JP3066624B2 - Human epidermal cell culture medium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium for culturing cells, particularly a basal medium suitable for culturing human epidermal cells and a medium for growing human epidermal cells prepared therefrom.

[0002]

2. Description of the Related Art Skin is located on the surface of a living body and is composed of an epidermis layer and a dermis layer, and plays an important role in protecting a living body and preventing invasion of foreign substances from the outside. Therefore, individuals with skin damage or the like are exposed to dangers such as leakage of bodily fluids and bacterial infections, particularly when skin damage is deep and widespread due to severe burns or burns,
It causes abnormalities in the living body and may lead to death in some cases.

[0003] As one of the methods for dealing with such skin damage, skin transplantation has been conventionally known, and in recent years, cultured skin transplantation using cultured epidermal cells has attracted attention [Naotaka Ishikura et al. J. Jpn.PRS), 7 , 10
86-1093 (1987); Norio Kumagai et al., Journal of the Nihon Keikai (J. Jpn. PRS), 8 , 574-585.
(1988); Norio Kumagai, Dermatology, 10 (12) ,
1088-1093 (1988); Norimitsu Kuroyanagi, Protein Nucleic Acid Enzyme, 36 (7) , 1443-1449 (199 ) .
1); Green, Nikkei Science, January 1992, pages 84-92, etc.].

[0004] Although the above-mentioned cultured skin transplantation has already been confirmed to be useful in clinical settings, there are still some problems with the technique for culturing human epidermal cells used for transplantation.

That is, as a method for culturing the human epidermal cells,
Are, for example, reports of Piterkou et al. (Arch. Dermatol.,12
Three, p.1541a, Nov., 1987]. Human epidermis
Vesicles can be continuously cultured for the first time in vitro
It was in 1975 when the method by Green et al. Was announced
The method is a method for controlling growth by radiation treatment.
Mouse 3T3 fibroblasts for human epidermis cell feeder lei
Characterized in that it is used as a layer (supporting cell layer)
[See JP-A-52-87291]. To this culture system
According to the addition of the epidermal growth factor EGF,
Has been shown to be able to extend from 50 to 140 cell generations,
Further, the culture medium is now being further improved.

[0006] On the other hand, it has been reported that the use of a feeder layer is not indispensable due to improvement and study of the medium, and a basal medium MCDB153 for culturing human epidermal cells was announced.
A medium prepared for growth of human epidermal cells comprising the basal medium, that is, insulin 5 μg / ml, hydrocortisone 0.5 μg / ml, ethanolamine 0.1 mM,
Phosphoethanolamine 0.1 mM, EGF 10 μg /
and a medium supplemented with 70 μg / ml of bovine pituitary extract BPE [Journal of TissueCulture M
ethods, Vol. 9 (2), p. 83 (1985)].

[0007] Further, Pitelkou, etc.
Histidine is about 4-fold higher in a growth medium consisting of
An improved medium containing the same amino acid so that isoleucine is about 51 times, methionine is about 4 times, phenylalanine is about 4 times, tryptophan is about 4 times and tyrosine is about 6 times is reported [Mayo Clin Proc., 61 , 771-
777 (1986)].

[0008] However, the above-mentioned culture media and culture techniques using the same are still unsatisfactory for applying them to cultured skin transplantation using human epidermal cells.

[0009]

DISCLOSURE OF THE INVENTION The present invention provides a novel method having a high cultivation efficiency and capable of culturing and growing a limited amount of human epidermal cells in a short time and in a large amount in the above-mentioned conventional technical level. It is an object of the present invention to provide a culture medium for the method, in particular, the above-mentioned technique which is sufficiently applicable to a cultured skin transplant.

[0010]

According to the present invention, in MCDB153 basal medium for tissue culture, histidine is about 3 times, isoleucine is about 50 to 100 times, methionine is about 6 to 12 times, and phenylalanine is about 6 to 12 times. ~ 12 fold, tryptophan about 3-6 fold and tyrosine about 10-20
A basal medium for culturing human epidermal cells, characterized in that it has been modified so as to have a doubled ratio, in particular, histidine is about 3 times, isoleucine is about 100 times, methionine is about 12 times, phenylalanine is about 12 times, tryptophan is about 12 times. The above-mentioned basal medium containing 6 times and about 20 times tyrosine, the above-mentioned medium suitable for growing human epidermal cells, and an effective amount of a growth promoting substance and a pharmacologically effective amount of an antibiotic for supporting the growth of human epidermal cells are further added. And a method for expanding human epidermal cells using the medium.

