KR19990042747A - Serum-free medium for cell culture - Google Patents

Abstract

The present invention relates to a cell culture medium containing ITS-complex, linoleic acid-BSA, glucagon, thrombin, tyrocalcitonin, and dexamethasone in RDF (RPMI1640: DMEM: Ham's F-12) mixed basal medium.

According to the present invention, a serum-free medium can be obtained that is precisely supplied with a component required by the cell, which shows growth close to the growth of human insulin cells in the serum-added medium without adding serum, and which can be produced at low cost.

Description

Serum-free medium for cell culture

The present invention relates to a serum-free medium for cell culture, and more particularly to a serum-free medium suitable for culturing immortalized insulin secreting human beta cell lines.

Unlike in vivo, in order to supply culture medium to artificially cultivate animal cells in vitro, it is necessary to satisfactorily satisfy the nutrients close to the biological conditions based on body fluids such as plasma or lymph and environmental conditions such as pH, temperature and osmotic pressure. . Therefore, the culture medium composition was determined based on the body fluid, and the determined culture medium was selected and serum was added to the cells at an appropriate ratio, and then used for cell culture. In this way, early in the development of the medium, Morgan (Morgan) et al. 1950 produced M199 medium based on the body fluid composition and cultured the primary chick (Morgan, et al., 1950). Subsequently, the main medium developed was Eagle basal medium (Eagle, 1955) used for culturing human and rat fetal cells, and culture medium used for culturing rat fetal cells (Dulbecco's modified eagles medium, Dulbecco, et al., 1959), and Hamman's F-12 (Ham, 1965) and Morore et al. RPMI-1640 (Moore et al., 1967) used to culture hamster tumor cells.

In cell culture, 5-20% of serum is added to the basal medium according to the nutritional requirements of the cells. Serum contains a variety of hormones in the insulin or polypeptide family that promote cell growth and function, growth factors that regulate cell division and function, and adhesion and diffusion factors such as fibronectin, transferrin as a binding protein, lipids and minerals. Many trace elements are included. Serum is also a buffer that regulates the osmotic pressure and pH of the culture, and has a viscous action that protects cells from proteolytic inhibition and physical shock (Griffiths, 1987). Therefore, a constant concentration of serum is added to the basal medium to form a culture medium. Animal cells can be cultured using serums containing these various functions. However, when industrially producing useful substances, which are proteins of a specific kind, using cell culture, there are some problems as follows.

First, since serum contains many different types of proteins, it is difficult to isolate and purify useful substances to be obtained after cell culture. Second, due to regional specificity and trace elements, the ingredients are different at the time of serum production (Lot.), And there are several components that are not identified. Therefore, it is difficult to supply exactly the components required by cells and growth inhibition phenomenon for specific cells. This may be observed. Third, since serum is expensive, production cost and experiment cost are high in industrial mass cultivation or general experiment of cells. Finally, it is a major source of mycoplasma or virus.

Due to the above problems, the development of a low serum medium with low serum concentration or a serum-free medium without using serum at all was required.

The types of low serum and serum free media currently on sale or under development include: lowering serum concentrations, partially adding serum substitutes, adding hormones or growth factors or purified proteins instead of serum, and chemical composition It is classified as the case of adding the revealed protein, and the most frequently used is the addition of hormones, growth factors or purified protein instead of serum. Serum-free media for specific cell lines have the opportunity to be sourced from manufacturers in addition to development in the laboratory (Ratafia, 1989). Among serum-free media, serum-free media have been developed that add selenite, transferrin, albumin and lecithin as cell growth promoters of growth hormone instead of serum (Hayashi et al., 1976) (Guilbert, 1976). ). For example, human rectal cancer cells (Murakami et al., 1980), serum-free medium for unicellular antibody producing cells (Wolfe et al., 1988) have been studied.

Meanwhile, studies on hormones and growth factors using rat islet tumor cell lines (Fong et al., 1981) have also been published. However, the nutritional requirements vary according to the specificity of the animal species or cell specific, so the results of experiments using rat islet tumor cell lines cannot be directly applied to the culture of human tumor cell lines. . Therefore, the present inventors have intensively studied to find a serum-free medium composition suitable for human insulin-secreting cell lines, and thus, the present inventors have completed the present invention by identifying a combination of basic media, essential hormones, and peptides that provide an efficient culture environment. .

