality cells with partially altered fibronectin or heterogeneous collagen
This is because a culture dish with a coating can also inhibit cell growth.   The presence of a substrate, e.g., a matrix component, results in suboptimal or more
In severe cases, such as when serum factors are present, when cell density is low, tissue
When establishing cells or when longer survival times are needed, cells
It seems that it can form a knee.   Therefore, in this specification, a novel medium and a medium for growing cells such as epithelial cells are described.
The system and system were described, and examples were given. The invention is described in the following non-limiting examples.
Will be more apparent.   The base medium mSBM used in the following reference examples contains the following components: Hydrocortisone 1.1 μM Insulin 5μg / ml Transferrin 5μg / ml Triiodothyronine 20pM Ethanolamine 1 × 10^-FourM O-phosphorylethanolamine 1 × 10^-FourM Adenine 0.18mM Progesterone 2 × 10^-9M Selenium 3 × 10^-8M Cholera toxin 9ng / ml Epidermal growth factor 10ng / ml Calcium-free DMEM 75% Ham F12 25%   Unless otherwise indicated, mSBM also contains 0.3% chelexed fetal bovine serum (cFBS). cFBS
Is not a necessary component of the culture medium, but can be stored frozen for a long period without losing the activity of cholera toxin.
As is traditionally used in cholera toxin supplements,
Also used here.   In the following Reference Examples, a medium called emSBM was also used. This medium contains the components
Includes varying concentrations and the following ingredients: Triiodothyronine 1.002 × 10^-8Increase to M Cholera toxin increased to 100ng / ml Bovine hypothalamus extract (BHE) 50 μg / ml   In Reference Example 7, strontium (Sr) was optionally added to the medium.
In some cases, no cAMP enhancer was added to the medium. In Example 3, the cAMP enhancer,
Cells were grown without addition of logesterone, serum or bovine hypothalamus extract. Implementation
Example 11 grows cells without EGF and Example 5 shows cells without hydrocortisone
Was grown.   When primary cultures are established in emSBM, cAMP enhancers, serum, progesterone,
Neither enhanced triiodothyronine nor BHE is required.   The aforementioned medium is usually produced by the following method. However, the components of the present invention are
It should be understood that they can be manufactured and combined in a conventional manner adapted to the specific properties. The present invention
Is a sterile medium. The sterilized components are sterilized after production by a usual method. Less than
In the examples below, a suitable sterilization process was used in each case. Stock of all ingredients
The solution can be stored at -20 ° C. However, nutrient sources can be stored at 4 ° C.   All stock solutions are prepared at a concentration of 500 times the final concentration. Hydrocortizo
(Sigma) was dissolved in absolute ethanol and phosphate buffered saline (PBS).
Dilute. Insulin, transferrin and triiodothyronine (all Sig
A stock solution (manufactured by ma) is prepared as follows. First, no triiodothyronine
Dissolve 2: 1 in water ethanol 1N HCl. Dilute insulin in diluted hydrochloric acid (about 0.1N),
Dissolve transferrin in water. Mix these three solutions and make them 500 times more concentrated with water
Dilute. Ethanolamine and O-phosphorylethanolamine (both from Sigma
Is diluted with water to a concentration 500 times and sterilized by filtration. Progesterone (Sigma) anhydrous
Dissolve in ethanol and dilute with water. Maintains activity when stored for long periods
For this purpose, bovine serum albumin (BSA) can be added. Selenium (Sigma) is ethanolamine
Prepare as in Cholera toxin is commercially available from Sigma under sterile conditions. this
Is diluted with water. EGF is a commercially available sterile product from Biochemical Techno-logies, Inc.
Is dissolved in PBS. Adenine is difficult to dissolve, but can be prepared by many methods known to those skilled in the art.
Can dissolve. Sterile calcium-free DMEM from J.R.Scientific or Hazelton
Obtain. Ham's F12 is obtained from M.A. Bioproducts. BHE (Collaborative Resea
rch or ECGS) dissolves in DMEM with low calcium concentration. Storage of EGF stock solution
BSA can be added to extend the storage time. DMEM and F12 combines with each other and adds the individual components to the medium. Immediately after preparation
Can be used or stored at 4 ° C. For frozen storage, add EGF until use
Don't do it. The medium obtained in this way is a sterile medium.   The activity of BHE and EGF is evaluated on a lot-by-lot basis. Including BHE or EGF
Growth in a medium containing these components and growth in a medium containing these components is about 0 to about 50 ng / ml.
