JPS60196186A - Serum-free culture medium composition containing low-molecular gelatin for adhesion-dependent cells - Google Patents

Abstract

PURPOSE:The titled medium composition that contains low-molecular-weight gelatin, thus enabling a large scale of cultivation of useful substance by selecting adhesion-depending cells which are capable of producing the objective substance. CONSTITUTION:The objective serum-free medium composition for mammarian adherent cells such as Fischer's medium, Parker's medium, etc., is allowed to contain low-molecular-weight gelatin of about 1,000-150,000 molecular weight. This kind of medium is combined with phosphorlipid, unsaturated fatty acids, ethanolamine, insulin, selene, transferrin or albumin. Mammarian adherent cells are transplanted into the serum-free synthetic culture medium containing gelatin of low molecular weight, extended as they adhere, then proliferated. The composition helps the proliferation of a variety of adherent cells.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a serum-free medium for adhesion-dependent mammalian cells containing low-molecular-weight gelatin, and a serum-free medium using the same for adhesion-dependent mammalian cells that adheres to a culture substrate surface, spreads, and develops a characteristic #II country-specific morphology. While maintaining
#I) Epithelial cells and fibroblasts derived from various tissues or organs are known as adhesion-dependent cells that divide and proliferate while forming an @ layer. on the other hand,
Conventionally, the culture of clear milk vIJ cells has been carried out using a medium containing a high concentration of fetal serum from decapitated horses, etc. However, in such a serum-containing medium, the quality of serum between lots varies However, there are various disadvantages such as difficulty in obtaining serum of the same quality, the serum itself being expensive, and difficulty in separating and purifying the target product from the cultured M cell culture. In recent years, inexpensive serum-free media have been studied. However, in a serum-free medium, phipronectin, which is a factor that promotes cell attachment and spread and is present in large amounts in serum, is removed from the medium when serum is not added, and therefore adhesion to the culture substrate is inhibited. For attachment-dependent cells, which are essential factors for cell proliferation, even if sufficient types and amounts of cell growth factors and nutritional components are present in the medium, 1. As a means of resolving this drawback of serum-free media, which results in significant inhibition of cell growth, high concentrations are added to the media to facilitate the attachment and spread of cells to the culture substrate. The cause of the addition of molecular collagen to the bottom and sides of the culture vessel is the presence of phipronectin, phytoin, etc., collagen, and sulfate polysaccharides, which are factors for cell adhesion and spreading to the surface of the microcarrier substrate (microcarrier), etc. , lectin,
Coating with polylysine or adjusting the surface charge of these culture substrates promotes cell attachment and spread and promotes cell proliferation.

For example, in serum-free culture of V79 cells, gelatin with a molecular weight of 100,000 to 200,000 heat-extracted from collagen, bovine serum albumin, insulin, pyruvate, hypoxanthine, and thymidine are dissolved in MEM, and dispensable amino acids are added. It is contained in 5F-F medium, which is a synthetic medium, and this medium is used to effect the adhesion of the cells to the culture substrate by gelatin (tissue culture research, collection of lecture abstracts of the 52nd research meeting of the Japanese Society for Textile Culture). , a method for improving the adhesion of No. 16 to 17 adhesion-dependent cells to a substrate is also known [Japanese Unexamined Patent Application Publication No. 71884/1984, Cell-Structure and e-Funk System". and
Function), 7, 245-252 (
1982)) 0 Similarly, a method using phipronectin to improve adhesion is also known (Japanese Unexamined Patent Publication No. 1983-1999).
Publication No. 186491, Ce1l 5structure
and Function, 7, 245-252.
(1982)).

As mentioned above, all of the conventional methods are expected to have an adhesion effect on the culture substrate surface in the serum-free culture method of adhesion-dependent cells. Coating methods such as gelatin or phipronectin have the disadvantage that the culture vessel coated with a cell adhesion promoting substance must be renewed each time culture is carried out, and is not suitable for high-density culture.On the other hand, the cell adhesion promoting substance coating microcarrier method is suitable for high-density culture, but
Microcarriers are expensive, and they can only be used once and cannot be used again. There were drawbacks that made it difficult.

