Method for preparing Jurkat cells on large scale by utilizing bioreactor

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a method for preparing Jurkat cells on a large scale by utilizing a bioreactor. The preparation method comprises the following steps: (1) cell resuscitation: resuscitating cells from liquid nitrogen; (2) carrying out cell multiplication culture: carrying out step-by-step amplification multiplication culture in T bottles and cell culture bags with different specifications; (3) large-scale culture of cells: performing large-scale culture by using a bioreactor; and (4) harvesting the cells: collecting the cell suspension, and centrifugally harvesting the cells. The preparation method overcomes the problem that a large number of thymus cells or human lymphocytes need to be used as immunogens when the anti-human T lymphocyte immune globulin is prepared, can simply and quickly prepare a large number of Jurkat cells, is used for preparing the anti-human T lymphocyte immune globulin, improves the quality, reduces the biological safety risk, and has a wide application prospect. The method is beneficial to industrial production and has a relatively good economic effect.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for large-scale preparation of Jurkat cells using a bioreactor. Background Technology

[0002] Jurkat cells are immortalized T lymphocytes derived from the peripheral blood of human patients with acute T-cell leukemia. They possess unlimited proliferative capacity and retain many of the biological characteristics and functions of T lymphocytes, expressing antigens such as CD3 on their surface. Due to their unique biological characteristics and ease of culture, Jurkat cells have been widely used in various fields such as immunology, oncology, and drug development. For example, Jurkat cells play an important role in studying T-cell receptor (TCR) signaling pathways, apoptosis mechanisms, HIV infection mechanisms, and in screening and testing new drugs for T-cell-related diseases.

[0003] Anti-human lymphocyte immunoglobulin is an immunosuppressive drug primarily used to prevent and treat organ transplant rejection and certain autoimmune diseases. It is extracted from the blood of healthy horses, pigs, or rabbits that have undergone multiple immunizations to produce specific antibodies against human lymphocytes. Traditional preparation of anti-human lymphocyte immunoglobulin typically uses thymus cells from stillborn infants induced in hospital or lymphocytes recovered from discarded leukocyte filter plates at blood banks. This process inevitably carries numerous risks, including biosafety and ethical concerns, and often results in raw material shortages.

[0004] Chinese patent application CN200710034501.1 discloses a method for preparing raw materials for anti-human lymphocyte immunoglobulin. The method describes the culture and preparation process of the Jurkat cell line as follows: Jurkat cells are seeded in culture flasks containing RPMI 1640 medium, and interleukin-2 is added to maintain stable CD molecule expression in the cell line. After three days, fresh medium is added for passage culture. Several days later, two-thirds of the culture medium in each flask is poured out, cells are harvested by centrifugation and counted. The remaining cells in the culture flasks are replenished with fresh medium according to the amount poured out, and culture continues for the next immunization. The Jurkat cell culture preparation described in this patent is too simplistic and has the following drawbacks: 1. There is no data on the Jurkat cell culture and preparation process, including cell count, culture flask specifications, culture volume, etc.; 2. The largest commonly used culture flask is only T225, and it is impossible to achieve large-scale cell preparation using culture flasks; 3. RPMI 1640 medium is a basal medium, and with only interleukin-2 added, its nutrients are simply insufficient to support the continuous passage and culture of Jurkat cell lines.

[0005] In the field of cell preparation, bioreactors have become key equipment due to their unique technological advantages. They enable large-scale cell culture, allowing for flexible adjustment of culture volumes according to demand, expanding from a few liters at the laboratory level to thousands of liters at the industrial production level, meeting the needs for large quantities of cells in various scenarios and effectively solving the problem of limited yields associated with traditional culture methods. Simultaneously, bioreactors possess the ability to precisely control the culture environment, enabling real-time monitoring and precise adjustment of key parameters such as temperature, pH, oxygen partial pressure, and nutrient concentration. This ensures cell growth in a stable microenvironment, significantly improving cell activity, function, and quality stability, and reducing batch-to-batch variability. This is crucial for ensuring the efficacy and safety of biopharmaceuticals and cell therapy products. Furthermore, bioreactors employ automated and intelligent operation modes, reducing human intervention and the risk of contamination. They also allow for the recording and traceability of various parameters during the culture process, meeting the quality standards of relevant industries. Moreover, some advanced bioreactors can simulate the in vivo physiological environment, which is beneficial for cell growth, differentiation, and functional expression, further improving the quality and efficiency of cell preparation and providing strong technical support for the development of biomedicine, cell therapy, and other fields. However, no method for large-scale culture of Jurkat cells has been reported, and any adjustment of any parameter may affect the proliferation of Jurkat cells or the expression of surface antigens.

[0006] Therefore, developing a method for large-scale preparation of Jurkat cells is urgently needed in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for large-scale preparation of Jurkat cells using a bioreactor.

[0008] This invention provides a method for large-scale preparation of Jurkat cells, comprising: taking Jurkat cells and sequentially performing amplification culture and large-scale culture; The amplification culture includes cell culture in a cell culture bag; the large-scale culture includes cell culture using a bioreactor.

[0009] Preferably, the amplification culture step includes: amplification culture sequentially in T25 flask, T225 flask and cell culture bag.

[0010] Preferably, the cell seeding density during the amplification culture is 5 × 10⁶ cells / year. 4 ~1.0×10 5 Cells / mL, culture volume in T225 flask is 80~100mL, culture volume in cell culture bag is 800~1000mL.

[0011] Preferably, the step of scaling up the culture includes: seeding Jurkat cells cultured in a cell culture bag into a bioreactor for further culture.

[0012] Preferably, the bioreactor is a stirred bioreactor.

[0013] Preferably, the conditions for large-scale cultivation are: cultivation temperature 36-38℃, stirring speed 60-80 rpm / min, and pH 7.2-7.6.

[0014] Preferably, the large-scale cultivation time is 3-5 days.

[0015] Preferably, the effective volume for cultivation in the bioreactor is ≤6L.

[0016] Preferably, during the large-scale cultivation process, the dissolved oxygen parameter of the bioreactor is 60% air saturation.

[0017] Preferably, the culture medium is RPMI 1640 medium containing 10% fetal bovine serum.

[0018] In this invention, RPMI 1640 medium containing 10% fetal bovine serum refers to RPMI 1640 medium containing 10% fetal bovine serum by volume.

[0019] This invention provides a method for large-scale preparation of Jurkat cells using a bioreactor by screening the steps and process parameters of amplification culture and large-scale culture. The preparation method includes the following steps: (1) Cell resuscitation: resuscitating cells from liquid nitrogen; (2) Cell amplification culture: amplifying and culturing cells in T-flasks and cell culture bags of different sizes in a step-by-step manner; (3) Large-scale cell culture: large-scale culture using a bioreactor; (4) Cell harvesting: collecting the cell suspension and harvesting the cells by centrifugation. This invention achieves the following beneficial effects: 1. Using Jurkat cells to prepare immunogens against human lymphocyte immunoglobulins has lower biosafety and ethical risks, and better quality control compared to fetal thymocytes and human lymphocytes.

[0020] 2. Compared with the preparation method of anti-human lymphocyte immunoglobulin raw material disclosed in Chinese patent application CN200710034501.1, the process provided by the present invention has the following advantages: (1) There are clear process parameters, and Jurkat cells can be prepared smoothly on a large scale according to the process; (2) The process of the present invention uses commercial bovine serum as Jurkat cell culture raw material, which has a greater possibility of industrialization compared with non-commercial cell culture interleukin-2; (3) The process of the present invention uses a bioreactor to culture Jurkat cells, which has the advantages of large-scale culture and production cost advantage compared with the culture flask used in the comparative patent.

[0021] 3. This invention has found that, compared with scaled-up culture in a 10-layer cell factory, Jurkat cells cultured in cell culture bags have a greater advantage in terms of proliferation rate.

[0022] 4. This invention has found that, compared with the scaled-up culture of a 40-layer cell factory, the Jurkat cells cultured in the bioreactor of Tianxinhe Biotechnology Co., Ltd. have advantages in terms of proliferation rate and positive expression rate of CD3 and CD4 molecules.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 The cell proliferation curves are obtained from the preparation methods of Example 1 and Comparative Example 1, in cell culture bags and 10-layer cell factories.

[0026] Figure 2 The cell proliferation curves obtained in the preparation methods of Example 1 and Comparative Example 2, cultured in a bioreactor and a 40-layer cell factory.

[0027] Figure 3 Flow cytometry results of positive expression rate (%) of CD molecules (CD3 and CD4) in cells cultured in a bioreactor.