Supports maximal growth as compared to a range of concentrations of EGF and BHE from about 0 to about 200 μg / ml.
Check the concentration.Reference Example 1: Implementation of primary culture   Foreskin and other tissues, including ears and abdomen, were obtained from routine biopsies and 50 mcg / ml
Wash with phosphate buffered saline (PBS) containing tamycin and 1.25meq fungizone for 2 minutes.
Was. The entire tissue sample is then washed with 95% ethanol for 1 minute to remove surface contaminants.
I left. Thereafter, the plate was washed again with a PBS-gentamicin fungizone solution. Subcutaneous tissue
Remove aseptically and rinse tissue with PBS-gentamicin fungizone solution.
Was subdivided into Using a collagenase-trypsin tissue mixture, subdivide the tissue into 3
Dissociated at 7 ° C. The components of the collagenase dissociation mixture used are as follows.
Collagenase 55.5mg / ml, trypsin 2.0mg / ml, glucose 0.375mg / ml, fungi
Dzone 1 meq / ml and gentamicin 50 mcg / ml in phosphate buffer. On the digest
Remove the supernatant, neutralize, and remove remaining digestion at 30-minute intervals until the tissue is completely dissociated.
The new enzyme was added to the part.   Cell fractions containing a mixed population of dissociated cells are pooled, counted and counted without EGF.
Total volume contains 10ml emSBM (5ug / cm^TwoT75 flask (coated with collagen)
2.6 × 10^FourPieces / cm^TwoWas plated. Such primary cultures become confluent in 7 to 17 days.
Was. One day after plating, aspirate emSBM and add a new emSBM containing 10 ng / ml EGF.
Was replaced with Plate cells every 3 days using emSBM containing EGF, assuming 0 days of plating
The medium was changed with. It takes 7-17 days for the primary culture to become confluent. At this point
Cells can be cultured further continuously as described in Reference Example 2 or frozen for later use.
And stored in liquid nitrogen. Frozen medium is 10% bovine as a base
Includes LCDMEM with baby serum and 10% dimethyl sulfoxide (DMSO). frozen
Cells are removed from the dish and counted as in Reference Example 2. However, spin down and settle again after counting, 2 × 10⁶~ 5 × 10⁶reconstitute in frozen medium at a density of / ml
And dispensed into 1.8 ml nunc cryovials. Pre-frozen for 18-24 hours
The vial is later transferred to a liquid nitrogen storage tank until needed. These experiments
Table 1 below shows the results. Cell viability was usually above 90%.   The meanings of the abbreviations used in Table 1 are as follows. "HEP" indicates human epithelial cells
Where "n" indicates neonatal foreskin cells and "NF" followed by a number indicates that it is obtained from a source other than foreskin.
Shows human epithelial cells. Reference Example 2: Effect of continuous passage on colony formation rate ("CFE") and growth rate   Human epithelial cells in a T75 flask obtained in the same manner as in
Place in a flow hood, aspirate the medium, and use a 2 ml / 60 mm dish or 5 ml / T75 trypsinbel.
Versene ((calcium or magnesium free, M.A.Bioproducts) 200
(balanced saline solution of 500 mg / l trypsin with velsen (EDTA) at 500 mg / l). about
The flask was incubated at 37 ° C. for 3 minutes or until cells appeared on the surface of the dish.
Then a 2 ml / 60 mm dish or 5 ml / T75 soybean trypsin inhibitor (phosphate buffered
2.5 mg / ml, GIBCO) was added to the solution. Use a pipette to grow cells
The adhered cells were washed off by spraying on the surface of the scoop. Pipette cell suspension into tube
Then, it was rotated at 1200 rpm for 5 minutes. Aspirate the supernatant, leaving only the cell pellet
did. Resuspend the pellet in an appropriate volume of mSBM without EGF for counting.
(Usually 5 ml is enough). 8833 cells / cm after counting cells^Two(2.5x10^Five/
60 mm dishes) were implanted in a final volume of 4 ml / 60 mm dishes in EGF-free mSBM. 10 cells
% CO_TwoAnd incubated at 37 ° C. 24 hours later 10 ng / ml EGF was added
. The medium was replaced with a fresh medium (containing EGF) every three days, considering the plating day to be day 0.