[0011] The basal medium of the present invention comprises MCDB for tissue culture.
It is characterized in that specific amino acids constituting the 153 basal medium are fortified. The MCDB153 basal medium itself for enriching amino acids is already known [see the report of Piterkou et al. And the references cited therein], and is also available as a commercial product. Among them, the specific amino acid composition to be modified according to the present invention is histidine 0.8
× 10 ^-4 M, isoleucine 0.15 × 10 ^-4 M, methionine 0.3 × 10 ^-4 M, phenylalanine 0.3 × 10
^-4 M, tryptophan 0.15 × 10 ^-4 M and tyrosine 0.15 × 10 ^-4 M.

In the present specification, the term “MCDB153 basal medium” refers not only to a medium having the same composition as that already reported including the amino acid composition but also to a medium having a composition substantially the same as this. Used as a generic term. That is, for example, the types and contents of amino acids, main inorganic ions, vitamins, predetermined organic compounds, trace elements and the like in the above-mentioned medium are determined by the M
The CDB153 basal medium can be appropriately changed within a range that does not substantially impair the essential characteristics. Each of these components can be appropriately used in the form of a salt or a hydrate.
Further, the medium is usually liquid, but may be in the form of a powder that is used by dissolving it in water at the time of use.In such a case, components such as a buffer and a pH adjuster are separately added as additives.
It can also be removed from powdered products. Furthermore, according to the study by the present inventors, it was confirmed that the disodium monohydrogen phosphate and sodium pyruvate contained in the MCDB153 basal medium were not important for the purpose of the present invention. Therefore, the presence or absence of such a composition in the medium of the present invention and the amount of the composition when the composition is combined can be determined arbitrarily.

As a specific example of the MCDB153 basal medium, for example, a medium having the composition shown in Table 1 below can be exemplified.

Table 1 L-alanine 8.91 mg / L L-arginine hydrochloride 210.67 L-asparagine monohydrate 15 L-aspartic acid 3.99 L-cysteine hydrochloride monohydrate 42.15 L- Glutamic acid 14.7 L-glutamine 876 Glycine 7.51 L-histidine hydrochloride monohydrate 16.77 L-isoleucine 1.97 L-leucine 65.6 L-lysine hydrochloride 18.27 L-methionine 4.48 L-phenylalanine 4.95 L-proline 34.54 L-serine 63.05 L-threonine 11.9 L-tryptophan 3.06 L-tyrosine 2.72 L-valine 35.15 biotin 0.015 folic acid 0.8 Inositol 21.62 Nicotinamide 0.037 Calcium pantothenate 0.48 Pyrid hydrochloride Syn 0.062 Riboflavin 0.038 HEPES 6700 Baking soda 1200 Thiamine hydrochloride 0.34 Cyanocobalamin 0.41 Choline bitartrate 25.33 Ribo acid 0.21 Putrescine dihydrochloride 0.16 Thymidine 0.73 Adenine sulfate 72.79 Sodium chloride 7597.2 Potassium chloride 111.84 Calcium chloride (anhydrous) 3.33 Magnesium chloride (anhydrous) 57.13 Disodium hydrogen phosphate 283.92 (anhydrous) Sodium pyruvate 55 Glucose (anhydrous) 1080.96 Phenol red 1 .17 Copper sulfate pentahydrate 0.00275 Ferrous sulfate heptahydrate 1.39 Manganese sulfate heptahydrate 0.00015 Ammonium molybdate tetrahydrate 0.00124 Nickel chloride hexahydrate 0.00012 Nato selenite Um 0.0052 Sodium silicate (anhydrous) 0.061 Stannous · dihydrate 0.000113 ammonium vanadate 0.00058 Zinc sulfate · 7 chloride dihydrate 0.144.

