JPH0123119B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing interferon, and more particularly to a method for producing interferon by treating animal cells grown on microcarriers containing positively charged chemical residues with an interferon-inducing agent. It is something. Interferon is produced by various viruses and double-stranded
A protein produced by animal cells when stimulated by an inducing agent such as RNA, and has the effect of suppressing the intracellular proliferation of viruses. The effects of interferon are non-specific with respect to virus species, but specific with respect to animal species. That is, interferon produced in cells of one animal species does not act on other animal species. In recent years, the therapeutic effects of interferon on certain viral diseases and even tumors have been recognized, and its potential as a medical drug is attracting attention. In order to apply interferon as a medicine, it is necessary to produce interferon in vitro using some human cells and to separate and purify it. Human
Until now, leukocytes separated from blood have often been used as cells for producing interferon, but diploid cells derived from fetuses or newborns are gradually replacing this. The supply of white blood cells must depend on an unspecified number of people, and there is a high risk that various lymphokines may be mixed in interferon preparations produced by white blood cells. Therefore, it is not always easy to monitor the safety of interferon preparations obtained from leukocytes. On the other hand, since diploid cells derived from a single individual can be cultured in large quantities, it is easier to confirm the safety of the obtained interferon preparation compared to cases derived from white blood cells. It is believed that Conventionally, methods for culturing tether-dependent cells such as diploid cells using Lou bottles or roller bottles are known, but it is quite difficult to culture large amounts of tether-dependent cells using this method. It is believed that That is, in this method, cells only grow in a single layer on the bottom of the roux bottle or the side of the roller bottle, so for mass culture, it is necessary to handle an extremely large number of roux bottles or roller bottles. This is because handling becomes extremely complicated. Recently, a culture method called microcarrier culture method suitable for mass culture of tether-dependent cells has been developed (Japanese Patent Application Laid-Open No. 53-62889). In this culture method, seed cells are inoculated into a medium in which microcarriers containing positively charged chemical residues (hereinafter simply referred to as microcarriers) are suspended and cultured in a suspended state. The cells attached to the microcarrier surface and proliferate there. The microcarrier culture method can be said to be a culture method particularly suitable for mass culture of tether-dependent cells because it is easy to increase the cell attachment surface area and it is also easy to handle. On the other hand, as a technique for producing interferon in cultured cells, cells are stimulated using an interferon inducer such as double-stranded RNA called the superinduction method, and then cells are treated with metabolic inhibitors such as cycloheximide and actinomycin D. The UV method is a method to further increase interferon production by treating cells with an interferon-inducing agent (US Patent No. 3,773,924). Methods such as methods for further enhancing eron production are known. The present inventors focused on the above-mentioned culture method and interferon production method as a method for producing a large amount of interferon derived from cultured cells such as diploid cells, and injected superinductor into cells cultured by the microcarrier culture method. Attempts have been made to produce interferon using the Shion method, but
In this method, only low units of interferon are often produced, and high units of interferon cannot be stably produced. An object of the present invention is to solve these problems and provide a method for producing interferon with remarkable effects that is suitable for mass production. The object of the present invention is to treat the microcarriers on which the cells have adhered and grown with an interferon-inducing agent when animal cells grown on microcarriers are treated with a glucocorticoid and a negatively charged water-soluble polymer. This is achieved by the production method. First, a method for culturing tether-dependent cells such as diploid cells using a microcarrier culture method, which is a premise of the method of the present invention, will be explained. The microcarriers are suspended, seed cells are inoculated into a medium containing serum at an appropriate concentration as required, and culture is carried out at an appropriate temperature with gentle stirring. As microcarriers, polysaccharides such as
An appropriate amount of amine residues are bonded to microparticles made of dextran, dextrin, starch, cellulose, their substituent derivatives, etc., or certain synthetic polymers, such as polyvinyl alcohol, hydroxy-substituted acrylates or methacrylates, polystyrene, etc. Those that have been made are used. The microcarrier concentration is determined by the microcarrier material,
1 ml of medium, depending on the type of cells to be cultured
The microcarrier surface area is approximately 10cm ² ~