Cells became confluent in 5-7 days, at which point they were passaged again.   Epidermal keratinocytes cultured in mSBM are highly mobile and form dispersed colonies
. Therefore, the colony formation rate is estimated as the plate efficiency. Total cells 24 hours after seeding
Plate efficiency, measured by number, was typically 30% to 35% at low passages (P: 3 or P: 4)
However, at this stage, the lag period is particularly short, and the plate efficiency is more than 100% in 24 hours.
Was frequently observed (Table 2).   Epidermal keratinocytes obtained from neonatal foreskin (cell lines FS16, B009 and B013, Table 2)
Throughout passage 6 (shown by other researchers to show a rapidly growing culture)
Initially maintained a population of small round cells (population doublings greater than 25 or cell numbers)
Increased by 6,400-333,000 times). Similarly, epidermal keratin cells obtained from a 2-year-old donor
Vesicles are maintained through 6 passages with population doublings of more than 24 or approximately 4,600 times the number of cells
Increased. Reference Example 3: Effect of calcium concentration   MSBM (0.08 mM calcium) human epidermal keratinocytes based on the method described above
Or Ca⁺⁺The cells were cultured in mSBM at a concentration of 1.8 mM with serum concentrations of 0.3% and 1.0%. Culture medium
When calcium was added, the morphology changed. In the presence of large amounts of calcium, cells
It grew in a very confluent colony, but still left a large amount of small proliferating cells. blood
The growth rate did not decrease appreciably when the washing concentration was kept below 1%. A lot of mosquitoes
Cell yields in confluent conditions due to stratification with
It is considerably higher in the cells with the addition of lucium.   Previously, the ability to vary calcium concentration while maintaining a growth culture
Experiments that were not possible, for example with known triggers of terminal differentiation in competent cells
The action of growth factors in relation to certain calcium and the differentiation, proliferation or growth of the growth factors
Studies on the induction of long inhibition can be performed. An example of such an experiment is shown in the table.
3 is shown. This experiment examined the effect of TGFβ on calcium. It was observed that the effect of TGFβ was reversible even in the presence of calcium. The above table shows the effects of calcium and TGFβ on human epidermal keratinocytes in mSBM.
Data from two separate experiments are shown.   a)% env-ionophore derived envelope%   b) After removing calcium and TGFβ and culturing in mSBM for another 48 hours,
Recovery was noted.   Note: TGFβ treatment and Ca⁺⁺% Difference in induced envelope showing the effect on growth is related to growth
There is no.   c) Medium contained 0.3% cFBS. The place where 5ng / ml TGFβ was added is indicated by ((+) T).
Where 1.8 mM calcium was added ((+) Ca⁺⁺).Example 1: Effect of serum concentration   Collagen containing mSBM using various concentrations of chelexed fetal bovine serum (cFBS)
28.3cm tissue culture dish coated with^TwoHuman epithelial cells (HEP) 1.7 × 10^FivePlaying pieces
And cultured as described above. The case where the cFBS concentration is 0% is the present invention. 24 hours flat
Plate efficiency, final cell yield, (known to be a small growing cell population (Barrando
n and Green,supra)) The percentage of cells in the 9-14 nM diameter range was determined and is shown in Table 4 below.
Increasing serum concentration did not significantly affect final cell yield, but plate efficiency
Has increased a little. However, increasing serum levels reduced the proportion of small proliferating cells
. This change in the cell population could also be seen by phase contrast microscopy.   Using various serum concentrations ranging from 0 to 5% or more, cells were cultured in mSBM and emSBM.
Can grow. However, increasing the serum concentration to only 1%, 9- Serum was either terminally differentiated or a cell population as indicated by the apparent loss of cells in the 14 nM range.
Has been found to promote the aging phenomenon. Kellexing calcium (chelexi
The same is true when ng) is removed from serum. Based on the above, serum can be used for cell growth and
Contains factors that directly or indirectly affect differentiation and are not related to calcium
It becomes clear that you are.Reference example 4 :Substrate effect   Substrates of type I collagen or fibronectin were first developed using mSBM or emSBM.
And has been found to be useful in the establishment of epidermal cells from
Have been. Collagen is usually used when culturing human epidermal keratinocytes using mSBM.