As a result of various studies on serum-free synthetic culture of adhesion-dependent mammalian cells, the present inventors discovered, quite unexpectedly, that low-molecular-weight gelatin with a molecular weight of approximately 1000 to 150'00 was added to the serum-free synthetic medium used. It was discovered that the cells could spread and proliferate well without any coating on the surface of the culture substrate.

The present invention was completed based on the above findings, and provides a serum-free medium composition for adhesion-dependent mammalian cells, which is a serum-free synthetic medium containing low-molecular-weight gelatin with a molecular weight of approximately 1,000 to 15,000 as an active ingredient. , and a molecular weight of about 100
An adhesion-dependent MB characterized by transplanting adhesion-dependent mammalian cells into a serum-free synthetic medium containing 0 to 15,000 low-molecular-weight gelatin as an active ingredient, and then allowing the cells to adhere, spread, and proliferate. This is a method of culturing cells.

Furthermore, in the present invention, a medium capable of using low-molecular-weight gelatin is obtained, which can be used for culturing the desired cells by adding it to the medium, and the culture operation can be performed very easily. Since the additive is low-molecular-weight gelatin, it has various advantages such as no gelatinization and separation and purification from the target product in the culture becomes extremely easy.

First, the low-molecular-weight gelatin used in the present invention has a molecular weight of about 1000 to 15000, regardless of the origin of gelatin, if it is collagen-containing tissue such as bone or skin of mammals, birds, amphitheaters, or fish, or returning canals. (as an average molecular weight).For example, for convenience, the average molecular weight as a commercial product is 700'0,9000.
(manufactured by Nippi Co., Ltd.). In order to obtain low-molecular gelatin with a molecular weight of approximately 1,000 to 15,000, collagen is extracted with a weak acid using a conventional method, or high-molecular gelatin is extracted with hot water from a collagen-containing tissue or 1l-i' organ using an acidic or alkaline method. , and hydrolyzed with vi enzyme and passed through a molecular sieve to obtain a molecular weight of approximately 000-15.
000 fraction is obtained and used as needed in the serum-free synthetic medium used for culture.
It may be added in an amount of 0.001 to 10 mg/ml. Furthermore, the amount of low-molecular-weight gelatin to be used may be adjusted appropriately depending on the nature of the tissue, return duct, adhesion-dependent cells derived from cancer or other disease foci, such as TRC-29 derived from renal cancer.
For the R strain, the optimal concentration range of low molecular weight gelatin in the final preparation culture composition is 0.01-0.1 mg/ml, and for the uterine cancer-derived HeLa strain, it is 0.01-0. 1 m9/rnl, and in the case of the 1D CK strain derived from Daiken, it is 1 to 10 m9/ml.

Further, the serum-free synthetic medium used for adjusting this medium is not particularly limited, and examples include White's medium, Fisher's
)'s medium, Parker's medium or its modified medium, Earle's medium or its modified medium, 'Waymouth's medium or its modified medium,
), Eagle's medium or its modified medium, Pack's medium, Ha
m) and modified media, Trowell
) media, mags, etc. Further examples include V-614', Burr's medium, and Shi:teba N150. N635. N703
．． N858゜M”l99, CMRL-1066,
CMRL-1415, NCTC as a medium for Arc
109°NCTC135, MB752/1 for Waymouse, M for BME, Eagle medium.
EM, Dulbetsko MEIV [, Shoklik, Alpha MEM, Huck's medium: N15. N16...
F7. FIO, F12° Trowell medium is T8. Matsukoi 5 is a medium for Matsukoi.
A. RPMI-1629, RPM as mole medium
I-4630, RPMI-1634, RPMI~164
0 or a mixed medium thereof. Also, for example, MEM medium, 199 medium, ... Mu medium, R
PMI-1640 medium, CMRL-1066 medium, NC
In the medium composition such as TC-109 medium, various amino acids, vitamins, inorganic salts, and other glucose are added and adjusted. Preferably, the compounds include ethanolamine, incolin, glucagon, somatostatin, and parathyroid hormone (PTf()).
, Thyroprotein Relieving Hormone (TRI)
), Luteining Hormone Φ Releasing Hormone) (Lf(-RH), Selenium, Transferrin, Albumin, Evidermal Gross Factor (EGF)
'), fibriblast cross factor (FGF
), prostaglandins such as grosstaglandin F
Prostaglandin E1.2α \ One or more compounds selected from triodothyronine, hydrocortiscin, glogesterone, testosterone, estradiol, lactalbumin, etc. may be added for adjustment. For example, intuurin is 0.1 to 10
μ9/ml, selenium is 10-2 to 1O- as selenic acid
3nR4XEGF 1~100n,! ;'A/.

F G F l ~10 njq/fnl, puo, X
Taglandin E□1-100 ng7fnl:, triyothothione 1-1100p, hydrocortiscin 1
0 to 1 OOnl'v3 Tran This solution is sterilized by filtration using a Millipore filter or the like, or rarely sterilized by heating to prepare a serum-free synthetic medium containing low-molecular-weight gelatin.

Next, when culturing cells in a serum-free synthetic medium containing such low-molecular-weight gelatin, A3 cell transplantation is usually performed as a cell @ suspension, and depending on necessity, a serum-free synthetic medium is used. The dilution is adjusted at

In the final adjustment at this time, the amount of low-molecular-weight seratin used may be adjusted to 0001 to IQII+/m13.

Furthermore, the adhesion-dependent mammalian cells to which the serum-free medium composition for adhesion-dependent mammalian cells containing low-molecular-weight seratin prepared as described above is subject to at least the culture substrate during cell division and proliferation. Any mammalian cell that requires attachment to a surface may be used, including attachment-dependent normal diploid primary or established cell lines derived from normal and fetal tissue, and attachment-dependent primary or established cells derived from cancer and other diseased tissues. Punishment++
Examples include cells selected from %.

Furthermore, rather than the culture of adhesion-dependent mammalian cells, it may be used depending on the desired laboratory level, small scale to large scale, for example, 500 rnl to 501 J or more of the medium level. Further, the cells may be cultured under conditions of 37° C., 51 CO 3 and 100% humidity for 96 hours or more. The adhesion-dependent cells used in this culture first exhibit stickiness and extension due to the effect of the low-molecular-weight gelatin used, and then proliferate.

In this culture, cell proliferation was at least as high as in the case of a serum-containing medium shown as a control in an example described later, indicating extremely good proliferation.

As described above, the attachment-dependent serum-free medium composition containing low-molecular-weight gelatin of the present invention exhibits a good culture effect on various attachment-dependent cells and has the ability to produce the desired substance. By selecting adhesion-dependent cells, it is possible to provide an extremely simple and good method for mass-cultivating useful substances.

Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited by these in any way.
The cells were dispersed by treatment with a Tris-HCl buffered saline solution (hereinafter referred to as TBS solution) containing /m6 at 37°C for 10 minutes, and then treated with a phosphate buffered saline solution containing no magnesium and calcium (hereinafter referred to as PBS(-) solution). ) and wash 3 times with serum-free RP.
A cell suspension was obtained by suspending the cells in MI-1640 medium to a concentration of 2 x 10'' Cef46 cells.