[0015] The basal medium of the present invention comprises the MCDB153.
The specific amino acids constituting the basal medium are fortified to a predetermined value, that is, histidine is increased about 3 times (weight times, hereinafter the same),
Isoleucine about 50-100 times, methionine about 6-
12 times, phenylalanine about 6 to 12 times, tryptophan about 3 to 6 times and tyrosine about 10 to 20 times, particularly preferably histidine about 3 times, isoleucine about 100 times, methionine about 12 times, The greatest feature is that the composition is modified so that phenylalanine is about 12 times, tryptophan is about 6 times, and tyrosine is about 20 times.

The basal medium of the present invention is very suitable as a medium for growing human epidermal cells based on the above characteristics, and the present invention provides a medium for growing human epidermal cells using such a basal medium. Also provide.

The preparation of the medium for growing human epidermal cells of the present invention can be performed in the same manner as in a general medium for cell culture.
That is, this is adjusted by adding an effective amount of a growth-promoting substance and a pharmacologically effective amount of an antibiotic, which are generally known to be used for supporting growth of human epidermal cells, to the basal medium of the present invention.

The growth-promoting substance is not particularly limited and may be any of various known substances, and the amount thereof can be arbitrarily set within a range effective for supporting cell growth. Of these, particularly preferred substances include, for example, growth promoting substances selected from the group consisting of insulin, hydrocortisone, ethanolamine and phosphoethanolamine. In addition, the growth of human epidermal cells
It is also known that bovine pituitary extract known as PE is effective [Journal of Tissue Culture Methods, V
ol. 9 (2), p. 83 (1985)], which can also be preferably added and compounded as the above-mentioned growth promoting substance. Further, according to the study of the present inventors, the BPE has a quantitative limitation in its availability,
Instead, it was confirmed that bovine cerebral-hypothalamic extract (BHE), which is easily available in larger quantities, similarly favorably supports the growth of human epidermal cells and is effective for long-term subculture. Therefore, in the present invention, such BHE can also be effectively used as the growth promoting substance. The above BH
E is an aqueous extract of bovine cerebral-hypothalamic tissue, which can be produced in the same manner using bovine cerebral-hypothalamic tissue instead of bovine pituitary tissue in BPE.

However, conventionally, epidermal growth factor (EGF),
Tumor growth factor (TGF) and hepatocyte growth factor (HGF)
Each of these substances also has a proliferative effect on human epidermal cells, and in the present invention, each of these factors can be used as a growth-promoting substance. Considering the application of cultured cells to cultured skin transplantation using a culture medium, some dangers are pointed out.In fact, according to the studies by the present inventors, when morphological abnormalities of cultured cells are observed Therefore, it is not particularly preferable, and the medium of the present invention can sufficiently exhibit the desired growth effect without using these growth factors.
Rather, it is preferable to avoid such addition and blending.

Particularly preferred growth media of the present invention include:
Less than 2 × 10 ⁻⁶ M, especially about 2 × 10 ⁻⁷ M insulin, 0.1-2.5 μg / ml, especially about 0.5 μm
g / ml hydrocortisone, 0-500 μM, especially about 100 μM ethanolamine, 0-2500 μM,
In particular, about 100 μM phosphoethanolamine and 25-
The MCDB153 basal medium to which 150 μg protein / ml of BPE and / or BHE is added and blended can be exemplified.

The antibiotics to be added to the growth medium of the present invention and the amount of the added antibiotics can also be in accordance with the conventional usage in this field. Typically, about 10 kanamycins
It is preferable to add and mix about 0 μg / ml.

The growth medium of the present invention may contain various other medium components commonly used in this field, as long as the characteristics thereof, particularly the effects specific to the basal medium of the present invention used are not impaired. Or components already added and blended can be added as appropriate. For example, amino acids, vitamins, trace elements, and the like contained in the basal medium of the present invention have a composition particularly suitable for preparation of a medium for growing human epidermal cells. One or more of these may be added as appropriate, as long as they are not impaired.

Hereinafter, the method of culturing human epidermal cells using the growth medium of the present invention will be described in detail. First, human epidermal cells (keratinocytes) for culturing are prepared by various methods known per se. Can be adjusted. The culturing can be performed in the same manner for both primary culturing and subculturing, and culturing can be basically performed according to a conventional method, for example, at 37 ° C. in a 5% carbon dioxide gas phase.
There is no restriction on the size of the culture dish, the culture substrate, the cell density at the time of seeding, the medium replacement time, and the like.