Used habitually. However, the actual substrate dependence and substrate lifetime
Investigations were performed to determine the effect on (Huspan).   Human epidermal keratinocytes grown as primary with emSBM using collagen substrate
The frozen stock of vesicles was prepared according to the procedure described above in the presence (+ CN) and absence (-C) of collagen.
N) and plated on mSBM. When confluency is reached, collagen + CN cells
The cells were subcultured in the presence and absence of cells (see Table 5A), and -CN cells were
In the presence and absence of the enzyme-subcultured into culture dishes (see Table 5B). General form, 4-hour plate
Efficiency and cell size distribution were tested at each passage. Flat plate efficiency exceeding 100%
Very short delay before resumption of growth that occurs when cells are subcultured in the medium of the present invention.
Means extra time. Plate cells from each condition, collect at the same time, and
I compared directly. The percentage of cells with a diameter of 9-14 mM indicates the percentage of proliferating cells in the population (
Barrandon and Green, supra).   Cells established from frozen primary cultures in the absence of collagen are cultured on collagen.
Slightly lower plating efficiency compared to cultured cells. Cells in the absence of collagen (-CN)
Furthermore, the plate efficiency was increased by subculture in culture dishes in the presence (+ CN) and absence (-CN) of collagen.
Was consistently better under the (+ CN) condition. However, -CN proliferating cells
It grew to a dense state and could be passaged (see Table 5B).   Maintain in the presence of collagen, then culture in the presence or absence of collagen
Cells passaged in the dishes showed various plating efficiencies, but use of collagen or
Had no obvious advantages or disadvantages in its use (see Table 5A).   Cells grown for 8 passages and passaged to collagen show reduced plate efficiency at 8 passages
(Equivalent to about 29.4 population doublings). Cells grown in the absence of collagen were p6-p7 (about 19
(Equivalent to ~ 22 population doublings).   High serum (5%) with 3T3 feeder cells (see, eg, Rheinwald and Green, supra)
Of epidermal cell lines established in p4 in the presence and absence of collagen from frozen stocks
No significant difference was observed in the plate efficiency when the plate was cultured in a culture dish. Furthermore,
The plate efficiency obtained in this experiment is significantly higher than when using cells grown in mSBM.
(Tables 5A and 5B).   Collagen is a cell population that falls in the diameter range of 9-14 mm, i.e. 7 passages (-CN) or 8
It was found not to affect the percentage of the passaged (+ CN) proliferating cell population (see Table 5C). Reference Example 5: Preparation of biological skin equivalent and keratin cell sheet   Using keratinocytes grown in mSBM in high serum (5%) and high calcium (1.8 mM)
To make a living skin equivalent. This procedure allows keratinocytes grown in MCDB 153
It should be noted that this cannot be performed if used. The epidermis maintains the basal layer
It developed normally with stratification and differentiation.   Skin prepared from keratinocytes grown in mSBM according to a conventional method (eg, patent method)
Biological skin equivalents were prepared by plating on valencies. Then put the skin equivalent in mSBM for 2 days
After growing, Ca⁺⁺Was added to form a layer. mSBM, 0.3% cFBS and Ca⁺⁺Skin inside
The equivalent was subsequently cultured to obtain a well-organized differentiated epidermal layer.   In addition, cells were established in mSBM on collagen and calcium concentration was reached when confluence was reached.
The concentration to 1.8 mM and use mSBM for 1.8 mM Ca⁺⁺Daily for 4 days, then
Suitable for transplantation, etc. by removing the adherent cell sheet using collagenase
A sheet of layered epidermal cells was prepared.Reference example 6 :Large-scale keratinocytes using collagen-coated microparticle carriers culture   Using an mSBM medium with a calcium concentration of 0.08 mM and a BHE concentration of 50 μg / ml,
Enrich human epidermal keratinocytes with cross-linked collagen microparticle carrier coated with collagen.
Plate cultivation was successful with efficiency. Disconnect using a 250 ml Belco culture vessel.
Stirring continuously (2 minutes every 8 minutes) at 30 rpm, small volume containing 0.5 g gel beads
5.1 x 10 in medium (75 ml)⁶Highly efficient (> 50%) seeding of human epidermal cells (HEP)
did it. Gel beads are equilibrated with mSBM, and bovine tendon collagen about 9.5 mg in 100 ml of pure water.
