JPS6313675B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for washing cells that have adhered and grown on the surface of a microcarrier. Recently, as the importance of mass culturing tether-dependent animal cells such as diploid cells has increased in connection with the production of certain vaccines and biological drugs such as interferon, the cultivation methods have also improved. We have made great progress. One example of such a culture method is a method in which cells are allowed to adhere to the surface of microcarriers, or microparticles of a suitable material, suspended in a medium, and are allowed to grow there, ie, a method generally referred to as a microcarrier culture method. Generally, in the process of culturing cells using such a method, and furthermore in the process of making the cultured cells produce interferon, for example, old media are used as in cases (a) and (b) below. It is essential to remove the cells and replace them with a fresh medium or a salt aqueous solution (hereinafter simply referred to as a washing solution) consisting of a buffer solution, that is, to wash the cells. (a) In general, when tethering-dependent cell proliferation reaches a point where the cell attachment surface is almost completely covered, the proliferation rate decreases markedly, and proliferation eventually stops. Therefore, in order to continue cell culture for the purpose of obtaining a larger amount of cells, etc., a so-called digestion treatment with proteolytic enzymes is performed to break the bonds between the cells and the cell attachment surface as well as the bonds between the cells.
It is necessary to prepare a single cell suspension and reinoculate it into a system with a new cell attachment surface, that is, to carry out a cell passaging operation. Trypsin is most preferably used as the protease, but in order to use trypsin effectively in a small amount, it is necessary to use proteins that inactivate trypsin, as well as proteins that are involved in adhesion between cells and adhesion between cells. ing
It is important to allow trypsin to act while the concentration of Ca ⁺⁺ ions is sufficiently reduced. On the other hand, it is well known that a medium used for cell culture inevitably contains proteins derived from serum, proteins derived from cellular metabolic products, and Ca ⁺⁺ ions. Therefore, proteins present in the culture medium prior to digestion during cell passaging, Ca ⁺⁺
In order to sufficiently remove ions, etc., it is necessary to wash the cells with a washing solution that does not contain proteins and Ca ⁺⁺ ions. (b) As a method for producing, for example, interferon in cultured cells, a method called the superinduction method is known. In this method, cells are treated with an interferon-inducing agent such as a double-stranded nucleic acid, and then a metabolic inhibitor such as cycloheximide or actinomycin D is applied, and then the medium is replaced with an interferon-producing medium to induce interferon. is produced in the medium. At this time, if the amount of interferon inducer or metabolic inhibitor remaining in the interferon production medium is too large, interferon production will be significantly inhibited. Furthermore, if a protein is present in the interferon production medium, the specific activity of the resulting interferon, ie, the interferon activity per protein weight, will decrease, increasing the burden of the subsequent purification process. Therefore, when replacing the medium with an interferon-producing medium, it is necessary to wash the cells with a protein-free washing solution to sufficiently remove interferon inducers, metabolic inhibitors, and even proteins contained in the medium. It is essential. A specific method for carrying out the above-mentioned cell washing in the microcarrier culture method involves, for example, settling the microcarriers on which cells have grown and grown, sucking up the supernatant medium, and then adding a washing solution and resuspending the microcarriers. There is a method of washing cells repeatedly. When such cell washing is carried out, some of the cells are detached from the microcarrier surface due to turbulence of the liquid as the culture medium and washing liquid are withdrawn and supplied. In the microcarrier culture method, such detachment of cells is particularly common due to the following reasons (a), (b), and (c), and in severe cases, it can reach 40 to 50%, which is a serious problem. That's a problem. (a) Especially when using hydrous microcarriers, and even when using non-hydroscopic microcarriers in general, it is difficult to wash the cells efficiently compared to, for example, a culture method using a Lou bottle or a roller bottle; Therefore, in order to perform sufficient cleaning, it is necessary to use a relatively large amount of cleaning liquid. (b) The microcarrier is fluidized and agitated with the flow of the medium and washing solution as it is removed and supplied, making it easy for cells to detach. (c) The microcarrier culture method is a culture method particularly suitable for large-scale culture, but on a large scale, the flow and turbulence of the liquid due to the extraction and supply of the medium and washing liquid become relatively intense. Cells detached from the microcarrier surface and floating in the washing solution can be recovered by methods such as filtration or centrifugation, but in cell culture, all such operations must be performed aseptically. Coupled with this, it requires a great deal of extra effort. Furthermore, in the case of interferon production, for example, even if cells once detached from the microcarrier surface are recovered, they no longer contribute to interferon production. An object of the present invention is to provide a method for washing cells grown on a microcarrier surface that can effectively prevent cells from being detached from the microcarrier surface during washing. The above objects of the present invention are very effectively achieved by a cell washing method comprising washing cells grown on the surface of a microcarrier with an aqueous salt solution containing a non-protein water-soluble polymeric substance. Cells grown on the surface of the microcarrier, which is the substance to be washed and subjected to the method of the present invention, can be appropriately obtained according to known microcarrier culture methods. As microcarriers, polysaccharide microparticles with positively charged chemical residues (e.g., diethylaminoethylated crosslinked dextran microparticles, dimethylaminopropylated polyacrylamide microparticles, etc.) are most commonly used, but synthetic polymers They may be microparticles (for example, polystyrene microparticles), inorganic microparticles (for example, glass microparticles), or microparticles or microscopic bodies made of a material that has an affinity for cells. The most common method is to culture microcarriers while suspended in a medium. A method in which the cells are cultured in this state is also used. Next, an example of a method for washing cells grown on the surface of a microcarrier will be described. First, the microcarriers with cells attached and grown on their surface are sedimented and the supernatant medium is sucked off. At this time, care must be taken so that the microcarriers with attached cells do not flow out with the flow of the supernatant medium. For this purpose, it is effective to provide a material at the suction port of the line for sucking up the supernatant medium or in the middle of the line. A saline solution or wash solution consisting of a washing medium or buffer is then added to resuspend the microcarriers. Repeat this operation as many times as necessary to perform cleaning. The above washing method uses a sedimentation separation method to separate the microcarriers with attached cells from the culture medium or washing solution, but the separation may also be performed using a filtration method or a combination of both methods. You can also do it with Furthermore, the above method is a so-called batch method in which the washing solution is added after removing the medium or washing solution.
A continuous method may be used in which removal of the culture medium or washing solution and supply of the washing solution are performed in parallel. The essence of the method of the present invention is to contain a non-protein water-soluble polymer substance at an appropriate concentration in the washing solution. This non-protein water-soluble polymer substance allows
Cells are prevented from detaching from the microcarrier surface due to liquid flow and turbulence that occur when the culture medium and washing solution are withdrawn and supplied. Specific examples of non-protein water-soluble polymer substances used in the present invention include naturally occurring water-soluble polysaccharides such as dextran and alginic acid, methyl cellulose, carboxymethyl cellulose,
Soluble starch, derivatives of natural polysaccharides such as hydroxyethyl starch powder, and polyvinylpyrrolidone,
Synthetic polymer substances such as polyoxyethylene-polyoxypropylene copolymer and the like can be mentioned. The preferred concentration of the non-protein water-soluble polymeric substance in the cell washing solution varies depending on the type of polymeric substance used, the type of microcarrier, and even the type of cells, and can be appropriately determined through experiments. However, it is generally appropriate to range from 0.0001 to 1% by weight, more preferably from 0.001 to 0.1% by weight, based on the cleaning liquid. There is no particular restriction on the molecular weight range of these polymeric substances as long as they are water-soluble. As the washing solution, an aqueous salt solution adjusted to an appropriate osmotic pressure is used, but a buffer such as a phosphate buffer, a medium containing certain nutrients, etc. are particularly preferably used. Next, examples of the effects of the present invention will be specifically explained using Examples. Example 1 4.6 x ¹⁰⁴ human diploid cells/cells were grown in Eagle's MEM medium containing 0.25% diethylaminoethylated cross-linked dextran microcarriers and 5% calf serum.
ml was inoculated and cultured for 6 days under stirring conditions at 37°C.
Once in the middle, the culture medium was replaced with fresh Eagle's MEM medium containing 5% calf serum. The number of cells on day 6 is
It was 5.6×10 ⁵ cases/ml. 200ml each of the suspension,
The contents were dispensed into four glass containers whose inner surfaces were treated with silicone, and after the microcarriers were allowed to settle, 170 ml of the supernatant medium was sucked out from each container. After that, the two
170 ml of PBS (Ca ⁺⁺ minus) was added to the remaining two bottles, and 170 ml of PBS (Ca ⁺⁺ minus) containing 0.05% methyl cellulose was added to the remaining two bottles to resuspend, and the number of cells was counted after each sample was sampled and washed once. Furthermore, the above operation was repeated twice, and the number of cells was counted after washing twice and washing three times. The difference between the average number of cells from two tubes under the same conditions and the initial cell number of 5.6 x ¹⁰⁵ cells/ml, that is, the ratio of the number of detached cells to the initial cell number, is
The number of detached cells when washed three times with PBS (Ca ⁺⁺ minus) containing 0.05% was about 40% of that when methylcellulose was not included. To count the number of cells, sample a small amount, let the microcarrier settle, suck up the supernatant, add the same amount of citric acid 0.1M and crystal violet 0.1% aqueous solution as the sucked supernatant, and hold at 37°C. After incubation for several hours, the number of cell nuclei was counted using a hemocytometer. Example 2 9.0 x ¹⁰⁴ human diploid cells/cells in Eagle's MEM medium containing 0.25% diethylaminoethylated cross-linked dextran microcarriers and 5% calf serum.
ml was inoculated and cultured at 37°C for 7 days. The culture medium was changed twice in the middle to new Eagle MEM containing 5% calf serum.
The medium was replaced. The number of cells on the 7th day was 7.3×10 ⁵ cells/
It was hot in ml. 200 ml of the suspension was dispensed into 9 glass containers whose inner surfaces were treated with silicone, and each container contained a non-protein water-soluble polymer substance of the type and concentration shown in Table 1 in the same manner as in Example 1.
After washing three times with PBS (Ca ⁺⁺ minus), the number of cells was counted in the same manner as in Example 1. The results are shown in Table-1. Furthermore, for numbers 1 and 4, the microcarriers were sedimented and the supernatant PBS (Ca ⁺⁺ minus) was sucked up, and then trypsin was applied to detach the cells from the microcarriers.