Accordingly, the present invention aims to produce a serum-free medium at a low cost, which is precisely formulated with the components required by the cells and provides a growth environment for immortalized human pancreatic beta cells equivalent to those in the serum-added medium.

1A to 1F are phase contrast micrographs (x100) of HB cells cultured for 3 days in a container coated with collagen and gelatin and an uncoated container, respectively.

2 and 3 are graphs showing the number of cells in hormone and growth factor combination RDF medium (each value represented by the mean of six experiments (+ S.E.M.)).

※ Explanation of code about main part of drawing

A: fetal bovine serum

B: RDF

C: ITS-complex

D: tyrocalcitonin + thrombin

E: linoleic acid-BSA + somatostatin

F: linoleic acid-BSA + somatostatin + thrombin

G: linoleic acid-BSA + glucagon + thrombin

H: linoleic acid-BSA + glucagon + somatostatin + thrombin

I: linoleic acid-BSA + tyrocalcytonin + somatostatin + thrombin

J: linoleic acid-BSA + glucagon + tyrocalcitonin

K: RDF / FCS (1%)

L: RDF (Basic Badge)

M: ITS-Complex + Linoleic Acid-BSA + Glucagon + Tyrocalcytonin + Somatostatin + Thrombin + Dexamethasone

N: linoleic acid-BSA + glucagon + tyrocalcytonin + somatostatin + thrombin + dexamethasone

O: ITS-complex + glucagon + tyrocalcitonin + somatostatin + thrombin + dexamethasone

P: ITS-complex + linoleic acid-BSA + tyrocalcytonin + somatostatin + thrombin + dexamethasone

Q: ITS-complex + linoleic acid-BSA + glucagon + somatostatin + thrombin + dexamethasone

R: ITS-complex + linoleic acid-BSA + glucagon + tyrocalcytonin + thrombin + dexamethasone

S: ITS-Complex + Linoleic Acid-BSA + Glucagon + Tyrocalcytonin + Somatostatin + Dexamethasone

T: ITS-Complex + Linoleic Acid-BSA + Glucagon + Tyrocalcytonin + Somatostatin + Thrombin

In order to achieve the object as described above, according to the present invention, cells containing ITS-complex, linoleic acid-BSA, glucagon, thrombin, tyrocalcitonin, dexamethasone in RDF (RPMI1640: DMEM: Ham's F-12) mixed basal medium Culture medium is provided.

The medium according to the present invention comprises 1) cellular adaptation of low serum medium, 2) selection of basal medium, 3) selection of adhesion components in adherent cells, 4) search for hormones and growth factors, 5) search for hormones and growth factors. Optimal concentration determinations, and 5) these were finally selected through a combination step.

Cell culture generally uses about 5 to 20% of serum in basal medium. For the adaptation of low serum medium to cell lines, normal cells can be added to serum by adding 2% of serum in basal medium. There is no change in cell morphology or intrinsic product that can occur as a result of the reduction, and in the case of tumor cells, there is no change in cell growth and function even if the serum addition rate is less than 2% (Doyle et al., 1993). ). Therefore, in the present invention, immortalized human pancreatic beta cells were used to adapt up to 2%.

In selecting a basal medium to be used as a serum-free medium, since the characteristics of the components are different depending on the basal medium to be researched and marketed, it is necessary to consider various factors such as amino acid types, vitamins, trace elements, nucleic acids, etc. The components were made into a single medium or a single medium was combined at an appropriate ratio to create a mixed basal medium and analyzed for cell adaptability in this basal medium.

The constituents of the basal medium vary depending on the type of medium, and are mainly about 50 kinds of components, with 13 to 20 kinds of amino acids used as a nitrogen source, and Ca ²⁺ , K ⁺ , Mg ² as inorganic salts. ⁺ , Na ⁺ , HPO _4, etc. are used to maintain ions and osmotic pressure in the medium, and in the case of vitamins, biotin, panthothenate, choline chloride, folic acid, Thiamine, vitamin B ₁₂ , and the like are contained in about 10 kinds, and are used for enzyme catalysis in a medium. Other substances include hypoxanthine, thymidine, a nucleic acid, glucose used as a carbon source, and linoleic acid, which functions as a cell membrane component. , Lipoic acid, and the like. Since these components have different effects on cell growth according to the cell type, the components of the medium should be identified to prepare single and mixed media and applied to the experiment.

The characteristics of the basal medium used in the present invention are as follows (Table 1).

