KR880002318B1 - Serum free culture media - Google Patents

Abstract

Serum-free media for proliferating animal and hum B-lymphoma cells and myeloma cells, and their hybridormas were prepd. Thus, a medium was prepd. contg. Cu 8.0 X 10-2 g/ml, Zn 0.4 X 10 g/ml, Fe 0.3 X 10 g/ml, inculin 300 g/ml, transferrin 300 g/ml, bovine serum albumin 200mg/ml, lactalbumin bydrolysate 700 mg/ml, glycerol 500 g/ml, carboxymethylcellulose 200 mg/ml, and B-cell growth factor 0.1 v/v %.

Description

Serum-free medium

1 is a comparison of growth curves comparing the growth of namalba cells in a control group cultured in a medium of normal serum concentration and a test group in which serum concentration was reduced to 3% and cultured in a medium containing B-cell growth factor. .

2 shows that the concentration of sperm was reduced to 1%, and the growth of Namalba cells cultured in a medium containing B-cell growth factor and a medium without B-cell growth factor and growth of Namalba cells cultured in a medium of normal serum concentration. There is also a comparison of the growth curves compared.

FIG. 3 is a comparison of the growth and proliferation of Namalba cells with the addition of each of the nine inorganic ions.

4 is a comparison of growth and proliferation of Namalba cells with the addition of hormones, growth factors and other components, respectively, compared to the control.

The present invention relates to serum-free media that can be used to culture lymphoma cells and myeloma cells and hybridoma cells derived therefrom.

The serum-free medium of the present invention was prepared by completely removing serum components from the conventional media components that have been used for the cultivation of these cells, and replacing them with inorganic ions, hormones, and other components. At the same time, it is possible to effectively produce the desired material. Serum most commonly used as an indispensable medium component for culturing mammalian cells is highly heterogeneous in composition depending on the species, age, and gender of its source (Proceedings of the Society for Experimental Biology and Mecidine 149, 344, 1975). ), Which contains components whose composition and function are not fully understood, and thus cannot control collectively the growth and maturation of specific cells to be cultured.

Therefore, research into specific media components effective for growth and maturation of specific cell populations is continuously conducted, and thus, the main role of serum in tissue culture is to supply hormones and growth factors required for the growth of the cells. According to (Control of proliferation in animal cells, p97, 1974 / proc Natl. Acad. Sci. USA 75, 901, 1978), cell culture in serum-free medium with serum and hormone replacement was successful.

(Methods in Enzymol. 45, 94, 1979 / Anal. Biochem. 114,422, 1981 & Nature, 259, 132, 1976 / proc.Natl.Acad.Sci. USA 75,901,1978 / Cells, tissue and organ cultures in neurobiology p619, 1977 / In vitro 14,24,1978). In recent years, there has been a growing interest in the industrial mass production of cell bacteria related to the production of monoclonal antibodies, and in part, it has been reported that hormones are used instead of serum for certain kinds of cells.

(Antimicrob. Agents Chemotherapy 18 (6), 930, 1980 / Proc. Natl. Acad. Sci. U.S.A. 79, 1158, 1982). However, conventional serum-free media used for lymphoma cells, myeloma cells, and hybridoma cells are known to require NS-1 (P3 / NS1 / 1-Ag4-1) cells and hybridoma cells derived therefrom that have high nutritional requirements. The range of cell types that can be applied is limited, and the cells grown in the serum-free medium are very susceptible to damage by physical contact, so there is a significant loss of cell viability in the cells grown at high density. . Therefore, in the case of mass production in which cells must be continuously circulated in order to maintain breathability and secure a certain space between cells, the yield of the desired substance is lowered.

In addition, Sp2 (Sp2 / 0-Ag 14) and its hybridoma cells had lower cell growth rate when grown in these serum-free medium than those in serum-added medium, and had a cloning efficiency of 1-3%. Significantly lower than 60-90% in this added medium. This slowing down of growth rate in hybridoma cells may act as a factor that causes various modifications in the production of monoclonal antibodies.

Therefore, in the present invention, by removing the serum components from the conventional media constituency to remove the uncertain influence (growth growth or growth inhibition) on the target cells, by preparing a synthetic medium consisting of the components of which the composition and function is clearly identified desired cells To allow for efficient growth and maturity.