[Table] After adding Eagle's MEM medium containing 5% calf serum to a total volume of 100 ml and stirring, the microcarriers were separated by sedimentation to obtain a cell suspension. Separately, prepare four spinner flasks containing 190 ml of Eagle MEM containing 0.25% microcarrier and 5% calf serum, and add 10 ml each of the cell suspension obtained from No. 1 to two spinner flasks (No. 4).
The resulting mixture was inoculated into the remaining two spinner flasks and cultured for 6 days at 37°C with stirring. Twice in the middle, the culture medium was replaced with a new medium containing 5% calf serum. The average number of cells in two spinner flasks inoculated from number 1 on the 4th day after inoculation was 2.7×10 ⁵ cells/
The average number of cells in the two spinner flasks inoculated from No. 4 was 3.3×10 ⁵ cells/ml. The respective values on the 6th day after inoculation were 6.7×10 ⁵ cells/ml and 8.4×10 ⁵ cells/ml.

[Brief explanation of the drawing]

The figure is a diagram showing the results of Example 1, where 1 is the result for the methyl cellulose coexistence system and 2 is the result for the non-protein water-soluble polymer substance-free system.

Claims (1)

[Claims] 1. A method for washing cells, characterized in that when cells grown on the surface of a microcarrier are washed with an aqueous salt solution, a non-protein water-soluble polymer substance is allowed to coexist in the aqueous solution.