A concentration range of 100 cm ² is preferred. The medium composition, serum concentration, etc. are appropriately selected depending on the type and type of cells to be cultured, cell concentration, etc. The inoculated cells attach to the surface of the microcarrier and begin to proliferate, and by continuing the culture at an appropriate temperature for approximately several to 10 days, the cells proliferate until they almost completely cover the surface of the microcarrier. During this time, supply oxygen, adjust pH, add nutrients, replace medium, etc., as necessary. Next, interferon production by cells cultured by the microcarrier culture method will be explained in detail. The cultured cells are incubated in a medium containing a low unit of interferon for about several hours to one day, if necessary, for the purpose of enhancing interferon production prior to interferon induction. Prior to interferon induction, the present inventors added a negatively charged water-soluble polymer and a glucocorticoid to a medium in which microcarriers to which cells had attached and proliferated were suspended, in order to enhance interferon production. It was found that a synergistic effect can be obtained. Examples of negatively charged water-soluble polymers include water-soluble polymers with carboxyl groups such as carboxymethyl cellulose (hereinafter abbreviated as CMC), polyalginate, polglutamate, and pectin, and phosphate groups such as cellulose phosphate. Aqueous polymers having a sulfate group such as dextran sulfate and heparin are preferably used. Since the suitable concentration and timing of addition of these water-soluble polymers differ depending on the type of water-soluble polymer used, appropriate conditions are selected for each. For example, in the case of CMC having carboxyl groups with weak ionic strength, 0.05% to 1%,
More preferably, an amount corresponding to a concentration of 0.1% or more and 0.5% or less is added approximately 5 hours or more, more preferably 10 hours or more before addition of the interferon inducing agent, and the ionic strength is In the case of dextran sulfate having strong sulfate groups, it is preferred to add a corresponding amount at a concentration of 0.0001% to 0.01% immediately before, or at most 1 hour before, the addition of the interferon-inducing agent. Glucocorticoids used include hydrocortisone, brednisolone, dexamethasone, and the like, but are not limited to these. The concentration of hydrocortisone, prednisolone or dexamethasone is usually 0.01-100.
Micromoles (μM) are used, more preferably 0.1 to 10 μM. Glucocorticoids are added 72 to 6 hours, preferably 48 hours, before addition of the interferon-inducing agent. At concentrations that enhance interferon production, glucocorticoids inhibit RNA synthesis without affecting DNA and protein synthesis.
Enhance synthesis. Although the interferon production-enhancing effect of glucocorticoids is sometimes observed in planar cultured cells, the effect appears more consistently in microcarrier cultured cells. That is, it is suitably used for mass production of interferon using microcarrier cultured cells. Glucocorticoids have the function of regulating the rate of cytopathic effects caused by treatment with interferon-inducing agents and allowing cells to relatively stably produce high units of interferon. Next, interferon induction treatment is performed using the so-called superinduction or UV method. That is, an interferon inducer is added to a medium in which microcarriers with attached and grown cells are suspended, and then, for example, in the case of the superinduction method, a metabolic inhibitor such as cycloheximide or actinomycin D is added at an appropriate concentration. After adding at an appropriate time schedule and incubating for an appropriate period, the old medium containing the above-mentioned added drugs is preferably replaced with a new medium, and the interferon is incubated in the medium, for example, by incubating for about one day. produce. Various interferon-inducing agents are known to induce interferon in cells, but in the method of the present invention, poly(I):poly
It is most effectively carried out using double-stranded RNA such as (C), poly(A):poly(U), etc. Next, examples of the present invention will be described. Example 1 Human normal diploid cells were placed in 1200 ml of Eagle MEM containing 5% calf serum and 0.3% cross-linked dextran microcarriers with diethylaminoethyl groups.
The cells were inoculated at a rate of 10 ⁵ cells/ml and cultured at 37° C. for 5 days with gentle stirring in a glass spinner flask. During the process, the culture medium was replaced twice with fresh Eagle's MEM medium containing 5% calf serum. The number of arriving cells was 0.9×10 ⁶ cells/ml. Next, 200 ml each of the medium containing the cells and microcarriers was dispensed into six small spinner flasks. Cells and microcarriers were sedimented, the supernatant medium was discarded, and two flasks were filled with 1 μM hydrocortisone and 5 μM calf serum.
Add another 2% new Eagle MEM medium containing 2%
Add fresh Eagle's MEM medium containing 1 μM prednisolone and 5% calf serum to the main flask, and add new Eagle's MEM medium containing 5% calf serum to the remaining two flasks in an amount equal to the discarded medium. Continue culturing for days. The cells and microcarriers were then sedimented again, the supernatant medium was discarded, and an equal volume of medium containing 100 international units/ml of interferon and 2% calf serum was added to the flask to which hydrocortisone or prednisolone had been previously added. Add hydrocortisone or prednisolone to the same concentration, and add 0.2% to each of the three flasks, one flask with hydrocortisone or prednisolone and one flask without glucocorticoid. to
CMC was added to the remaining three tubes, and CMC was not added to the remaining three tubes, and the culture was continued at 37° C. for about 20 hours. next
polyI: polyC at 50μg/ml, cycloheximide
Add to all flasks at 10μg/ml,
After incubating at 37°C for 4 hours, adding actinomycin D at 4 μg/ml and incubating at 37°C for 1 hour, the microcarriers with attached cells were sedimented, the supernatant was discarded, and Eagle MEM supplemented with 0.2% CMC was added. Add the same amount of medium as discarded,
Furthermore, this operation was repeated once more. The final added medium was incubated at 37°C for approximately 48 hours.
After 24 hours had passed, about 2 ml of the culture supernatant was taken out as a specimen for interferon titer measurement. up to 24 hours and in the final medium used.
The amount of interferon produced up to 48 hours
Measured by CPE inhibition method using FL cells and V. stomatitis virus,
Converted to international units. The results are shown in Table 1.