Synthetic serum-free medium of the present invention is a transferrin, insulin, human serum albumin, bovine serum albumin (Anal. Biochem. 102, 255-270), which has been shown to have an effect on cell growth and maturation. , 1980 / The Journal of Experimental Medicine 147,923-933, 1978) and monoethanol amine (Proc. Natl. Acad. Sci. USA 79, 1158-1162, 1982), lactalbumin hydrolysate, glycerol, Carboxymethyl cellulose (CMC), B-cell growth factor and inorganic ions are used as common ingredients to secure a range that can be commonly applied, and various components are added according to the specificity of specific cells. By excluding or excluding them, each cell can grow and mature in an optimal state.

In addition, the synthetic serum-free medium of the present invention contains essentially no impurities derived from serum, thereby allowing easy and rapid separation of high-purity cell products.

Therefore, in the case of mass production, it is possible to reduce the cost of the separation and purification process, and to eliminate all problems that may be caused by contamination of serum proteins in the medical aspect.

The synthetic serum-free medium of the present invention can be applied to a wider variety of B-lymphoma cells, myeloma cells and hybridoma cells as compared to the conventional serum-free medium which has been used for culturing lymphoma cells, myeloma cells and hybridoma cells. Can be. In particular, the synthetic medium of the present invention is hybrid myocytes derived from mouse myeloma cells, NSO and Sp2, and their fusion partners, human myeloma RPMI 8226, a human myeloma cell, and human lymphoma cells. Remarkable growth and maturation is seen in Namalba cells and hybridoma cells derived from them, demonstrating that the synthetic serum-free medium of the present invention fully satisfies the different nutritional requirements of the various cells described above. will be.

The various cell groups described above showed similar cell growth rates when cultured in the medium of the present invention, and the amount of antibody produced from each hybridoma cell was higher than that when cultured in the serum medium. 120%), wherein the antibody produced had no modification of the selectivity of the original monoclonal antibody. In addition, the cloning efficiency was 10-12% higher than that in the conventional serum-free medium, and showed excellent resistance (cell survival rate of 96% or more) to the mechanical struts than the cells cultured in the serum medium. This was induced by adjusting the osmotic pressure, which was not considered in the conventional serum-free medium, to a suitable level (328-390 mosm / l), which is remarkable when industrially mass producing the desired cells using the synthetic serum-free medium of the present invention. It can bring about an improvement in productivity.

In the present invention, the primary cell line was used as a human lymphoma cell Namalba cells, serum-free medium application test for human myeloma RPMI 8226, NSO, Sp2, 548, 46, 20 (monoclonal antibody production for human interferon Cell lines) and the like.

In the present invention, after establishing the Namalba cells, it was found that the B-cell growth factor affects the growth of these cells, and the appropriate concentration was determined. This resulted in long-term culturing of namalba cells in medium containing serum concentration and containing B-cell saint factor.

Under these conditions, each of the inorganic salts, hormones, and other components was added to the medium, and then the growth of the cells was compared with the control group (Namalba cells grown in the medium of normal serum concentration) to determine the final component and its appropriate concentration. Serum-free medium was prepared by adding each component at an appropriate concentration to RPMI 1640 medium containing no serum. Long-term culturing of Namalba cells in this serum-free medium resulted in no effect on cell division time, morphology and interferon production in cultures longer than 6 months. Application tests also confirmed that this medium can be used for the cells described above.

Example 1

Circulation test of Namalba cells by B-cell saint factor (BCGF)

Namalba cells grow normally in RPMI 1640 medium containing 7% NBCS (New Born Calf Serum). The time of division of namalba cells exhibiting this normal growth is 24 hours under optimal conditions, and the cell concentration at this time is It will reach 2-3 × 10 ⁶ cells / ml.

In this example, the Namalba cells were normally cultured until the extra logarithmic growth phase, and the cultures were centrifuged at 1500 × g for 10 minutes at room temperature to harvest the cells. Then, 20 ml of RPMI 1640 medium containing 3% NBCS was added to the culture vessel so that the cell concentration was about 2.0 × 10 ⁴ cells / ml. B-cell growth factor (BCGF) was added to adjust to a final concentration of 0.2% (v / v) and compared to an incubator at 37 ° C supplied with 5% carbon dioxide.

Cells grown in RPMI1640 medium containing 7% NBCS were used as a control, samples were taken at regular intervals from the incubator and stained with trypan blue, and then the number of living cells was measured using hemocytometa.

At this time, 20 cells were passaged and purified to the same number in the test and control groups. As a result, the cells were almost similar to the medium containing 7% NBCS even in the medium containing 3% NBCS. Could grow. (Figure 1)

Therefore, the cells can be grown normally only by adding B-cell growth factor to the medium whose serum concentration is lowered from 7% to 3%, which is an essential component for the growth of B-cell lineage.

The serum concentration was reduced using only the B-cell growth factor by the above-described method, and almost normal growth was observed even at a serum concentration of at least 1% (FIG. 2).

In other words, almost no cells were grown in the medium with the serum concentration lowered to 1%, but in the medium in which the B-cell growth factor was added to the medium with the serum concentration lowered to 1%, the final concentration was 0.2v / v%. It grew normally.

Therefore, by using B-cell growth factor, Namalba cells can be grown normally at a serum concentration of up to 1%.

Example 2

Determination of the composition and optimal concentration of inorganic ions and hormones that affect the growth of Namalba cells.

In order to culture Namalba cells using serum-free medium, it is necessary to determine the composition and concentration of inorganic ions, hormones and other growth factors essential for this.

Therefore, it was found out of the 23 components listed in Table 1 only to affect the growth of the Namalba cells was used to prepare a serum-free medium.

TABLE 1

First, the effects of inorganic ions on the growth of Namalba cells were examined for each of the nine inorganic ions.

Namalba cells were grown to normal logarithmic diet in RPMI 1640 medium containing 3% NBCS, and then cells were harvested by centrifugation at 1500 × g for 10 minutes at room temperature. The cell concentration was adjusted to 2 × 10 ⁵ cells / ml, and 7 ml each of the T-25 culture vessel was added to the B-cell growth factor at 0.1v / v%.

Each of the nine inorganic ions was added to the culture vessel at a proper concentration shown in Table 1, and then cultured in a 37 ° C. incubator fed with 5% carbon dioxide. After 3 and 5 days, a certain amount of culture was taken from the incubator to measure the number of living cells. As a result, it was found that Cu, Zu and Fe influenced the growth of Namalba cells (Figure 3).

The thirteen components of Table 1, which consist of hormones, growth factors and other compounds, were also examined in the above-described manner, indicating that transferrin, insulin, bovine serum albumin, lactalbumin hydrolysate, glycerol and carboxymethyl cellulose were present in Namalba cells. It can be seen that it affects the growth (figure 4).

The minimum requirement for the nine components that affect the growth of the Namalba cells (the amount that does not cause a drastic change in the growth of the Namalba cells) was determined by the method described above, and is contained in Table 2.

TABLE 2

Example 3

Preparation of Synthetic Serum-Free Medium for Normal Culture of Namalba Cells.

Using the results obtained in Example 1 and Example 2 described above, a serum-free medium capable of normally growing namalba cells was prepared.

Serum was completely removed from the RPMI 1640 medium components, followed by Cu (0.8 ng / ml), Zn (40 ng / ml), Fe (30 ng / ml), insulin (3 µg / ml), transferrin (3 µg / ml), Bovine serum albumin (2 mg / ml), lactalbumin hydrolysate (7 mg / ml), glycerol (5 µg / ml) and carboxy methyl cellulose (2 µg / ml) were added (Table 2) and B-cell growth Factor was added at a concentration of 0.1v / v%. Cells were harvested by centrifuging purified Namalba cells in RPMI 1640 medium containing 3% NBCS for 10 minutes at 1500 × g, and then using the RPMI 1640 medium (Table 2 instead of serum and B-cell growth factor). Medium) to be added at a concentration of 2-3 × 10 ⁵ cells / ml. When the total volume was 500 ml, it was placed in a spinner flask and incubated in a 37 ° C. incubator supplied with 5% carbon dioxide. Cultures were taken at regular time intervals and the number of viable cells was measured by trypan blue dye exclusion using a hemocytometa. As a result, the dividing time for more than 20 passages was 18-20 hours. The number of living cells was also 96%. In addition, the growth and proliferation of the cells did not change even after prolonged cultivation. The growth and proliferation were similar to those cultured in a medium with a serum concentration of 7%.

Example 4

Application tests on synthetic serum-free medium of animal and human B-cells and comparison of cell products.

In this example, the synthetic serum-free medium used for culturing Namalba cells was used to express cells of animals and human B-cell lines similar to those of Namalba cells (NSO, Sp2, 548, 46, 20, human Myeloma RPMI 8226). It was also tested for application to.

As a result of testing in the same manner as in Example 3, all cell lines used were confirmed to grow and proliferate almost normally as Namalba cells. The titer and production of the substances produced and secreted by cells grown in serum-free medium were measured by biological assays and immunological radiometric methods.

-인터페론 생성량을 측정하기 위하여, 다음과 같은 방법으로

-인터페론을 유발시켰다. 5%이산화탄소가 공급되는 37℃의 배양기에서 세포 농도 2×10⁵cells/㎖이 되도록 나말바 세포를 배양한 후, 나트륨-부티레이트를 최종 농도 1mM이 되도록 첨가하고 24시간 동안 배양하였다. 그런다음, 비루스(Hemaglutin virus of Japan, HVJ)를 50HAU/㎖의 농도로 가하고 계속하여 2시간 배양한 다음, 배양액을 실온에서 1500×g로 10분간 원심 분리하여 세포들을 수확하였다.Of Namalba cells cultured in serum-free medium

In order to measure the amount of interferon production,

Triggered interferon. After incubating the Namalba cells at a cell concentration of 2 × 10 ⁵ cells / ml in a 37 ° C. incubator with 5% carbon dioxide, sodium-butyrate was added to a final concentration of 1 mM and incubated for 24 hours. Then, virus (Hemaglutin virus of Japan, HVJ) was added at a concentration of 50 HAU / mL and incubated for 2 hours, and the cultures were harvested by centrifugation at 1500 x g for 10 minutes at room temperature.

-인터페론의 용액을 얻었다. 이 용액을 생물학적 분석법 및 면역학적 방사선 분석법으로 분석하여

-인터페론의 역가를 측정하였다.Fresh serum-free medium was added thereto, followed by incubation for 5 hours, and the incubator temperature was lowered to 28 ° C., followed by incubation for 17 hours. Centrifugation of the culture in the above manner to separate the supernatant,

A solution of interferon was obtained. This solution is analyzed by biological analysis and immunological radiation analysis

The titer of interferon was measured.

-인터페론의 여가 측정은 다음과 같이 행 하였다. 96구멍 플레이트에 조

-인터페론 용액과 표준

-인터페론 용액(Standard

-IFN solution)을 50㎕씩 넣고 10분간 자외선을 조사한 다음, 2배 계열 회석하고 4×10⁵cells/44ℓ 농도의 WISH세포를 100㎕씩 각각 가한 다음, 5%이산화탄소가 공급되는 배양기에서 22-24시간동안 배양하였다. 생리식염수를 함유한 인산완충 용액(PBS)10044ℓ를 가하여 1회 세척하여 96구멍 플레이트내의 내용물을 완전히 제거하고, 100-1000 TCID₅₀의 바시쿨라 스토마티티스 비루스(Vacicular Stomatitis virus, VSV)를 세포 대조군을 제외한 모든 구멍에 100㎕씩 가하고 다시 20-24시간 배양하여 비루스를 공격하게 하였다. 그런다음, 내용물을 제거하고 0.02%뉴트랄 레드 100㎕씩을 가하여 세포를 염색하였다. 30분 경과후레 상기한 방법으로 내용물을 제거하고, 10분간 자외선을 조사하여 비루스를 불활성화 하였다. 그런다음 세포가 함유하고 있는 뉴트랄 레드를 추출하여 545nm에서의 흡광도를 측정하여 표준

-인터페론 용액과 비교함으로서 조

-인터페론 용액의 역가를 측정하였다.By biological analysis

The leisure measurement of interferon was performed as follows. 96 hole plate

Interferon solution and standard

Interferon Solution (Standard

50 μl of IFN solution was added and irradiated with UV light for 10 minutes, followed by 2-fold dilution, 100 μl of 4 × 10 ⁵ cells / 44 L of WISH cells, respectively, and then in a incubator supplied with 5% carbon dioxide. Incubated for 24 hours. 10044 l of phosphate buffer solution (PBS) containing saline was added and washed once to completely remove the contents in the 96-hole plate, and 100-1000 TCID ₅₀ of Vacicular Stomatitis virus (VSV) was used as a cell control. 100 μl was added to all pores except for incubation for another 20-24 hours to attack the virus. Then, the contents were removed and cells were stained by adding 100 µl of 0.02% neutral red. After 30 minutes, the contents were removed by the method described above, and the virus was inactivated by irradiating with ultraviolet rays for 10 minutes. Then, extract the neutral red contained in the cell and measure the absorbance at 545 nm.

By comparing with an interferon solution

The titer of the interferon solution was measured.

-인터페론의 역가를 민역학적 방사선 분석법으로도 측정하였다(표 2).Generated

The titer of interferon was also measured by kinetic radioanalysis (Table 2).

-인터페론의 역가를 생물학적 분석법 및 면역학적 분석법으로 측정한 결과, 시험군과 대조군에서 거의 비슷한 활성(5×10³IU/㎖)이 나타났다.Produced and secreted from Namalba cells

The titer of interferon was measured by biological assays and immunological assays and showed almost similar activity (5 × 10 ³ IU / ml) in the test and control groups.

-인터페론의 역가 비교Produced and separated from Namalba cells by virus-induced

Comparison of titers of interferon

-인터페론의 생성에 아무런 영향도 미치지 않음을 알 수 있었다. 548, 46, 20세포주에 의하여 생성된

-인터페론에 대한 단일클론항체와 사람 마이에로마 RPMI 8226에 의하여 생성된 람다사술(IgG lambda-type light chain)을 면역학적 방사선 분석법을 사용하여 정량하였다.Thus, culturing in serum-free medium is associated with growth of Namalba cells.

It was found that no effect on the production of interferon. Generated by 548, 46, 20 cell lines

Monoclonal antibodies against interferon and lambda-type light chains generated by human Myeroma RPMI 8226 were quantified using immunological radioanalysis.

TABLE 2

-인터페론에 대한 단일 콜론 항체의 생산량은 대조군과 비교하여 시험군에서 약20%증가하였다. 사람 마이엘로마 RPMI 8226은 시험군에서도 대조군에서와 거의 유사한 성장과 증식을 하나, 람다 사슬의 생성량은 약간 적었다.By 548.46.20 cell line

The production of single colon antibody against interferon increased about 20% in the test group compared to the control. Human Myeloma RPMI 8226 showed similar growth and proliferation in the test group, but slightly less lambda chains.

Comparison of the amount of antibody isolated from the 548.46.20 cell line and human Myeloma RPMI 8226

Myeloma-derived NSOs and Sp2 cells derived from mice that do not produce and secrete antibodies are widely used as fusion spouses. These cells also grew and expanded almost normally in serum-free medium of the present invention. In addition, no significant delay in rupture time and changes in morphology were observed, and cloning efficiency was 10-12% higher than that in conventional serum-free medium.

Claims (5)

In the medium used for culturing animal and human B-lymphoma cells and myeloma cells and hybridomas derived therefrom, each component that affects growth and proliferation instead of the serum components of the conventional medium, namely CU Serum-free medium, comprising Zn, Fe, insulin, transferrin, bovine serum albumin lactalbumin hydrolysate, glycerol, carboxymethyl cellulose and B-cell growth factor at appropriate concentrations. The serum-free medium of claim 1, wherein the B-lymphoma cells, myeloma cells, and hybridoma cells are cell lines derived from humans. The serum-free medium of claim 1 comprising B-cell growth factor (BCGF), glycerol, carboxymethyl cellulose and lactalbumin hydrolysate in place of the serum component. The method according to claim 1, wherein Cu 8.0 × 10 ⁻² μg / ml, Zn 0.4 × 10 ¹ μg / ml, Fe 0.3 × 10 ¹ μg / ml, insulin 300 μg / ml transferrin 300 μg / ml, bovine serum albumin 200 mg / Ml, lactalbumin hydrolysate 700mg / ml, glycerol 500g / ml, carboxymethylcellulose 200mg / ml and B-cell growth factor at 0.1v / v% characterized in that the serum-free medium. A method for culturing a cell line of a B-cell lineage, comprising culturing a cell line of a B-cell lineage by adding 1-3% of serum to a serum-free medium of claim 3.

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