Coated overnight, then rinsed first with phosphate buffered saline and then free of calcium.
It was prepared by rinsing with fresh DMEM. One week later, try to remove trypsin-E
Collected by DTA, 2.1 × 10⁷Cells were obtained. Use standard culture conditions and mSBM to
These cells were plated in a T25 culture flask and harvested and counted 24 hours later.
When the efficiency was determined, it was found to exceed 100%.   This procedure was repeated using the keratinocyte cell line HEP 047 at passage 5. Medium 150m
l Gel beads inside (surface area approx.^Two) Cells in each of two stirred flasks containing 0.25 g
6 × 10⁶Was plated. Cell yields from the two flasks after 6.2 days were 6.2 x 107 and 5.
6 × 10⁷Met. Thus, an average of 10-fold increase in cell number could be reached.   In another experiment, cell biology was performed to facilitate passage without the use of proteases.
It has been found that movement between doses is effective. Spherical collar for cell line HEP 038 at passage 4
Plated on Gen-coated gel beads. Next, irregularly shaped collagen-coated gelatin
The beads were added to the culture after 4 days. Two more days later, the beads were examined microscopically. Fine
Vesicles are attached to both spherical gel beads and irregularly shaped gelatin beads
Was observed. Reference Example 7: Addition of a divalent cation substitute for calcium in mSBM   Improving cell proliferation and size distribution in human epidermal keratinocytes at passage 4
1 mM strontium and 3 mM magnesium in mSBM +/- bovine hypothalamus extract
Compared. The table below shows the results from the three cultures.   Similarly, the strontium dose response was determined in another keratin cell line. 1mM Sr
1.7 x 10 cell yield⁶From 2.1 × 10⁶It was found to increase. 9-14μm distribution is 2
It increased from 0% to 26%. According to yet another strain in another experiment, a cell yield of 1 mM Sr was 1
.45 × 10⁶From 2.66 × 10⁶Doubling time reduced from 33.9 hours to 28.1 hours
Was. Example 9: Establishment of primary culture in mSBM using explant by-product   The following modified mSBM composition: Hydrocortisone 1.1 μM Insulin 5μg / ml Transferrin 5μg / ml Triiodothyronine 20pM Ethanolamine 1 × 10^-FourM O-phosphorylethanolamine 1 × 10^-FourM Adenine 0.18mM Selenium 5.26 × 10^-8M Epidermal growth factor 10ng / ml Ca⁺⁺75% without DMEM Hams-F-12 25% Strontium 1mM Was used to establish primary explant cultures. Modification of progesterone (cAMP level
And can eliminate cAMP enhancer and promote growth in mSBM
In that strontium (Reference Example 7), which was found to have   Peel off fat and subcutaneous tissue from neonatal foreskin, 1-2 mm^TwoSubdivided into pieces. Bovine thalamus
Explant culture in the above medium with or without lower extract (BHE) (50 μg / ml)
I established things. The case where BHE is not added is the present invention.   The yield from explant plate by-products is shown in Table 9A.   Next, the cells were placed in a culture dish having a diameter of 100 mm at 3 × 10^Fivecell
/ Passaged on a dish. The yield from the secondary culture (collected after about one week) is shown in Table 9B. Example 3: Primary in the absence of exogenous cAMP enhancer, serum or bovine hypothalamus extract Proliferation of normal human epidermal cells   1mM SrCl without progesterone_TwoWith modified mSBM basal medium containing collagen
Human epidermal cells of three different origins can be obtained from skin explant by-products in coated tissue culture dishes.
Established. Cholera toxin, a powerful cAMP enhancer, chelexed newborn
Explants were established in the presence and absence of bovine serum and bovine hypothalamic extract,
The effects of these three substances on the establishment of primary culture and subculture were examined. Bovine serum
And the case where no bovine hypothalamus extract is contained is the present invention.   At all conditions tested, cells differentiated as explant by-products. Cell yield of 7 under all conditions
Continuous passage was monitored. FIGS. 1 and 2 show the results obtained when all cells under the given conditions were passaged.
The yield / passage calculated as the number of cells obtained is shown. S and K in FIGS. 1 and 2
X represents serum (0.3%), CT represents cholera toxin (9 ng / ml), BEH represents bovine hypothalamus
Partial extract (50 μg / ml).   Serum, cholera toxin, and bovine hypothalamus extract were both primary and normal humans
It was found that it was not required for serial passage of keratinocytes. Including these three substances According to no control culture, only 1 × 10 after 34 days of culture and 6 days of continuous passage^TenThe yield of
Obtained. In addition, according to the control conditions, serum alone and in the presence of serum and cholera toxin
It showed improved growth compared to established and expanded cultures.   Neither bovine hypothalamic extract or cholera toxin used in the absence of serum
Bovine hypothalamus extract significantly improved cell yield, although vesicle yield could be increased
And increased the number of passages where population doubling was maintained linearly
. In addition, bovine hypothalamic extract has significantly more negative serum effects than cholera toxin.
It was found to equilibrate. Bovine hypothalamus extract in the presence or absence of serum
When present, cholera toxin has been found to have no beneficial effect.   Therefore, any of serum, bovine hypothalamus extract or cyclic AMP enhancer,
It is not necessary for the establishment and continuous passage of human keratinocytes.Example 4: Culture and passage in the absence of EGF   Keratinocyte B063 was established and grown by adding BHE to the mSBM of Example 2.
, Were frozen for storage after 2 passages. Thaw these cells and culture in the absence of BHE
1x10 in the ground, 60mm culture dish^Five/ Seeded in a dish. Half of the culture dishes are 10 hours after seeding 2 hours after seeding.
EGF was added to ng / ml. Cells are harvested and counted after 3 passages and grown in the absence of EGF.
Was recultured and grown again to confluence. It is clear from the data (Table 11)
As needed, keratinocytes can grow in mSBM in the absence of EGF, if desired.
, May be comparable to growth in the presence of EGF. Example 5: Growth in the absence of hydrocortisone   If minimal supplementation is desired, hydrocortisone is not required, in which case
It is better not to do so.   This was demonstrated with hydrocortisone at 0, 0.04, 0.2, 0.4 or 0.8 μg / ml in the examples.
1.7 × 10 in medium 2^FiveTo seed 3 passages of B063 keratin cell line on 3 / 60mm dish More proven. Twenty-four hours later, plates are collected, the average plate efficiency is determined, and an additional 5
After a day, the growth rate was determined. Cell yields were comparable under all conditions. Between these decisions
The doubling time was calculated for 4 days (Table 12).   The above examples have been described for the purpose of illustration, and in view of the above description,
It is understood that various modifications as expected are included in the spirit and scope of the present invention.
I want to be understood.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 and 2 are graphs showing the experimental results of Example 3 in terms of cell yield and culture period.

Claims (1)

Claims: (1) a) about 0.5 to about 50 μg / ml insulin or about 10 ⁻¹⁰ to 10 ⁻⁸ M insulin-like growth factor; b) about 0.05 to about 50 μg / ml transferrin; c)
And triiodothyronine about 2~200pM, d) from about 10 ^-6 to about 10 ^-2 M ethanolamine or O- phosphoryl - at least one of ethanolamine, e) from about 0.005 to about 2.
0 mM calcium and f) DMEM, IDMEM, MEM, M199, RPMI 1640, Ham's F12, Ham '
s A chemically defined cell culture medium comprising a nutrient source selected from at least one of F10, NCTC 109 and NCTC 135, and containing no undefined mammalian hormones. (2) The culture medium according to claim 1, wherein the nutrient source comprises 75% calcium-free DMEM and 25% Ham's F-12. (3) The cell culture medium according to claim 1 or 2, further comprising about 0.04 to about 4 µg / ml of hydrocortisone. (4) The composition further comprising about 1 to about 50 ng / ml of epidermal growth factor.
The cell culture medium according to any one of claims 1 to 3. (5) In addition, a cyclic AMP level enhancer selected from at least one of cholera toxin, forescoline, isoproterenol, methyl isobutylxanthine, dibutyryl c-AMP, theophylline, caffeine and B. pertussis toxin is used in an amount of about 10%. ^-9
The cell culture medium according to any one of claims 1 to 4, wherein the cell culture medium contains from about 10 ^-3 M to about 10 ^-3 M. (6) The composition of claim 1, further comprising about 0.02 to about 2.0 mM adenine.
5. The culture medium according to any one of 5.