The culture medium was prepared using 20 nF;
Serum-free RPMI containing Collaborative Co., Ltd. ~1.6
40 medium with 0.002 m9/ml of low-molecular gelatin (manufactured by Nitsubi Co., Ltd.) with an average molecular weight of 7000, and 0.0
2mg/ml! , 0.2 mry/mg, 2m97m1
．． The gelatin-containing medium was prepared by dissolving the mixture at a concentration of 10 my/me (W/y) and sterilizing it by filtration using a Millipore filter of 0.22 mm/art. In addition, as control media, gelatin-free RP M I-1640 medium and 10 FC8-containing RP + VI I-1640 medium were also prepared at the same time.

1 ml of each culture medium was placed in a 35 mm diameter plastic petri dish (Corning Co., Ltd. No. 25000) and was quantified by a colorimetric method based on the Lowry method to calculate the amount of proliferating cells.

The results of the culture are shown in FIG. Low molecular weight gelatin 001-0
．． It can be seen that the proliferation amount of cells cultured in a medium containing 7 ml of 1 mf is equal to or higher than that of cells cultured in a 10% FC8-containing medium as a control, as shown by the bar graph at the right end of the figure.

Example 2 Average molecular weight 7000.9000.13000.1950
Gelatin of 0.30000.75000 (Co., Ltd.
Nitsupi #) in PBS(-) solution 10""ml (W/
v) After dissolving to a certain concentration, the solution was sterilized in an autoclave at 121°C for 15 minutes. It serum RPfVII-164
0 medium-c O,l rv7. A gelatin-containing medium was prepared by diluting it to a concentration of . The TRC-29R cell line was seeded at 2 x 105 cells/shear, and the growth was compared with a final medium volume of 2. As a result, the average molecular weight was around 9000 (molecular weight 7000-130).
00) cells proliferated well in a medium containing low-molecular-weight gelatin.

Example 3 A medium in which 1Q% FCS-containing Dulbecco MEM medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) and 10% FC8-containing MuF12 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.) were mixed at a ratio of 1:1 (hereinafter referred to as 10% FC8-containing DM)
HeLa cells cultured for 4 days in E/ medium) were treated with Nagase enzyme at 0.3 u/l.
Cells were dispersed by treatment with a TBS solution containing TBS at 37°C for 5 minutes, washed three times with a PBS(-) solution, and then heated with serum DMF2/
Suspend cells in F12 medium 5 x 10'/1nl
A concentration of HeLa cell suspension was prepared.

Preparation of medium: 104/, z insulin, 2 Q nI
ShioIEGF, 5Ag'/ml! h Lanceferrin, 1100n Hydrocortisone, 100n,! 9
zQ! F G F (both ml, 20 m9/m'
A gelatin-containing medium was prepared by dissolving the gelatin to a concentration of 100 ml and sterilizing it by filtration using a 0.22 μm Millipore filter.

In addition, as control media, a gelatin-free DME/F12 medium and a 10% F''C8'') medium were also prepared at the same time.

After dispensing 1 ml of each medium into a plastic Petri dish with a diameter of 35 mm (-y-=: No. 25000 manufactured by yg), the HeLa cell suspension was mixed 5x, 104
Seed to form cells/shears, final medium volume 2ml
And so. After culturing in an incubator at 37°C, 5% CO2, and 100% humidity for 7 days, the protein content of the proliferating cells was quantified as the amount of proliferating cells.

The culture results are shown in Figure 2. White circles plot the amount of I4 cells cultured in serum-free medium containing gelatin at each concentration. The bar graph on the right side shows the amount of cell proliferation when the same cells were cultured under the same conditions in a medium containing 10% FC8 as a control.

Example 4 Daiken cancer-derived MDCK strain cultured in X2/F12 medium containing FC8 for 4 days was treated with Nagase Enzyme Q, 3"7ml
The cells were dispersed by IVI treatment twice for 10 minutes at 37°C with a TBS solution containing the same, washed three times with a PBS(-) solution, and then suspended in serum-free DME/F12 medium to 5 × 1♂ cells/m.
1 of cell suspensions were prepared.

Media regulation lOμg/rnl insulin, 0.01
nM/d, 0.2 m9/ml, 2 mti/ml, 2 o
Dissolve to a concentration of m9/y, 40η-/ml,
A gelatin-containing medium was prepared by filtering and sterilizing the cells using a 0.22 μm Millipore filter. In addition, as a control medium, gelatin-free D''''/F12 medium volume 0: lo%F CS
A Ti-containing DMFVFI2 medium was also prepared at the same time. Each culture medium was placed in a plastic petri dish with a diameter of 35 mm (Corning '4JN).

25,000), and the above MDCK cell suspension was seeded at 5 x 10' cells/shear to give a final medium volume of 2 ml. 37℃, 5chi CO
□, After culturing for 5 days in a colander under 100% humidity conditions, the protein content of the cells was quantified as the amount of proliferating cells.

Culture result f: shown in FIG. White circles plot the amount of cell proliferation cultured in serum-free medium containing gelatin at each concentration. The bar graph on the far right is DME/F containing 10% FC8 as a control.
This is the result of #A cell proliferation when the same cells were cultured under the same conditions in 12 medium and A cells were cultured under the same conditions.

[Brief explanation of the drawing]

Figure 2 shows the curve of the amount of cell protein produced by cell culture against the concentration of low-molecular-weight gelatin in the adhesion-dependent serum-free medium using the HeLa strain, and Figure 3 shows the curve of the amount of cell protein produced by cell culture using the Daiken cancer-derived MDCK strain. A curve showing the amount of cell protein produced by cell culture versus the concentration of low-molecular-weight gelatin in the adhesion-dependent serum-free medium used is shown. Patent applicant Director of the Agency of Industrial Science and Technology Figure 1 Final gelatin concentration mg/ml Figure 2 OTO'10' 10-2]

Claims (1)

Scope of Claims: (1) A serum-free medium composition for adhesion-dependent mammalian cells, which is a serum-free synthetic medium containing low-molecular-weight gelatin having a molecular weight of about 1,000 to 15,000 as an active ingredient. (2) Molecular weight approximately 1000-15000 (7) Low molecules; Raya's medium, Parker's medium, Erle's medium, Weymouth's medium, Eagle's medium, Pack's medium, Mu's medium, Trowell's medium, McCoy's medium. Claim 1, which is a medium, Morneau's medium, Williams medium E medium, a modified medium thereof, or a mixed medium thereof.
Compositions as described in Section. 4) In a serum-free synthetic medium, phospholipids, fatty acids, amine compounds, insulin, selenium, transferrin,
The composition according to claim 3, which contains one or more compounds selected from albumin, Echthermal Gross Fist Factor, Finrelast E Gloss Factor, prostaglandin, triiodothyronine, and hydrocortiscin. . (5) Transplanting adhesion-dependent mammalian follicles into a serum-free synthetic medium containing low-molecular-weight gelatin with a molecular weight of about 1,000 to 15,000 as an active ingredient, and then allowing the cells to attach and spread to grow and culture. A method for culturing adhesion-dependent mammalian cells, characterized by: (6) The content of low-molecular gelatin with a molecular weight of about 1000-15000 is 0.001-10 /
6. The culture method according to claim 5, wherein the amount of rnl. (A patent claim in which the serum-free synthetic medium is White's medium, Finn's medium, Ham's medium, Trowell's medium, McCoy's medium, Morneau's medium, Williams Medium E medium or a modified medium thereof, or a mixed medium thereof. The cultivation method according to item 5. (8) In the serum-free synthetic medium, phospholipids, fatty acids, amine compounds, insulin, selenium, transferrin, albumin, avitermal cross factor, fibriblast gross φ factor, grosstaglan The culture method according to claim 7, which comprises containing one or more compounds selected from Gin, triodothyronine, and hydrocortiscin. 5. A method of culturing a cell which requires attachment to a medium substrate surface during division and proliferation.