Medium composition comparison (RPMI-1640, DMEM, Ham's F-12) Badge Type Badge Type RPMI-1640 DMEM Ham's F-12 Amino acids (species) 20 15 20 Organic salt (species) 5 7 9 Other ingredients (species) 3 2 8 Vitamin species 11 8 10

RPMI-1640 contains 20 amino acids and 11 vitamins, DMEM is a high concentration glucose medium containing a large amount of amino acids and vitamins compared to other media, and Ham's F-12 is an inorganic salt and sodium Other components include pyruvate, hypoxanthine and putrescine. Compared by component, amino acid RPMI-1640 is more than 5 kinds of DMEM, while the concentration is about 2 to 4 times less, and F-12 is 20 kinds like RPMI-1640, but the concentration is about 2 times less. . Other components include various types of hippoxanthin, lipoic acid, putrescine, thymidine, etc. in F-12 medium compared to three kinds of RPMI-1640 and two kinds of DMEM, and 11 kinds of vitamins are RPMI-1640. Most of them, F-12 is added in trace amount, DMEM is added at a concentration of about four times than RPMI-1640.

Since serum is not added to the serum-free medium, there are no adhesion and diffusion factors, fibronectin (Gold, 1979), fetuin (Fisher, et al., 1958), serum spreading (Honsik, 1989), and the like. . Therefore, in the culture of adherent cells, since the cells do not adhere and do not grow, an adhesion factor may be added to the culture medium or an adhesion function may be imparted to the culture vessel surface. There are various types of commercially available (Sigma, Gibco), but as the addition form fibronectin, collagen is mainly used for application of the culture vessel.

Since serum is not added to the serum-free medium, hormones or growth factors suitable for the proliferation of specific cells should be added to the chosen basal medium (Gospodarowicz, et al., 1976; Mather, et al., 1979; Murakami, 1980; Henry, 1981).

To examine some of the roles of hormones and growth factors, epidermal growth factor (EGF) regulates DNA replication in fibroblasts (D. Barnes et al., 1977) and prostaglandin is DNA replication. (AM Otto, 1982), cortisol plays a role in stimulating the production of growth factors (Bridson, 1969), insulin, EGF, glucocorticoids, and cholera toxin have a mitogenic effect (Mckeehan, 1984). And most growth factors and hormones are known as mitogen polypeptides that promote DNA synthesis in cells or are involved in cell proliferation (Lazar, 1987). EGF sometimes inhibits the growth of human epidermoid carcinoma in serum-free medium cultures (D.W.Barnes, 1982).

Serum contains a small amount of various hormones and growth factors, so select factors that may affect cell lines and add them to the culture. Common uses are insulin, transferrin, selenium (Guilbert, 1976) and the like, and commercially available as insulin-transferrin-sodium selenite (ITS Complex).

Therefore, in the present invention, using the immortalized human pancreatic beta cell line to investigate the effects of the basal medium, hormones and growth factors, etc. on the growth of human insulin cells, and to select the appropriate concentration of the selected components, these experimental results Serum-free medium is obtained by mixing whole elements on the basis of. The process is described in more detail as follows.

Example 1

Adaptation of Low Serum Medium

Immortalized human pancreatic beta cells [Dr. Roger James (obtained from the University of Leichester)] was incubated in a medium containing 10% (v / v) FCS (fetal bovine serum) and gradually lowered the serum concentration to 7.5%, 5%, 2% and 1%. Adapted to cells. Cell desorption was performed using trypsin / EDTA (GIBCO) at a concentration of 1 mg / ml, neutralized trypsin with RPMI / FCS at high serum concentrations, and soy trypsin inhibitor (Sigma) at 5% or less, and medium flask (25 cm). ² , Falcon) were cultured as 1x10 ⁵ cells / 6-7ml and cells were passaged at weekly intervals.

Cells adapted to low serum medium were frozen and preserved using cell freezing medium (Sigma, C6295).

Human pancreatic beta cells (HB-cells) cultured in 10% RPMI / FCS were cultured in 1 × 10 ⁵ cells in a medium flask (25 cm 2), and the serum concentration was gradually increased to 7.5%, 5%, 2.5%, 1%. Initial incubation with adaptation through three passages. After culturing HB-cells (1 × 10 5 cells / well) of triplicate 12 well plates (Nunc) for 7 days and measuring the number of cells, FCS containing 10%, 7.5%, 5%, 2.5%, 1% The number of HB-cells cultured in RPMI / FCS culture (3 ml) was shown in Table 2. In the table, the number of cells cultured with 10% FCS showed a slowing rate of about 73% at 5% FCS, and about 7% slow growth at 1% adaptive cells.

Cell count following initial passage of low serum medium FCS concentration (%) Cell number 10 7.5 5 2.5 One Cell No. (x10 ⁵ ) 2.48 ± 0.15 1.82 ± 0.16 1.20 ± 0.05 0.42 ± 0.04 0.18 ± 0.01

Cell numbers were measured after 5 days of incubation in low serum medium using cells adapted to each step from 10% to 1% of FCS. HB-cells ( ⁵ × 10 ⁵ cells / well) in a duplicate 25cm ² medium flask (Falcon) were incubated for 5 days and cell counts were determined to include 10%, 7.5%, 5%, 2.5%, and 1% FCS. The number of HB-cells cultured in RPMI / FCS culture (3 ml) was shown in Table 3. In the table, almost similar cell growth was seen at serum concentrations up to FCS 2.5% in FCS 10% medium. This generally indicates that the adaptive cell lines at each stage were adapted to low serum medium, and even in FCS 1% medium, the adaptive cell lines showed about 53% growth compared to 10% adaptive cell lines, compared to 7% of the initial adaptive cells. It shows a fairly adapted result.

Cell count following initial passage of low serum medium FCS concentration (%) Cell number 10 7.5 5 2.5 One Cell No. (x10 ⁵ ) 1.44 ± 0.20 1.02 ± 0.02 1.30 ± 0.15 1.12 ± 0.14 0.76 ± 0.05

Example 2

Screening of the base badge

RPMI-1640 (R), Ham's F-12 (F), DMEM (D), manufactured by Gibco, and H-Max (Hybri-Max, Sigma), a serum-free medium, were used as a basal medium. RPMI (RPMI-1640, R), F-12 (Ham's F-12, F), DMEM (D), D / F (DMEM: F-12), R / D (RPMI-1640: Ham's F-12), R / F (RPMI-1640: Ham's F-12), and RDF (RPMI-1640: DMEM: Ham's F-12) mixed 1: 1 to 1 , 25 mM HEPES, 1.2 mg / ml sodium bicarbonate and the antibiotic penicillin / streptomycin were added and screened for basal medium using cells adapted to FCS (1%). Comparative medium was used RPMI / FCS (RPMI-1640, sodium pyruvate, L-glutamine, HEPES buffer, FCS), RPMI / SF (RPMI-1640, sodium pyruvate, L-glutamine, HEPES buffer).

After culturing FCS (1%) adaptive cells, they were desorbed with trypsin and neutralized by the same amount of trypsin inhibitor, followed by centrifugation to obtain the number of cells to be tested. This was incubated overnight with 2 ml of culture medium on a 12 well plate (Nunc), 2 ml of medium was completely removed after the cells were attached, and the cultured cells were washed 2-3 times with PBS (Gibco), followed by R, D, and F. Cells were incubated for 5 days by adding 3 ml of R / D, D / F, R / F and RDF, and measured by tryphan blue exclusion.

In basal medium screening, 8 types of RPMI / FCS (0.5%), RPMI / SF, and titration ratios were searched. FCS (0.5%) and FCS (1%) adaptive cells were added daily with 2 ml of RPMI / FCS (0.5%) and RPMI / FCS (1%) cultures in a 12 well plate at an initial cell concentration of 5x10 ⁴ cells / well. Incubated twice, washed twice with PBS, and then added 3 ml of basal medium to measure cells after triplicate culture for 7 days for FCS (0.5%)-adapted cells and 5 days for FCS (1%)-adapted cells. . As a result (Table 4), when cells adapted to 0.5% serum were cultured at 5 × 10 ⁴ cells / well for 7 days, RDF medium had more cells than H-max, a serum-free comparison medium, except DF and RDF. The basal medium was counted less than the initial seeded cell number. Comparing the best RDF medium of the basal medium with 0.5% added serum, the growth rate was about 55% or more. In addition, RDF was the best of the basal medium and the growth was similar to H-max, the comparative medium, even when inoculated at 5x10 ⁴ cells / well in cells adapted to 1.0% serum. 12 degrees lower than RDF but contributed to cell growth rate than other media.

Screening of Basal Cultures Serum Concentration and Adaptive Cell Lines 0.5 1.0 Incubation date 7 5 Initially inoculated cells (x10 ⁴ ) 5.0 5.0 FCS concentration (%) basal medium Cell number (x10 ⁴ ) Cell number (x10 ⁴ ) H-MaxF-12DMEMRPMID / FR / DR / FRDFRPMI / FCSRPMI / SF 3.88 ± 0.222.23 ± 0.512.38 ± 0.331.13 ± 0.315.05 ± 0.302.60 ± 0.152.92 ± 0.546.13 ± 0.3211.17 ± 0.921.22 ± 0.04 14.00 ± 0.889.15 ± 1.106.50 ± 0.581.70 ± 0.2311.40 ± 1.157.93 ± 0.526.78 ± 0.6213.22 ± 0.1921.17 ± 0.964.75 ± 0.20

Example 3

Cell adhesion component search

Serum contains many substances that can attach and spread to cells. However, since serum is not added in the serum-free medium, such a substance should be added depending on the cells. Therefore, when proliferating adherent cells, it is possible to directly or indirectly induce cell adhesion, diffusion, morphology, differentiation and cell motility by applying a container with adhesion impurity or by adding an attachment substance to the cell culture.

Among the various commercially available adhesion factors, four types of collagen (Sigma), fibronectin (Sigma), gelatin (Sigma) and collagen (CLR), which are mainly used, are applied to the cell culture container or applied to the culture medium. The adhesion of the cells was examined by the addition.

Collagen Type I (C1809, Sigma) prepared a collagen solution of 0.01% (w / v) in 0.1M acetic acid, stirred for about 4 hours until it melted at room temperature, and then chloroform was about 10% of this solution. Was added, and allowed to stand and react overnight at 4 ° C. The solution was separated and dispensed into a culture vessel at 10 mg / cm ² , incubated overnight at 4 ° C., and sterilized immediately by ultraviolet rays to be used for experiments. Collagen (CLR) was diluted to 10 mg / cm ² , dispensed into culture vessels, and used for experiments under ultraviolet light. Gelatin solution (2%) was dissolved at 37 ℃, pressurized, sterilized and applied to the culture vessel at 0.1 to 0.2 mg / cm ² completely dried and used in the experiment, fibronectin was added directly to the medium to induce cell culture. .

As an addition method of the culture medium, fibronectin (Sigma) was added to 0.5 μg, 1 μg, 1.5 μg, 2 μg, 2.5 μg, 5 μg per ml to test cell attachment (Table 5). FCS (0.5%) adaptive cell lines were incubated overnight in RDF / FCS (0.5%) 2ml medium in 12 well plates with 1 × 10 ⁴ cells / well initial cell inoculation number and washed twice with PBS and fibronectin 0.5 μg / ml 1 Cells were measured after incubating with 3 ml of RDF / SF containing 3 μg, 1.5 μg, 2 μg, 2.5 μg, and 5 μg for 10 days in triplicate with each concentration. As a result, fibronectin continued to help cell growth except that fibronectin was not added with RDF / SF as a control medium and 0.5 μg / mg was added (1.0 μg / ml to 5.0 μg / ml).

Fibronectin Requirement on Cell Adhesion Fibronectin Concentration (㎍ / ml) Cell No. (x10 ⁴ ) RDF / SF only RDF / SF + 0.5RDF / SF + 1.0RDF / SF + 1.5RDF / SF + 2.0RDF / SF + 2.5RDF / SF + 5.0 2.67 ± 0.341.50 ± 0.493.47 ± 0.254.13 ± 0.294.23 ± 0.216.80 ± 0.627.10 ± 0.35

For gelatin (Sigma), 0.2% solution was coated on a 12 well plate at 0.5 ml, 1 ml, 2 ml, 3 ml per well, then FCS (1%) adaptive cell lines were dispensed at 5 × 10 ⁴ cells / well, and RDF / SF RDF / FCS (1%) medium was added and incubated with triplicate for 7 days, and the cell number was measured (Table 6). As a result, when comparing RDF / SF and RDF / FCS (1%), gelatin did not contribute to cell adhesion in RDF / SF medium, and cells were suspended even in RDF / FCS, which inhibited the FCS addition. Seemed.

Gelatin Coating Requirement on Cell Adhesion Gelatin (0.2%) / well RDF SF (cell No. x 10 ³ ) RDF FCS (1%) (cell No. x 10 ³ ) 0.5ml1ml2ml3ml 2.00 ± 0.404.84 ± 0.667.50 ± 0.366.00 ± 0.94 2.84 ± 0.7820.00 ± 2.4824.66 ± 1.3626.84 ± 1.40

In the case of collagen, a 12-well plate, which is a coating container, was coated with Sigma collagen solution 1, 3, 10 mg / cm ² , CLR collagen 10 mg / cm ² , and a container which was not applied as a control was used. FCS (1%)-adapted cells were dispensed at 5 × 10 ⁴ cells / well and cultured for 7 days with triplicate by addition of RDF / SF and RDF / FCS (1%) medium, and cell number was measured (Table 7). As a result, in the case of RDF / SF medium, it was similar to that of 1 µg / ml in non-coating, and when applied at a higher concentration, the adhesion of the cells was maintained and the number of cells gradually increased. It appeared to affect cell attachment and growth more than CLR products. In the case of RDF / FCS, due to the serum added culture medium, there was no difference in the role of collagen between Sigma and CLR products, with and without collagen applied.

Collagen Requirement on Cell Adhesion Collagen (µg / ml) RDF SF (cell No. x 10 ⁴ ) RDF FCS (1%) (cell No. x 10 ⁵ ) Non-Coating Coated Sigma (1.0) Sigma (3.0) Sigma (10.0) CLR (10.0) 1.07 ± 0.271.02 ± 0.201.58 ± 0.301.73 ± 0.351.33 ± 0.37 2.96 ± 0.572.97 ± 0.523.09 ± 2.122.58 ± 1.372.97 ± 0.52

Example 4

Collagen was added to 0.1 M acetic acid to obtain a 0.01% (w / w) collagen solution. Then it was stirred for 1 to 3 hours at room temperature until dissolved. The collagen solution was transferred to a glass bottle with screw caps to form a chloroform layer carefully from the bottom. The well plate was coated with 10 μg of collagen per well and dried overnight at 24 ° C. using UV light in a sterile tissue culture hood. Gelatin solution (2%, w / v) was prepared and sterilized by autoclaving at 121 ° C., 15 psi for 30 minutes. The wells were coated with 2 ml 2% gelatin solution per well and dried for at least 2 hours. Dispense 5 x 10 ⁴ cells into uncoated wells, the collagen coated wells and gelatin coated wells, add RDF / SF and RDF / FCS medium, respectively, and incubate for 3 days to incubate for 3 days. The result was obtained. For non-coated RDF / SF, only a few cells were attached as expected and agglomerated for non-coated RDF / FCS. When coated with gelatin (1c and 1d), 1c (RDF / SF) was flocculated suspended and 1d (RDF / FCS) had some cells attached in a wide distribution. When coated with collagen (1e and 1f), 1e (RDF / SF) adhered and proliferated slightly cells, and 1f (RDF / FCS) agglomerated.

Example 5

Hormone and Growth Factor Search

Serum-free medium contains cells that can only grow if hormones or growth factors present in the serum are added. Specific hormones or growth factors specific to human insulin species cells were used for screening according to the recommended amount of 28 species purchased from Sigma.

Cells were grown at 5 × 10 ⁴ cells / well and grown, then hormones and growth factors were added at recommended concentrations and cell proliferation was observed 3 days later.

Using cell lines adapted to FCS (1%), cell growth and inhibitory factors were searched using RDF as a basal medium for 28 Sigma cell media products, hormones and growth factors (Table 8). Hormones and growth factors were searched for growth stimulation in a series of HB-cell tripletate plates described in the material & method. As a result, 24 of 28 species helped cell growth, and 4 species were found to inhibit the cells. Among the hormones, ITS-complexes (Insulin-, linoleic acid-BSA, glucagon, tyrocalcytonin acted significantly on cell growth, and aldosterone, thyroxine, estradiol and sodium selenite acted as inhibitors. Factors were highest and overall growth factor requirements, whereas insulin did not function significantly unlike other cells.

Growth Responsiveness of Human Insulin Species to Hormones and Growth Factors Added amount (concentration, / ml) Relative cell number / plate Fetal bovine serum [1% (v / v)] ITS-complex (5 μg) linoleic acid-BSA (1 mg) glucagon (10 μg) tyrocalcitonin (50 ng) somatostatin (50 ng) thrombin (100 ng) dexamethasone (500 ng) hydro Cortisone (5 μg) Transferrin (Human) (100 μg) Epidermal Growth Factor (10 ng) Insulin (20 μg) Tyrotropin Hormone (10 ng) Platelet-Induced Growth Factor (10 ng) 1.006.15 ± 0.772.98 ± 0.092.98 ± 0.022.34 ± 0.822.03 ± 0.301.88 ± 0.071.87 ± 0.071.79 ± 0.401.67 ± 0.451.65 ± 1.201.57 ± 0.171.53 ± 0.611. 46 ± 0.361.43 ± 0.88

Growth Responsiveness of Human Insulin Species to Hormones and Growth Factors Added amount (concentration, / ml) Relative cell number / plate Prostaglandin F2 (500 mg) Prostaglandin D2 (500 ng) Nerve Growth Factor (10 ng) Prostaglandin E1 (500 ng) Fibroblast Growth Factor (100 ng) Progesterone (20 ng) Neurotensin (200 ng) Prostaglandin E2 (500 ng) Triiodotyroin (250 ng) Putrescine (16.1 μg) leuthelation hormone (40 ng) aldosterone (10 μg) thyroxine (50 ng) estradiol (10 ng) sodium selenite (2 μg) 1.38 ± 0.441.32 ± 0.711.31 ± 0.121.27 ± 1.001.22 ± 0.401.22 ± 0.131.21 ± 0.181.10 ± 0.101.10 ± 0.061.04 ± 0.771.03 ± 0.610.93 ± 0.300.83 ± 0.300.80 ± 0.130.74 ± 0.24

Example 6

Determination of optimal concentrations of hormones and growth factors

Of the 28 species used for hormone and growth factor screening, 7 species of ITS complex, linoleic acid, glucagon, tyrocalcytonin, somatostatin, thrombin, and dexamethasone, which are more effective than other components in cell growth, were incubated at the recommended concentration up and down four levels. The growth of the cells was observed by addition to the medium (Table 9).

Concentrations of hormones and growth factors Hormone / growth factor Concentration (/ ml) ITS mixed linoleic acid-BSA glucagon tyrocalcytonin somatostatin thrombin dexamethasone 0.5μg0.1mg1μg5μg5ng10ng50ng 2.5μg0.5mg5μg25μg25ng50ng250ng 5μg1mg10μg50μg50ng100ng500ng 10μg2mg20μg100μg100ng200ng1mg

Seven types of ITS complex, linoleic acid-BSA, glucagon, tyrocalcytonin, somatostatin, thrombin, and dexamethasone, which affect cell growth than other components in hormone and growth factor screening, are shown in RDF culture medium as shown in Table 10 below. Was added. HB cells cultured in RDF medium for 3 days were screened with triplicates and hormones and growth factors at appropriate concentrations for cell growth. The result is as follows.

Optimal Concentration Screening for Cell Growth of Hormones and Growth Factors Hormones & Growth Factors Concentration (/ ml) Cell number (x10 ⁴ ) Insulin-Transferrin-Selenium Complex 0.5 µg 2.5 µg 5 µg 10 µg 2.54 ± 0.303.73 ± 0.753.11 ± 0.423.13 ± 0.21 Linoleic Acid-BSA 0.1mg0.5mg1mg2mg 2.84 ± 0.823.29 ± 0.443.03 ± 0.133.13 ± 0.23 Glucagon 1 µg 5 µg 10 µg 20 µg 2.23 ± 0.252.52 ± 0.162.51 ± 0.343.03 ± 0.24 Tyrocalcitonin 5 µg 25 µg 50 µg 100 µg 2.86 ± 0.314.48 ± 0.463.60 ± 0.893.44 ± 0.29 Somatostatin 5ng25ng50ng100ng 3.10 ± 0.093.08 ± 0.253.08 ± 0.362.61 ± 0.12 Thrombin 10ng50ng100ng200ng 2.18 ± 0.112.56 ± 0.342.84 ± 0.293.32 ± 0.31 Dexamethasone 50ng250ng500ng1mg 2.42 ± 0.192.56 ± 0.132.52 ± 0.182.37 ± 0.34

Table 10 shows the results of determining the concentration of the hormones and growth factors added to the medium of the present invention through the above experiment.

Application concentrations of hormones and growth factors on human pancreatic beta cells Added amount (concentration, / ml) Range (concentration, / ml) Fetal serum ITS-complex linoleic acid-BSA glucagontyrocalcytonin somatostatin thrombin dexamethasone hydrocortisonetransferrin (human) epidermal growth factor insulin tyrotropin hormone platelet-induced growth factor --5-500μg1-100mg0.3-500μg1-500ng0.3-500ng0.1-100μg0.5-50μg0.4-100μg0.1-100μg0.05-10μg0.1-10mg0.03-10μg0.05-10μg

Applied Concentrations of Hormones and Growth Factors on Human Insulinoma Cells Added amount (concentration, / ml) Range (concentration, / ml) Prostaglandin F2 Prostaglandin D2 Nerve Growth Factor Prostaglandin E1 Fibroblast Growth Factor Progesterone Neurotensin Prostaglandin E2 Triiodotyroin Putrescine Ryutening Hormone Aldosterone Thyroxine Estradiol Sodium Selenate 0.25-10μg0.25-10μg0.05-50μg0.25-10μg0.5-50μg0.01-10μg0.1-200μg0.25-10μg0.1-10μg0.1-10mM0.02-50μg ---- -: Shows inhibitory activity.

Example 7

Combination of Hormones and Growth Factors

5 × 10 ⁵ cells per flask were placed in a 25 cm ² culture flask (Falcon) and cultured in medium containing 1% FCS. After incubation overnight, the 1% serum culture medium was replaced with a mixed medium in which all seven ingredients were added to the RDF basal medium or six ingredients except one. Cells were cultured in a mixed medium for 5 days, respectively, and counted using a hemocytometer.

Example 8

Combination of Hormones and Growth Factors

1 × 10 ⁵ cells per flask were placed in a 25 cm ² culture flask (Falcon) and cultured in medium containing 1% FCS. After overnight incubation, the 1% serum culture medium was replaced with a mixed medium in which seven components were added to the RDF basal medium, either by addition or by one factor, subtracted or totally diluted (respectively single, 2, 3, 4, 5). , Using mixed media combining 6, 7 factors). The seven components are ITS-complex (2.5 μg / ml), linoleic acid-BSA (0.5 mg / ml), glucagon (20 μg / ml), tyrocalcytonin (25 μg / ml), somatostatin (5 ng / ml), thrombin (200 ng / ml), dexamethasone (250 ng / ml). Cells were cultured in mixed media for 5 days each and counted using a hemocytometer. 3 shows a combination showing good growth of the insulin-secreting cell line for each condition.

According to the present invention, a serum-free medium can be obtained that is precisely supplied with a component required by the cell, which shows growth close to the growth of human insulin cells in the serum-added medium without adding serum, and which can be produced at low cost.

Claims (4)

A medium for cell culture in which at least one of ITS-complex, linoleic acid-BSA, glucagon, thrombin, tyrocalcitonin, and dexamethasone is included in an RDF (RPMI1640: DMEM: Ham's F-12) mixed basal medium. The method according to claim 1, wherein the ITS-complex, linoleic acid-BSA, glucagon, tyrocalcytonin, somatostatin, thrombin and dexamethasone are 5-500 μg, 1-100 mg, 0.3-500 μg, 1-500 ng, 0.3-500 ng per ml of medium, respectively. Cell culture medium, characterized in that it is included in the range of 0.1-100μg, and 0.5-50μg. 3. Hydrocortisone, transferrin, epidermal growth factor, insulin, tyrotropin hormone, platelet-induced growth factor, prostaglandin F2, prostaglandin D2, nerve growth factor, prostaglandin E1, fibroblast growth according to claim 1 or 2 Factor, progesterone, neurotensin, prostaglandin E2, triiodotyroin, putrescine, luteinizing hormone further comprises a cell culture medium. 4. The method of claim 3 wherein the hydrocortisone, transferrin (human), epidermal growth factor, insulin, tyrotropin hormone, platelet-induced growth factor, prostaglandin F2, prostaglandin D2, nerve growth factor, prostaglandin E1, fibroblast growth Factors, progesterone, neurotensin, prostaglandin E2, triiodotyroin, putrescine and luteinizing hormone are 0.4-100 μg, 0.1-100 μg, 0.05-10 μg, 0.1-10 mg, 0.03-10 μg, 0.05 Included in the range of -10 μg, 0.25-10 μg, 0.25-10 μg, 0.05-50 μg, 0.25-10 μg, 0.5-50 μg, 0.01-10 μg, 0.1-200 μg, 0.25-10 μg, 0.1-10 μg, 0.1-10 mM and 0.02-50 μg Cell culture medium, characterized in that.