[Table] Example 2 Human normal diploid cells were grown 1.5x in Eagle MEM1.4 containing 5% calf serum and 0.3% cross-linked dextran microcarriers with diethylaminoethyl groups.
The cells were inoculated at a rate of 10 ⁵ cells/ml and cultured at 37° C. for 4 days with gentle stirring in a glass spinner flask. Once in the middle, the culture medium was replaced with fresh Eagle's MEM medium containing 5% calf serum. The number of cells reached was 6.8×10 ⁵ cells/ml. Next, the medium containing the cells and microcarriers
200ml each was dispensed into seven small spinner flasks. Cells and microcarriers were sedimented, the supernatant medium was discarded, and hydrocortisone at the specified concentration was added to some spinner flasks at 37°C.
Continue culturing for 1 day. Sediment the cells and microcarriers again and discard the supernatant medium. Interferon 100 international units/ml, calf serum 2%, CMC 0.2%.
The same concentration of hydrocortisone as previously added was added to the flasks to which hydrocortisone had been added at the predetermined concentrations.
The culture was continued for approximately 20 hours. Next, polyI:polyC was added at 50 μg/ml and cycloheximide at 10 μg/ml to all flasks, and incubated at 37°C for 4 hours. Actinomycin D, 4 μg/ml was added to each flask for 1 hour at 37°C.
After incubation for an hour, the microcarriers with attached cells were sedimented, the supernatant was discarded, and 0.2%
The same amount of Eagle's MEM medium supplemented with CMC was added to the discarded amount, and this operation was repeated once more. The final added medium was incubated at 37°C for approximately 48 hours. The amount of interferon produced up to 48 hours in the final medium used was measured by a CPE inhibition method using FL cells and V. stomatitis virus, and converted to international units. The results are shown in the attached drawings.

[Brief explanation of drawings]

The attached drawings show the results of Example 2.
The horizontal axis shows hydrocortisone concentration (μM), and the vertical axis shows interferon titer (international units/ml).

Claims (1)

[Claims] 1 When animal cells grown on microcarriers with positively charged chemical residues are treated with an interferon-inducing agent, the microcarriers on which cells have attached and grown are treated with glucocorticoids and negatively charged water-soluble polymers. A method for producing interferon, which comprises: