Method for large-scale high-density culture of human diploid cells

Abstract

The invention provides a method for large-scale high-density culture of human diploid cells. The method comprises the following steps: carrying out multiplication culture on the human diploid cells by using a microcarrier and a bioreactor; digesting the cell suspension with the microcarrier by using a digestion tank; and inoculating the cell suspension and the microcarrier into a flaky carrier and a bioreactor or a swing bed reactor for continuous culture to obtain the large-scale human diploid cells. The density of the human diploid cells cultured by the method can reach 300 * 10 < 4 >-500 * 10 < 4 > cells / mL, the culture scale can reach 50-100 L / batch, and the human diploid cells have high consistency and can meet market requirements.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for large-scale high-density culture of human diploid cells by combining microcarriers and sheet carriers. Background Art

[0002] Human diploid cells derived from human tissues are identical to the human genome, have no exogenous factors, and have no potential tumorigenicity. Human diploid cells are susceptible to a variety of viruses. The prepared human diploid vaccines have good immunogenicity and safety, and are suitable for industrial production of vaccines. Currently, the human diploid cells used for vaccine production on the market mainly include MRC-5, WI-38, 2BS, KMB-17, etc. Human diploid cells are limited-generation cells, and the general working generation is within 40-60 generations. Usually, after exceeding the working generation, the cells will age and degenerate, and the generation interval will become larger and the toxin production capacity will decrease. In industry, it is necessary to expand and use cells within a limited generation.

[0003] Human diploid cells grow relatively slowly and have high requirements for the environment. Currently, most manufacturers use static culture technology to produce diploid vaccines. This method is relatively simple to operate, but it is difficult to increase cell density and expand culture scale. In addition, this technology has a high production cost and low yield, and method improvements are urgently needed.

[0004] Human diploid cells have a low tolerance to fluid shear force during culture. Most bioreactors on the market have relatively large shear forces, making them difficult to use for large-scale culture. The lifting impeller has a smaller shear force than other impellers, such as the Rushton impeller and the pitched-blade impeller. Bioreactors equipped with this type of impeller can meet the culture needs of human diploid cells, such as the 310 cell culture bioreactor from NBS, USA. Compared with static culture technology, this method can greatly increase the culture density of human diploid cells, but the volume of bioreactors equipped with lifting impellers is limited. The culture volume of the 310 cell culture bioreactor from NBS, USA, which is currently commonly used on the market, is 14L, which is difficult to scale up.

[0005] Therefore, there is a need to seek methods for large-scale and high-density culture of human diploid cells. Summary of the invention

[0006] In order to solve the problem that it is difficult to increase cell density and expand culture scale in existing human diploid cell culture, the present invention provides a method for large-scale high-density culture of human diploid cells, wherein the method uses planar 2D culture and microcarrier culture for preliminary cell expansion, and uses sheet carrier culture for large-scale production.

[0007] In some embodiments, the present invention provides a method for large-scale high-density culture of human diploid cells, comprising the following steps:

[0008] (1) Proliferation of diploid cells using microcarriers: The microcarriers are placed in a bioreactor for pre-culture, then inoculated with a cell suspension, and perfusion culture is performed after inoculation;

[0009] (2) Cell digestion: connect the digestion tank to the bioreactor, transfer the cells cultured in step (1), wash the cells with buffer using the perfusion function, drain the excess buffer using the perfusion function after washing, add trypsin digestion solution, and digest. When more than 90% of the cells fall off the microcarriers, it indicates that the digestion stage is completed;

[0010] (3) Cultivating diploid cells using a sheet carrier: Pre-culture a cell culture bag filled with a sheet carrier using a cell growth medium. After the pre-culture is completed, inoculate the human diploid cells digested in step (2) for cell culture.

[0011] In some embodiments, the human diploid cells include human diploid cell lines of MRC-5 cell line, WI-38 cell line, 2BS cell line, KMB-17 cell line, and SLF-1 cell line.

[0012] In some embodiments, the human diploid cells are MRC-5 cell lines, which are derived from embryonic lung tissue and are susceptible to human viruses, such as human poliovirus type I, herpes simplex virus, and vesicular stomatitis, and can be used for efficacy tests, host transfection, cytotoxicity, and virus extinction tests.

[0013] In some embodiments, the human diploid cells are SLF-1 cell lines, which have vigorous growth and a long life span, up to 67 generations on average, are pure and pollution-free, have simple culture methods, and are sensitive to a variety of viruses.

[0014] In some embodiments, the method further comprises reviving the human diploid cells and adding cell culture fluid for culturing before using the microcarriers to expand the diploid cells;

[0015] Wherein, the cell culture fluid comprises, by volume: basal culture medium 84%-92%, 3% glutamine 1.0%-3.0%, 7.5% sodium bicarbonate 2%-3% (other carbon sources and nitrogen sources are also applicable), and bovine serum 5%-10%.

[0016] Preferably, the basal culture medium is selected from DMEM or MEM.

[0017] In some embodiments, the method further comprises planarly expanding the human diploid cells to prepare a cell suspension after thawing the human diploid cells and before expanding the diploid cells using microcarriers;

[0018] Among them, planar subculture is carried out according to a surface area ratio of 1:(6-10), for example, planar subculture can be carried out according to a surface area ratio of 1:6, 1:7, 1:8, 1:9 or 1:10, preferably 1:8.

[0019] In some embodiments, in step (1), the microcarrier is a Cytodex I microcarrier, and the bioreactor is a bioreactor equipped with a cell-lifting stirring impeller.

[0020] In some embodiments, in step (1), the rotation speed of the reactor in the pre-culture stage is 40-80 rpm, the temperature is 37±0.5°C, and the pH is 7.2-7.4.

[0021] In some embodiments, in step (1), the cell density in the reactor after inoculation is controlled at 600,000-800,000 cells / mL.

[0022] In some embodiments, in step (1), the rotation speed of the reactor during the perfusion culture stage is 40-80 rpm, the temperature is 37±0.5°C, the pH is 7.2-7.4, and the dissolved oxygen is 40%-80%.

[0023] In some embodiments, in step (2), the trypsin digestion solution comprises, by volume: 0.01 mol / L phosphate buffer 71.9%-75.2%, 1% trypsin 24.0%-26.0%, 4% EDTA·2Na 0.6%-1.6%, and 1 mol / L NaOH 0.2%-0.5%.

[0024] In some embodiments, in step (2), during the process of transferring the human diploid cells adherently grown on the microcarriers after digestion to the sheet carriers, the free cells and the microcarriers are not separated and are transferred together to the sheet carriers for the next step of culture.

[0025] In the method of the present application, when digesting cells, the culture medium is replaced by perfusion, which can achieve one-way in and one-way out without sedimentation, thus avoiding significant damage to the cells caused by sedimentation. In addition, the present application does not involve a re-suspension process and does not require sieve filtration, thus reducing cell loss.

[0026] The present application adopts the perfusion method, and about 5-6 volumes of growth fluid can be replaced, which can keep the cell viability at about 99%.

[0027] In some embodiments, in step (3), in the large-scale culture of human diploid cells, the filling amount of the sheet carrier is 30-60 g / L, and / or the reactor used in the cell culture stage is a fixed bed or basket bioreactor or a swing bed reactor.

[0028] Unlike microcarriers, sheet carriers have a three-dimensional growth space and can provide more growth area per unit weight; preferably, it is easier to expand the culture volume using a corresponding bioreactor. For example, the culture volume of a fixed bed, basket bioreactor or swing bed reactor can reach 40-100L, which can meet the needs of large-scale high-density culture of human diploid cells.

[0029] In some embodiments, in step (3), the human diploid cells digested in step (2) are inoculated at a density of 400,000-600,000 cells / mL.

[0030] In some embodiments, in step (3), the angle of the reactor during the cell culture stage is 6°-9°, the speed is 10-15 rpm, the temperature is 37±0.5°C, the pH is 7.2-7.4, and the dissolved oxygen is 40%-80%.

[0031] In some embodiments, the present invention provides a method for large-scale high-density culture of human diploid cells, comprising the following steps: reviving human diploid cells; expanding the culture of human diploid cells in cell culture flasks and cell factories and preparing a cell suspension; further amplifying and culturing human diploid cells using microcarriers and bioreactors; further amplifying the cultured human diploid cells with microcarriers and using a digestion tank to digest the cell suspension with microcarriers; and connecting the cell suspension and microcarriers together to a sheet carrier and a bioreactor or a swinging bed reactor for continued culture to obtain large-scale human diploid cells.

[0032] In some embodiments, the present invention provides a method for large-scale high-density culture of human diploid cells, comprising the following steps:

[0033] A. Resuscitation of human diploid cells

[0034] Place the frozen human diploid cells in a 36-39°C water bath and stir rapidly for 1-2 minutes for recovery. Transfer the recovered cells to a cell culture flask, add DMEM or MEM cell culture medium, mix well, and place flat in a 36-38°C carbon dioxide incubator for culture.

[0035] The formula of the cell culture fluid is: 84-92 vol% DMEM or MEM, 1.0-3.0 vol% 3% glutamine, 2-3 vol% 7.5% sodium bicarbonate and 5-10 vol% newborn calf blood, and the total of each component is 100%.

[0036] B. Planar expansion of human diploid cells and preparation of cell suspension

[0037] After the revived human diploid cells have grown all over the cell culture flask, they are then subcultured on the plane. First, discard the original cell culture medium, then rinse the cell attachment growth surface twice with phosphate buffered saline (PBS), then add 0.5%-2% trypsin digestion solution to completely cover the cell attachment growth surface and immediately discard it, place the cell culture flask in a 36-38°C incubator for 5-10 minutes, and when the cells appear frosted glass-like, tap the cell culture flask, and after the cells slide down like quicksand, add the cell culture medium to terminate the digestion, and perform plane subculture at a surface area ratio of about 1:8.

[0038] Planar expansion of human diploid cells, subculture specifications and procedures Figure 1 shown.

[0039] The purpose of this stage is to provide enough cells for the subsequent culture steps. The subculture specifications and number can be determined according to the culture volume of the bioreactor used in the subsequent steps, but it should not be too close to the limiting working generation of the cells used.

[0040] C. Further expansion of diploid cells using microcarriers

[0041] C1. Bioreactor Preparation

[0042] The microcarriers were loaded into a bioreactor equipped with a lifting stirring paddle at a filling volume of 18 g / L, and 0.01 mol / L phosphate buffer was added until the pH electrode and dissolved oxygen electrode probe of the bioreactor were submerged, and then sterilized by wet heat at 121°C for 45 minutes. After sterilization, the microcarriers were placed in a laminar flow hood to cool for 2-3 hours, and then the excess 0.01 mol / L phosphate buffer in the reactor was released, and the cell culture fluid was added until the overflow port of the stirring paddle was submerged for pre-culture. The pre-culture stage should last for more than 1 day.

[0043] The reactor parameters in the pre-culture stage were set as follows: rotation speed: 40-80 rpm, temperature: 37±0.5°C, pH: 7.2-7.4.

[0044] C2. Human diploid cell bioreactor culture

[0045] After the pre-culture is completed, the excess cell growth liquid in the reactor is discharged, and the cell suspension prepared by the planar amplified human diploid cells obtained in step B is inoculated. After inoculation, the cell density in the reactor is controlled at 500,000-800,000 cells / mL. The perfusion culture of the reactor is started 4-6 hours after the inoculation is completed, and samples are taken and observed regularly every day until the human diploid cells basically cover the surface of the microcarrier.

[0046] The reactor parameters during the culture phase were set as follows: rotation speed: 40-80 rpm, temperature: 37±0.5°C, pH: 7.2-7.4, dissolved oxygen: 40%-80%.

[0047] The purpose and effect of this stage are: human diploid cells are large in size, and it is difficult to reach a high cell density through planar culture digestion. Using microcarriers to culture human diploid cells can significantly increase cell density, reduce the use of cell factories, provide a cell basis for expanding production scale, and effectively reduce costs.

[0048] D. Preparation of cell suspension

[0049] D1. Preparation of cell digestion tank

[0050] This step requires a cell digestion tank equipped with an inclined blade paddle, which is required to have perfusion and heating functions, as well as cell access pipes, 0.01 mol / L phosphate buffer access pipes, pancreatic enzyme access pipes, perfusion outlet pipes equipped with filter screens, and surface ventilation pipes. After the digestion tank is assembled, it is sterilized with wet heat at 121°C for 30 minutes. After sterilization, it is left to stand for two hours under laminar flow to cool down, and then connected to the control cabinet for use.

[0051] D2. Cell Digestion

[0052] The digestion tank is connected to the bioreactor for human diploid cell culture through the cell access pipe, and then the cells are transferred in. While keeping the volume of the cell suspension in the digestion tank unchanged, the cells are washed with 0.01mol / L phosphate buffer through the perfusion function. After the cell growth liquid in the cell suspension is completely replaced, the color of the cell suspension will turn white. In this process, the 0.01mol / L phosphate buffer access pipe should be connected to the bottom of the digestion tank. After the cleaning is completed, the stirring function is turned off and the excess 0.01mol / L phosphate buffer is discharged through the perfusion function, and then the remaining volume is supplemented with 0.25% trypsin digestion solution of the same volume, and the stirring is turned on. The temperature is controlled at 37°C for digestion. After 5 minutes of digestion, samples are taken every two minutes to observe the cell digestion. More than 90% of the cells fall off the microcarriers, indicating that the digestion stage is completed. After the digestion is completed, the cells in the digestion tank are discharged into the prepared cell growth liquid with the microcarriers to terminate the digestion and sample and count to measure the cell density after the digestion is completed.

[0053] The 0.25% trypsin digestion solution includes:

[0054]

[0055] E. Large-scale cultivation of diploid cells using sheet carriers

[0056] Fix the cell culture bag into the swing bed reactor, connect the pH electrode and dissolved oxygen electrode as well as the liquid inlet and outlet pipes, and use the recommended culture volume (including but not limited to 1L-1.4L, 10L-14L, 50L-70L or 100L-140L) and an equal amount of cell growth solution to pre-culture the irradiated and sterilized cell culture bag filled with sheet carriers, and fully infiltrate the sheet carriers for 6-12 hours. After the pre-culture, the cell growth solution in the cell culture bag is discharged, and the above-mentioned digested human diploid cells mixed with microcarriers are inoculated at a density of 400,000-600,000 cells / mL, and then the cell growth solution is added to the recommended culture volume of the cell culture bag to start cell culture. During the culture process, the culture medium is taken daily to test the sugar consumption. After the daily sugar consumption level is stable, the cells basically grow all over the sheet carriers, and the density reaches 300×10 4 -500×10 4 cells / mL, and subsequent toxin inoculation and toxin production operations can be carried out as required.

[0057] The formula of the cell growth solution is as follows:

[0058]

[0059] Preferably, the basal culture medium is selected from DMEM or MEM.

[0060] The reactor parameters during the cell culture stage were set as follows: angle: 6°-9°, speed: 10-15 rpm, temperature: 37±0.5°C, pH: 7.2-7.4, dissolved oxygen: 40%-80%.

[0061] The purpose and effect of this stage are: using sheet carriers to culture human diploid cells digested from microcarriers can effectively eliminate the interference of the remaining microcarriers on the subsequent cell culture when the cells and microcarriers are not separated after digestion, and there is no problem of cell reattachment, and the digestion recovery rate is high. In addition, the sheet carrier culture and expansion of human diploid cells can ensure that the cells are more stably attached to the inside of the sheet carrier, and the subsequent harvest time for toxin production and other processes is longer and the turbidity of the harvest liquid is lower.

[0062] The beneficial effects of the present invention are as follows:

[0063] The method of the present invention uses planar 2D culture and microcarrier culture for preliminary cell expansion, and uses sheet carrier culture for large-scale production, which solves the problem that it is difficult to increase cell density and expand the scale of culture in existing human diploid cell culture. The present invention uses cell culture square bottles and cell factories to expand the culture of human diploid cells and prepare cell suspensions; uses microcarriers and bioreactors to further expand and culture human diploid cells; the microcarriers further expand and culture human diploid cells and use digestion tanks to digest the cell suspension with microcarriers; the cell suspension and microcarriers are connected together with sheet carriers and bioreactors or swing bed reactors for continued culture, and large-scale high-density human diploid cells are obtained.

[0064] In the method of the present invention, microcarriers are used to amplify diploid cells in the first-level culture, and sheet carriers are used in the second-level culture for cell toxin fermentation. The inventor surprisingly found in the research process that such a culture sequence can greatly improve the digestibility of cells, and there is no need to remove the carrier in the middle from the first-level microcarrier culture to the second-level sheet carrier culture. If the sheet carrier culture is performed first and then the microcarrier culture is performed, the process of removing the carrier is involved, and the applicable cell types are relatively small, which is not suitable for the diploid cells of the present application. In addition, due to the different culture sequences, the digestion results are particularly different.

[0065] The advantage of the present application is that diploid cells are transferred to sheet carriers for culture after fermentation through microcarrier culture. Since the pore sizes of microcarriers, sheet carriers and cells are different, the microcarriers are microspheres with extremely small particle sizes, and cells grow by attaching to the surface, which makes cell digestion convenient. The sheet carriers are sheet-shaped, similar to fiber holes, and have larger pore sizes than cells. Cells can enter the interior of the sheet carriers and grow by attaching to the walls. Therefore, from the primary microcarrier culture to the secondary sheet carrier culture, a perfusion method is adopted. After cell digestion, the cells are separated from the microcarriers and connected to the second stage. There is no need to remove the microcarriers, and the cell digestion rate is high, and the cell recovery rate is also high.

[0066] If microcarriers are used in the secondary stage to culture cells for toxin production, the cells will easily fall off, but sheet carriers are different. The use of sheet carriers in the secondary stage unexpectedly achieves a long-lasting and stable toxin production effect.

[0067] Generally, diploid cells are relatively large. If the process of removing microcarriers is required, the digestion tank needs to be modified, such as using a fermentation tank for modification. In the digestion step, a screen needs to be used for filtration, which causes great damage to the cells. The method of the present application does not require the step of removing microcarriers. Therefore, a digestion tank with a specific modified structure is not required. Ordinary commercially available digestion tanks can also be used, thereby reducing damage to the cells.

[0068] The human diploid cell density cultured by the method of the present invention can reach 300×10 4 -500×104 cells / mL, the culture scale can reach 50-100L / batch, and it has high consistency, which can meet market demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Flow chart for 1:8 planar subculture expansion of human diploid cells.

[0070] Figure 2 This is a growth curve of SLF-1 cells cultured using microcarriers in Example 1.

[0071] Figure 3 This is a growth curve of SLF-1 cells cultured using a sheet carrier in Example 1.

[0072] Figure 4 This is a growth curve of MRC-5 cells cultured using microcarriers in Example 2.

[0073] Figure 5 This is a growth curve of MRC-5 cells cultured using a sheet carrier in Example 2.

[0074] Figure 6 This is a growth curve of 2BS cells cultured using microcarriers in Example 3.

[0075] Figure 7 This is a growth curve of 2BS cells cultured using sheet carriers in Example 3. DETAILED DESCRIPTION

[0076] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0077] definition

[0078] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0079] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0080] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0081] The numerical range herein includes both endpoints and also includes any numerical value between the two endpoints.

[0082] DMEM: Dulbecco's modified eagle medium, purchased from Invitrogen Gibco (GIBCO).

[0083] MEM: Minimum Essential Medium, purchased from Invitrogen Gibco (GIBCO).

[0084] SLF-1 cells: from Beijing Sinovac Biotech Co., Ltd.

[0085] MRC-5 cells: from Beijing Sinovac Biotech Co., Ltd.

[0086] 2BS cells: from Beijing Sinovac Biotech Co., Ltd.

[0087] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0088] Unless otherwise specified, the reagents, instruments, materials, etc. used in the examples can be obtained commercially.

[0089] Example 1. Large-scale high-density culture of human diploid cells, SLF-1 cells.

[0090] A. Resuscitation of human SLF-1 cells

[0091] The frozen SLF-1 cells were placed in a 37°C water bath and stirred rapidly for 1 minute for recovery. The recovered cells were transferred to a cell culture flask, MEM cell culture medium was added, and the cells were mixed and placed flat in a 37°C carbon dioxide incubator for culture. The cell culture medium used contained 2 vol% of 3% glutamine, 3 vol% of 7.5% sodium bicarbonate and 10 vol% newborn calf serum.

[0092] B. Planar expansion of SLF-1 cells and preparation of cell suspension

[0093] After the revived SLF-1 cells have grown all over the cell culture flask, they are subcultured on a flat surface. First, discard the original cell culture medium, then rinse the cell attachment growth surface twice with phosphate buffered saline (PBS), then add 1% trypsin digestion solution to completely cover the cell attachment growth surface and immediately discard it. Place the cell culture flask in a 37°C incubator for 5 minutes. After the cells appear frosted, tap the cell culture flask. After the cells slide down like quicksand, add cell culture solution to terminate digestion, and perform flat subculture at a surface area ratio of about 1:8. After the culture specifications reach 4 40-layer cell factories, the next stage of operation can be carried out.

[0094] C. Further expansion of SLF-1 cells using microcarriers

[0095] C1. Bioreactor Preparation

[0096] Microcarriers (GE's CytodexⅠ microcarriers) were loaded into a bioreactor (14L 310 bioreactor from NBS, USA) at a filling volume of 18 g / L, and 0.01 mol / L phosphate buffer was added until the pH electrode and dissolved oxygen electrode probe of the bioreactor were submerged, and then sterilized by wet heat at 121°C for 45 minutes. After sterilization, the cells were placed in a laminar flow hood to cool for 2 hours, and then the excess 0.01 mol / L phosphate buffer in the reactor was released, and cell culture fluid was added until the overflow port of the stirring paddle was submerged for pre-culture. The pre-culture was carried out for 1 day, and the subsequent operations were carried out after confirming that the bioreactor culture medium was not contaminated the next day.

[0097] C2, SLF-1 bioreactor culture

[0098] After the pre-culture, the excess cell growth medium in the bioreactor was drained and the cell suspension prepared by the planar amplification of SLF-1 cells in step B was inoculated. After inoculation, the cell density in the reactor was controlled at 500,000 cells / mL. The perfusion culture of the reactor was started 6 hours after the inoculation, and samples were taken regularly for observation every day.

[0099] Figure 2 This is the growth curve of SLF-1 cells cultured on microcarriers. The inoculation density is 500,000 cells / mL. The cell growth is relatively slow in the first three days, and the cell density increases to 2 million cells / mL on the third day. The cell growth rate increases significantly on the 4th and 5th days. Microscopic observation shows that the cells are basically full on the 5th day, and the final density is about 6 million / mL. Subsequent operations are performed after the SLF-1 cells basically cover the surface of the microcarrier.

[0100] D. Preparation of cell suspension

[0101] D1. Preparation of cell digestion tank

[0102] This step requires a cell digestion tank equipped with an inclined blade paddle, which is required to have perfusion and heating functions, as well as cell access pipes, 0.01 mol / L phosphate buffer access pipes, pancreatic enzyme access pipes, perfusion outlet pipes equipped with filter screens, and surface ventilation pipes. After the digestion tank is assembled, it is sterilized with wet heat at 121°C for 30 minutes. After sterilization, it is left to stand for two hours under laminar flow to cool down, and then connected to the control cabinet for use.

[0103] D2. Cell Digestion

[0104] The digestion tank is connected to the SLF-1 cell culture bioreactor through the cell access pipe, and then the cells are transferred in. While keeping the volume of the cell suspension in the digestion tank unchanged, the cells are washed with 0.01mol / L phosphate buffer through the perfusion function. After the cell growth liquid in the cell suspension is completely replaced, the color of the cell suspension will turn white. In this process, the 0.01mol / L phosphate buffer access pipe should be connected to the bottom of the digestion tank. After the cleaning is completed, the stirring function is turned off and the excess 0.01mol / L phosphate buffer is discharged through the perfusion function, and then the remaining volume is supplemented with 0.25% trypsin digestion solution of the same volume, and the stirring is turned on. The temperature is controlled at 37℃ for digestion. After 5 minutes of digestion, samples are taken every two minutes to observe the cell digestion. More than 90% of the cells fall off the microcarriers, indicating that the digestion stage is completed. After the digestion is completed, the cells in the digestion tank are discharged into the prepared cell growth liquid with the microcarriers to terminate the digestion and sample and count to measure the cell density after the digestion is completed.

[0105] E. Scale-up of SLF-1 cells using sheet carriers

[0106] Use 100L cell growth solution to pre-culture a cell culture bag (SKC0206-0200RGPS12KX, Wuhan Saikecheng Technology Co., Ltd.) pre-filled with 4000g sheet carriers sterilized by irradiation, and fully infiltrate the sheet carrier for 12 hours. After the pre-culture, the cell growth solution in the cell culture bag is discharged, and the SLF-1 cells mixed with microcarriers after the above digestion are accessed at a density of 600,000 cells / mL, and then the cell growth solution is added to 100L to start cell culture. During the culture process, the sugar consumption can be detected by taking the culture solution daily. After the daily sugar consumption level is stable, the cells are basically covered with sheet carriers. In the process research stage, part of the carrier can also be taken by aseptic operation for direct counting.

[0107] Figure 3This is the growth curve of SLF-1 cells cultured using sheet carriers. The growth curve is relatively flat, and the growth rate is basically maintained at a relatively constant rate in the first 4 days, with the daily density increasing by about 1.6-1.8 times. On the 4th day, the cell density reached a plateau of about 3.5 million / mL, and on the 5th day, the cell density was about 3.4 million / mL, which basically remained stable, and the subsequent inoculation and production operations can be carried out as required.

[0108] Example 2. Large-scale high-density culture of human diploid cells, MRC-5 cells.

[0109] A. Resuscitation of human MRC-5 cells:

[0110] The frozen MRC-5 cells were placed in a 37°C water bath and stirred rapidly for 1 minute for recovery. The recovered cells were transferred to a cell culture flask, MEM cell culture medium was added, and the cells were mixed and placed flat in a 37°C carbon dioxide incubator for culture. The cell culture medium used contained 2 vol% of 3% glutamine, 3 vol% of 7.5% sodium bicarbonate, and 10 vol% newborn calf serum.

[0111] B. Planar expansion of MRC-5 cells and preparation of cell suspension

[0112] After the revived MRC-5 cells have grown all over the cell culture flask, they are subcultured on a flat surface. First, discard the original cell culture medium, then rinse the cell attachment growth surface twice with phosphate buffered saline (PBS), then add 1% trypsin digestion solution to completely cover the cell attachment growth surface and immediately discard it. Place the cell culture flask in a 37°C incubator for 5 minutes. After the cells appear frosted, tap the cell culture flask. After the cells slide down like quicksand, add cell culture solution to terminate digestion, and perform flat subculture at a surface area ratio of about 1:8. After the culture specifications reach 4 40-layer cell factories, the next stage of operation can be carried out.

[0113] C. Further expansion of MRC-5 cells using microcarriers

[0114] C1. Bioreactor Preparation

[0115] Microcarriers (GE's CytodexⅠ microcarriers) were loaded into a bioreactor (14L 310 bioreactor from NBS, USA) at a filling volume of 18 g / L, and 0.01 mol / L phosphate buffer was added until the pH electrode and dissolved oxygen electrode probe of the bioreactor were submerged, and then sterilized by wet heat at 121°C for 45 minutes. After sterilization, the cells were placed in a laminar flow hood to cool for 2 hours, and then the excess 0.01 mol / L phosphate buffer in the reactor was released, and cell culture fluid was added until the overflow port of the stirring paddle was submerged for pre-culture. The pre-culture was carried out for 1 day, and the subsequent operations were carried out after confirming that the bioreactor culture medium was not contaminated the next day.

[0116] C2, MRC-5 bioreactor culture

[0117] After the pre-culture, the excess cell growth medium in the bioreactor was drained and the cell suspension prepared by the planar expansion of MRC-5 cells in step B was inoculated. After inoculation, the cell density in the reactor was controlled at 500,000 cells / mL. The perfusion culture of the reactor was started 6 hours after the inoculation, and samples were taken regularly every day for observation.

[0118] Figure 4 This is the growth curve of MRC-5 cells cultured on microcarriers. The inoculation density is 500,000 cells / mL. The curve is generally flat, and the cells are logarithmically expanded. The third day is the middle logarithmic phase, and the fifth day is close to the end logarithmic phase. Microscopic observation shows that the cells basically cover the surface of the microcarriers, and the final density is about 6.1 million / mL, and subsequent operations are carried out.

[0119] D. Preparation of cell suspension

[0120] D1. Preparation of cell digestion tank

[0121] This step requires a cell digestion tank equipped with an inclined blade paddle, which is required to have perfusion and heating functions, as well as cell access pipes, 0.01 mol / L phosphate buffer access pipes, pancreatic enzyme access pipes, perfusion outlet pipes equipped with filter screens, and surface ventilation pipes. After the digestion tank is assembled, it is sterilized with wet heat at 121°C for 30 minutes. After sterilization, it is left to stand for two hours under laminar flow to cool down, and then connected to the control cabinet for use.

[0122] D2. Cell Digestion

[0123] The digestion tank is connected to the MRC-5 cell culture bioreactor through the cell access pipe, and then the cells are transferred in. While keeping the volume of the cell suspension in the digestion tank unchanged, the cells are washed with 0.01mol / L phosphate buffer through the perfusion function. After the cell growth liquid in the cell suspension is completely replaced, the color of the cell suspension will turn white. In this process, the 0.01mol / L phosphate buffer access pipe should be connected to the bottom of the digestion tank. After the cleaning is completed, the stirring function is turned off and the excess 0.01mol / L phosphate buffer is discharged through the perfusion function, and then the remaining volume is supplemented with 0.25% trypsin digestion solution of the same volume, and the stirring is turned on. The temperature is controlled at 37°C for digestion. After 5 minutes of digestion, samples are taken every two minutes to observe the cell digestion. More than 90% of the cells fall off the microcarriers, indicating that the digestion stage is completed. After the digestion is completed, the cells in the digestion tank are discharged into the prepared cell growth liquid with the microcarriers to terminate the digestion and sample and count to measure the cell density after the digestion is completed.

[0124] E. Scale-up of MRC-5 cells using sheet carriers

[0125] Use 100L cell growth solution to pre-culture a cell culture bag (SKC0206-0200RGPS12KX, Wuhan Saikecheng Technology Co., Ltd.) pre-filled with 4000g sheet carriers sterilized by irradiation, and fully infiltrate the sheet carrier for 12 hours. After the pre-culture, the cell growth solution in the cell culture bag is discharged, and the MRC-5 cells mixed with microcarriers after the above digestion are accessed at a density of 500,000 cells / mL, and then the cell growth solution is added to 100L to start cell culture. During the culture process, the sugar consumption can be detected by taking the culture solution daily, and the cells are basically covered with sheet carriers after the daily sugar consumption level is stable. In the process research stage, some carriers can also be taken by aseptic operation for direct counting.

[0126] Figure 5 This is the growth curve of MRC-5 cells cultured using sheet carriers. The growth curve is relatively flat, with logarithmic growth in the first 5 days, and the density expanded to 3.7 million / mL. On the 6th day, the cell density was about 4 million / mL, which basically remained stable, and the subsequent inoculation and toxin production operations can be carried out as needed.

[0127] Example 3. Large-scale high-density culture of human diploid cells, 2BS cells.

[0128] A. Resuscitation of human 2BS cells:

[0129] The frozen 2BS cells were placed in a 37°C water bath and stirred rapidly for 1 minute for recovery. The recovered cells were transferred to a cell culture flask, MEM cell culture medium was added, and the cells were mixed and placed flat in a 37°C carbon dioxide incubator for culture. The cell culture medium used contained 2 vol% of 3% glutamine, 3 vol% of 7.5% sodium bicarbonate, and 10 vol% newborn calf serum.

[0130] B. Planar expansion of 2BS cells and preparation of cell suspension

[0131] After the revived 2BS cells have grown all over the cell culture flask, they are subcultured on a flat surface. First, discard the original cell culture medium, then rinse the cell attachment growth surface twice with phosphate buffered saline (PBS), then add 1% trypsin digestion solution to completely cover the cell attachment growth surface and immediately discard it. Place the cell culture flask in a 37°C incubator for 5 minutes. After the cells appear frosted glass, tap the cell culture flask. After the cells slide down like quicksand, add cell culture solution to terminate digestion, and perform flat subculture at a surface area ratio of about 1:8. After the culture specifications reach 4 40-layer cell factories, the next stage of operation can be carried out.

[0132] C. Further expansion of 2BS cells using microcarriers

[0133] C1. Bioreactor Preparation

[0134] Microcarriers (GE's CytodexⅠ microcarriers) were loaded into a bioreactor (14L 310 bioreactor from NBS, USA) at a filling volume of 18 g / L, and 0.01 mol / L phosphate buffer was added until the pH electrode and dissolved oxygen electrode probe of the bioreactor were submerged, and then sterilized by wet heat at 121°C for 45 minutes. After sterilization, the cells were placed in a laminar flow hood to cool for 2 hours, and then the excess 0.01 mol / L phosphate buffer in the reactor was released, and cell culture fluid was added until the overflow port of the stirring paddle was submerged for pre-culture. The pre-culture was carried out for 1 day, and the subsequent operations were carried out after confirming that the bioreactor culture medium was not contaminated the next day.

[0135] C2, 2BS bioreactor culture

[0136] After the pre-culture, the excess cell growth medium in the bioreactor was drained and the cell suspension prepared by the planar expansion of 2BS cells in step B was inoculated. After inoculation, the cell density in the reactor was controlled at 500,000 cells / mL. The perfusion culture of the reactor was started 6 hours after the inoculation, and samples were taken regularly for observation every day.

[0137] Figure 6 This is the growth curve of 2BS cells cultured on microcarriers. The inoculation density is 500,000 cells / mL. The curve is generally flat, and the cell proliferation is obvious. The cell growth trend is basically the same in the first 4 days. Microscopic observation shows that on the 4th day, the cells basically cover the surface of the microcarriers, and the final density is about 5 million / mL, and subsequent operations are carried out.

[0138] D. Preparation of cell suspension

[0139] D1. Preparation of cell digestion tank

[0140] This step requires a cell digestion tank equipped with an inclined blade paddle, which is required to have perfusion and heating functions, as well as cell access pipes, 0.01 mol / L phosphate buffer access pipes, pancreatic enzyme access pipes, perfusion outlet pipes equipped with filter screens, and surface ventilation pipes. After the digestion tank is assembled, it is sterilized with wet heat at 121°C for 30 minutes. After sterilization, it is left to stand for two hours under laminar flow to cool down, and then connected to the control cabinet for use.

[0141] D2. Cell Digestion

[0142] The digestion tank is connected to the 2BS cell culture bioreactor through the cell access pipe, and then the cells are transferred in. While keeping the volume of the cell suspension in the digestion tank unchanged, the cells are washed with 0.01mol / L phosphate buffer through the perfusion function. After the cell growth liquid in the cell suspension is completely replaced, the color of the cell suspension will turn white. In this process, the 0.01mol / L phosphate buffer access pipe should be connected to the bottom of the digestion tank. After the cleaning is completed, turn off the stirring function and discharge the excess 0.01mol / L phosphate buffer through the perfusion function, then add the same volume of 0.25% trypsin digestion solution as the remaining volume, turn on the stirring, control the temperature at 37℃ for digestion, and take samples every two minutes after 5 minutes of digestion to observe the cell digestion. More than 90% of the cells fall off the microcarriers, indicating that the digestion stage is completed. After the digestion is completed, the cells in the digestion tank are discharged into the prepared cell growth liquid with the microcarriers to terminate the digestion and sample and count to measure the cell density after the digestion is completed.

[0143] E. Scale-up of 2BS cells using sheet carriers

[0144] Use 100L cell growth solution to pre-culture a cell culture bag (SKC0206-0200RGPS12KX, Wuhan Saikecheng Technology Co., Ltd.) pre-filled with 4000g sheet carriers sterilized by irradiation, and fully infiltrate the sheet carrier for 12 hours. After the pre-culture, the cell growth solution in the cell culture bag is discharged, and the 2BS cells mixed with microcarriers after the above digestion are accessed at a density of 600,000 cells / mL, and then the cell growth solution is added to 100L to start cell culture. During the culture process, the sugar consumption can be detected by taking the culture solution daily. After the daily sugar consumption level is stable, the cells are basically covered with sheet carriers. In the process research stage, part of the carrier can also be taken by aseptic operation for direct counting.

[0145] Figure 7 This is the growth curve of 2BS cells cultured using sheet carriers. The growth curve is relatively flat, with logarithmic growth in the first 5 days, and the density expanded to 3.5 million / mL. On the 6th day, the cell density was about 3.7 million / mL, which basically remained stable, and the subsequent inoculation and toxin production operations can be carried out as needed.

[0146] Comparative Example 1. Cultivation of human diploid MRC-5 cells

[0147] Experimental method: According to the method in the prior art, human diploid cells are first cultured using sheet carriers and then cultured using microcarriers. In this process, sheet carriers are used to amplify human diploid cells on a large scale and increase the inoculation density and inoculation amount of subsequent culture. This process is difficult to digest cells, has a low recovery rate, and has poor activity of recovered cells. The experimental results of different culture sequences compared with the process of switching from microcarriers to sheet carriers in this application are shown in Table 1 below.

[0148] Table 1. Culture results of different culture sequences

[0149]

[0150] From the results in Table 1, it can be seen that the method of the present invention uses microcarriers for primary culture and sheet carriers for secondary culture, and has good cell digestion effect, high digestion rate, high cell recovery rate, small cell loss, good persistence of toxin production, and high cell survival rate, which is significantly different from the method of the prior art (p value <0.01).

[0151] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for large-scale high-density culture of human diploid cells, characterized in that: The following steps are involved: (1) Proliferation of diploid cells using microcarriers: The microcarriers are placed in a bioreactor for pre-culture, then inoculated with a cell suspension, and perfusion culture is performed after inoculation; (2) Cell digestion: connect the digestion tank to the bioreactor, transfer the cells cultured in step (1), wash the cells with buffer by perfusion, drain the excess buffer by perfusion after washing, add trypsin digestion solution, and digest. When more than 90% of the cells fall off the microcarriers, it indicates that the digestion stage is completed; (3) Cultivating diploid cells using a sheet carrier: Pre-culture a cell culture bag filled with a sheet carrier using a cell growth medium. After the pre-culture is completed, inoculate the human diploid cells digested in step (2) for cell culture.

2. The method according to claim 1, characterized in that The human diploid cells include MRC-5 cell line, WI-38 cell line, 2BS cell line, KMB-17 cell line and SLF-1 cell line.

3. The method according to claim 1, characterized in that The method further comprises resuscitating the human diploid cells and adding cell culture fluid for culturing before using the microcarriers to amplify the diploid cells; Wherein, the cell culture fluid comprises, by volume: basal culture medium 84%-92%, 3% glutamine 1.0%-3.0%, 7.5% sodium bicarbonate 2%-3%, and bovine serum 5%-10%; Preferably, the basal culture medium is selected from DMEM or MEM.

4. The method according to claim 3, characterized in that The method further comprises planarly amplifying the human diploid cells to prepare a cell suspension after thawing the human diploid cells and before amplifying the diploid cells using microcarriers; Among them, plane subculture is carried out according to the surface area ratio of 1:(6-10).

5. The method according to claim 1, characterized in that In step (1), the microcarrier is a CytodexⅠ microcarrier, and the bioreactor is a bioreactor equipped with a cell-lifting stirring paddle.

6. The method according to claim 1, characterized in that In step (1), the speed of the reactor in the pre-culture stage is 40-80 rpm, the temperature is 37±0.5°C, and the pH is 7.2-7.4; and / or During the perfusion culture stage, the rotation speed of the reactor is 40-80 rpm, the temperature is 37±0.5° C., the pH is 7.2-7.4, and the dissolved oxygen is 40%-80%.

7. The method according to claim 1, characterized in that In step (2), the trypsin digestion solution comprises, by volume: 0.01 mol / L phosphate buffer 71.9%-75.2%, 1% trypsin 24.0%-26.0%, 4% EDTA·2Na 0.6%-1.6%, and 1 mol / L NaOH 0.2%-0.5%.

8. The method according to claim 1, characterized in that: In step (2), the human diploid cells adherently grown on the microcarriers are transferred to the sheet carriers after digestion without separating the free cells and the microcarriers, and are transferred together to the sheet carriers for the next step of culture.

9. The method according to claim 1, characterized in that: In step (3), the filling amount of the sheet carrier is 30-60 g / L, and / or the reactor used in the cell culture stage is a fixed bed or basket bioreactor or a swing bed reactor.

10. The method according to claim 1, characterized in that In step (3), during the cell culture phase, the reactor has an angle of 6°-9°, a speed of 10-15 rpm, a temperature of 37±0.5°C, a pH of 7.2-7.4, and a dissolved oxygen of 40%-80%.

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