More specifically, the culture is carried out in a culture dish coated with collagen as a culture substrate, particularly type I collagen, and the cell density at the time of seeding is about 2.5 to 5 × 1.
It is preferably carried out under a condition of about 0 ³ cells / cm ² , and the desired good cell growth can be achieved by changing the medium once every two days. The proliferating human epidermal cells thus obtained are not constant depending on the age of the donor of the cells, the condition of the cells, and the like, but generally increase to about twice the number of cells after about 24 hours of culture.

According to the use of the growth medium of the present invention, excellent growth of human epidermal cells is achieved as described above, and the grown cells are excellent in adhesion rate and have no abnormality in cell morphology. There is an advantage that application to cultured skin transplantation can be performed very advantageously.

This cultured skin transplantation can be performed by preparing a sheet of the proliferating human epidermal cells obtained according to the present invention, and the sheet can be grown, for example, into a confluent cell obtained as described above. The layers of human epidermal cells can be further cultured in a layered medium to layer the epidermal cells into layers and differentiate into a tissue morphology similar to that of living epidermis. The layered medium can be prepared from a normal basal medium, including the basal medium of the present invention, and has a calcium concentration of about 0.5.
Adjusted to 4-2.0 mM, and fetal calf serum (FCS)
Is preferably added. By culturing in the above-mentioned stratified medium for about one week, epidermal cells are multi-layered into 5 to 6 layers, and basal cells having proliferative ability are observed in the lower layer, and differentiated keratinocytes are observed in the upper layer. The thus prepared human epidermal cell sheet can be used for cultured skin transplantation in the same manner as a known sheet of this type.

[0027]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[0028]

Example 1 Preparation of Basal Medium of the Present Invention (1) In a Far Eastern MCDB 153 medium (manufactured by Far Eastern Pharmaceutical Co., Ltd., the composition of which is shown in Table 1 above), the following amino acids were prepared as follows. A predetermined amount of each amino acid (parenthesis indicates the molar concentration of each amino acid) was additionally added to prepare a basal medium of the present invention.

L-histidine hydrochloride monohydrate 50.31 mg / l (2.4 × 10 ⁻⁴ M) L-isoleucine 98.39 mg / l (7.5 × 10 ⁻⁴ M) L-methionine ^{86mg / l (1.8 × 10 -4} M) L- phenylalanine ^{29.74mg / l (1.8 × 10 -4} M) L- tryptophan ^{9.19mg / l (0.45 × 10 -4} M) L -Tyrosine 27.18 mg / l (1.5 × 10 ⁻⁴ M) The medium thus obtained was dissolved in water at the time of use, and further 6700
mg / l HEPES and 1200 mg / l baking soda are used. Hereinafter, this is referred to as “basal medium 1”.

(2) In the same manner as described above, each amino acid was additionally blended so as to have the following amino acid composition to prepare a basal medium.

L-histidine hydrochloride monohydrate 50.31 mg / l (2.4 × 10 ⁻⁴ M) L-isoleucine 196.78 mg / l (15 × 10 ⁻⁴ M) L-methionine 53.72 mg / 1 (3.6 × 10 ⁻⁴ M) L-phenylalanine 59.48 mg / l (3.6 × 10 ⁻⁴ M) L-tryptophan 18.38 mg / l (0.9 × 10 ⁻⁴ M) L-tyrosine 54.36 mg / l (3.0 × 10 ⁻⁴ M) The medium thus obtained was dissolved in water at the time of use, and further dissolved in 6700
mg / l HEPES and 1200 mg / l baking soda are used. Hereinafter, this is referred to as “basal medium 2”.

[0032]

Example 2 Preparation of Growth Medium of the Present Invention Using each of the basal mediums 1 and 2 prepared in Example 1, a medium for human epidermal cell growth was prepared as follows.

That is, after adjusting the pH to 7.2 with 6N NaOH, each of the growth media was prepared by adding the following growth promoting substances and antibiotics to each of the filter-sterilized basal media 1 and 2, respectively. Hereinafter, these are referred to as “growth medium 1” and “growth medium 2”, respectively, corresponding to the basal medium used.

Insulin 2 × 10 ⁻⁷ M hydrocortisone 0.5 μg / ml ethanolamine 100 μM phosphoethanolamine 100 μM BHE (adjusted in Reference Example 1) 100 μg protein amount / ml kanamycin 0.1 g / l

[0035]

Example 3 Culture of Human Epidermal Cells The cell concentration was 1-2 × 10 ⁵ cells / m according to Reference Example 2 described below.
5 ml of human epidermal cell suspension adjusted to be 1 l
Is 6 cm coated with collagen (type I)
The seeds were inoculated on a culture dish having a diameter (manufactured by Corning Incorporated), and primary culture was performed using each of the culture media 1 and 2. The culture is 37
C. under 5% CO 2 gas phase and start culturing 20-24
The medium was exchanged after time and every other day thereafter. Since the epidermal cells grew well to a full culture dish (confluent) in 12 days, the cells were subcultured and subcultured for the second cell generation.

[0036] The passage treatment was performed by removing the culture solution and using Dulbecco P
After washing twice with BS (-) (manufactured by Nissui), 0.01 w / v
Dulbecco's PBS (-) containing% EDTA and 0.125% trypsin was added, and incubated at 37 ° C for 5 minutes. After confirming that the epidermal cells were detached from the culture dish, 30 w / v% FCS (bovine A DME culture medium (manufactured by Nissui) containing fetal serum) was added, and an epidermal cell suspension was obtained by pipetting. The precipitate obtained by centrifugation at 4 ° C. and 1,000 rpm for 5 minutes was used to remove a small amount of culture medium 1 or 2. By suspending the suspension.

The subculture is performed using a suspension adjusted to a cell concentration of 3 to 7 × 10 ⁴ cells / ml.
It carried out similarly to the said primary culture.

The epidermal cells proliferated well to a full culture dish in one week. Further, by repeating the subculture and the subculture in the same manner, human epidermal cells could be proliferated well.

In the above culture, the effective amount of each of the growth promoting substances contained in the growth medium was examined using the basal medium 2, and as a result, within the compounding range disclosed in the present specification, especially the growth medium 2 was adopted. It has been confirmed that particularly favorable growth results can be obtained at the added amounts.

[0040]

Example 4 Comparative culture test of human epidermal cells According to Examples 1 and 2, Far Eastern MCDB153 medium or its improved medium reported by Pitherkou etc. [Mayo Clin. Pro
c., 61 , 771-777 (1986)] was prepared as a comparative medium. Hereinafter, these are referred to as “Far East medium” and “Piterkou medium”, respectively.

The difference between each medium and the growth mediums 1 and 2 of the present invention lies in the amounts of the six amino acids, which can be compared with the molar concentration of each amino acid in the Far Eastern medium as a reference (1). Table 2 below.

[0042]

[Table 2]

Human epidermal cells were cultured and expanded in the same manner as in Example 3 in a 35 mm 6-well culture dish coated with collagen (type I) using each of the above four types of media. The epidermal cells were seeded at 5 × 10 ⁴ cells / well, and one group using each medium was tested in 6 wells.

On the fourth day of the culture, the cells in the wells of each group were treated in accordance with the above-described subculturing treatment to obtain a cell suspension, and the number of these cells was counted using a Coulter counter (manufactured by Coulter Electronics). It was measured.

FIG. 1 shows the results.

In the figure, the vertical axis represents the cell number (cell / c).
m ² ), and the abscissa indicates each medium used (: a Far Eastern medium as a control, a medium 1 for growth of the present invention, a medium 2 for growth of the present invention, and a piterkou medium as a control).

As shown in the figure, the growth media 1 and 2 of the present invention were
Based on the difference in the amounts of the six amino acids constituting the above, human epidermal cells can be favorably proliferated and their cultivation efficiency is excellent, as compared to each of the control media. .

[0048]

[Reference Example 1] Preparation of bovine cerebrum-hypothalamus extract BHE A cerebral-hypothalamic tissue site centered on the hypothalamus was extracted from bovine brain tissue, and coagulated blood and membrane components were removed. It was washed 3-4 times with saline, stored frozen at -80 ° C, thawed at 4 ° C before use, and subjected to subsequent operations.

About 300 g of the thawed tissue was pulverized for 3 to 4 minutes in 500 ml of a cooled physiological saline with a Polytron homogenizer (manufactured by Kinetics Co., Ltd.).
After finely pulverizing for 2 minutes, the mixture was centrifuged at 4 ° C. and 38,000 × g for 40 minutes to obtain a supernatant. Streptomycin sulfate (manufactured by Meiji Seika) is added to the supernatant to a final concentration of 0.5 w / v%, and the mixture is stirred at 4 ° C. for 18 hours and centrifuged at 38,000 × g for 60 minutes to obtain a supernatant. Was.

The obtained supernatant was subjected to pressure filtration sterilization with a sterilized filter (manufactured by Millipore) having a pore size of 0.22 μm in a clean bench to obtain an extract of about 10 mg protein / ml. This was dispensed in 5 ml portions, stored at -80 ° C, thawed at room temperature before use, and used as BHE.

[0051]

[Reference Example 2] Preparation of human epidermal cells Unnecessary discarded skin tissue pieces 1 x 1 cm at the time of skin graft surgery or skin tissue removal surgery were transferred to a petri dish, washed 2-3 times in cooled Dulbecco's PBS, and the fat layer was removed. Resected. This was immersed in a 75% ethanol solution containing 0.05% chlorhexidine gluconate for about 30 seconds to sterilize the surface of the skin piece, and further washed once or twice with a DME medium. The dermis was removed as much as possible with sterile small surgical scissors and cut into strips of 0.2 to 0.3 mm × 0.5 mm. A sterilization treatment was performed again by dipping in a 75% ethanol solution containing 0.05% chlorhexidine gluconate for about 20 seconds, and the pretreatment was completed by washing once or twice with a DME medium.

[0052] A small piece of skin pretreated as described above was used at 250 U / m.
After immersing in a DME medium containing 1 l of dispase (manufactured by Godo Shusei Co., Ltd.) at 4 ° C. overnight (about 24 hours), the skin layer and the dermis layer are divided by tweezers, and the epidermis layer is 0.25 w / v%.
Dulbecco's PBS containing trypsin solution (manufactured by Gibco)
Immerse in (-) at 37 ° C for 10 minutes, 30v / v% FCS
A DME medium containing (fetal bovine serum) was added, and a cell suspension in which epidermal cells were dispersed was obtained by pipetting. This was centrifuged at 4 ° C. and 1000 rpm for 5 minutes, and the sediment was suspended in a small amount of growth medium 2 to prepare human epidermal cells.

[0053]

[Reference Example 3] Preparation of sheeted cells In Example 3, the confluent cells were
After washing with BS (-), the medium was replaced with a layering medium (DME medium containing 10% FCS and 3.3 g / l HEPES). The medium was replaced every other day, and after 7 to 8 days, the stratified cells were washed once each with PBS (-) and DME medium. 37% in DME medium containing 0.75% collagenase
Enzyme treatment was carried out at 40 ° C. for 40 to 60 minutes until the periphery was slightly peeled off, and each was washed once with PBS (−) and a preservation solution (L-15 medium). Fill the preservation solution, aspirate until the cell sheet is slightly immersed, and support (Vesquitin W)
, And peeled together to obtain a cell sheet.

[Brief description of the drawings]

FIG. 1 is a graph showing the human epidermal cell proliferation effect of the medium of the present invention in a human epidermal cell proliferation test according to Example 4.

Claims (5)

(57) [Claims] 1. An MCDB153 basal medium for tissue culture, wherein histidine is about three times and isoleucine is about 50 to 50%.
A human epidermal cell characterized by being modified so as to be 100 times, methionine about 6 to 12 times, phenylalanine about 6 to 12 times, tryptophan about 3 to 6 times and tyrosine about 10 to 20 times. Basal medium for culture. 2. Histidine about 3 times, isoleucine about 100 times, methionine about 12 times, phenylalanine about 12 times, tryptophan about 6 times and tyrosine about 2 times.
2. The basal medium according to claim 1, wherein the basal medium is 0-fold. 3. The medium according to claim 1, which is used for growing human epidermal cells. 4. The method of claim 3, further comprising an effective amount of a growth-promoting substance and a pharmacologically effective amount of an antibiotic to support the proliferation of human epidermal cells.
The medium according to 1. 5. A method for growing human epidermal cells using the medium according to claim 3.