[0028] Figure 4 Flow cytometry results of the positive rate (%) of CD molecules (CD3 and CD4) expression in cells cultured in a 40-layer cell factory. Detailed Implementation

[0029] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0030] The 10% fetal bovine serum was produced by Lanzhou Rongye Biotechnology Co., Ltd., model number RYS-KF81; the RPMI 1640 medium was produced by Tianxinhe Biotechnology Co., Ltd., serial number 501050; the T25 flask was produced by Corning Incorporated, USA, serial number 410639; the T225 flask was produced by Corning Incorporated, USA, serial number 431082; the cell culture bag was a breathable suspension cell culture bag produced by Corning Incorporated, USA, serial number 8861020; the bioreactor was produced by Tianxinhe Biotechnology Co., Ltd., with a volume of 7.5L, model number TXHBIO-7.5S-TJ-G.

[0031] Example 1: Method for large-scale preparation of Jurkat cells The method for large-scale preparation of Jurkat cells in this embodiment specifically includes the following steps: Step 1: Resuscitation of Jurkat cells 1. Preparation before the experiment 1.1 Preheat the water bath to 37°C.

[0032] 1.2 Place the RPMI 1640 medium containing 10% fetal bovine serum into a water bath and preheat it to 37°C.

[0033] 1.3 Fill an explosion-proof cryopreservation container with 2 / 3 of its volume with water, place it in a water bath, and preheat it to 37°C.

[0034] 1.4 Wear protective clothing, gloves, and goggles correctly to prevent cryopreservation tubes from exploding or liquid nitrogen from coming into contact with the skin.

[0035] 2. Rapid thawing of cells 2.1 Quickly locate the target cryopreservation tube from the liquid nitrogen tank to avoid prolonged exposure.

[0036] 2.2 Confirm that the information on the cryopreservation tube label is correct (cell name, cryopreservation date, passage number, etc.).

[0037] 2.3 Place the cryovials into an explosion-proof cryovial container and immerse them in a 37°C water bath. Gently shake continuously to promote even thawing. Thawing time should be controlled within 1-2 minutes, until the ice crystals completely disappear. Avoid prolonged high temperatures.

[0038] 2.4 Immediately after thawing, wipe the surface of the cryovial with 75% alcohol to sterilize it.

[0039] 3. Cell centrifugation and resuspending 3.1 Slowly transfer the thawed cell suspension into a 15 mL centrifuge tube.

[0040] 3.2 Add 5 mL of preheated complete culture medium dropwise to reduce DMSO toxicity.

[0041] 3.3 Transfer the centrifuge tubes to the centrifuge, and after equilibration, centrifuge at 1000 rpm for 5 minutes.

[0042] 3.4 After centrifugation, gently pour out and discard the supernatant, avoiding contact with the precipitate.

[0043] 4. Cell seeding and culture 4.1 Add 10 mL of RPMI 1640 medium containing 10% fetal bovine serum to a centrifuge tube and gently pipette (avoiding air bubbles) until the cells are evenly dispersed.

[0044] 4.2 Transfer the cell suspension into a T25 culture flask.

[0045] 4.3 Place the T25 culture flasks in a 37℃, 5% CO2 incubator for incubation.

[0046] Step 2: Jurkat cell expansion culture 1. Cell observation and counting 1.1 Observe under a microscope daily. The suspended cells should be relatively uniformly dispersed, without large clumps, and without any abnormal morphology indicating contamination or poor condition.

[0047] 1.2 Gently shake the T25 flask to suspend the cells evenly, avoiding violent blowing that could cause damage.

[0048] 1.3 Pipette 100 μL of cell suspension into a 1.5 mL EP tube and pipette using a 100 μL pipette tip to disperse the cells more evenly.

[0049] 1.4 Take a small amount of cell suspension, add it to a cell counting chamber, transfer it to a cell counter, and operate according to the instrument's instruction manual to directly read the viable cell density. The cell density should reach 8 × 10⁻⁶ cells / day. 5 ~1.2×10 6 When the number of cells / mL is reached, subculture amplification is performed.

[0050] 2. Amplification inoculation in T225 flasks 2.1 Add 90 mL of RPMI 1640 medium containing 10% fetal bovine serum to bottle T225 and preheat T225 in an incubator at 37°C.

[0051] 2.2 Dilute the cell suspension in the T25 cell flask to 5 × 10⁻⁶. 5 Cells / mL, take 10mL and transfer it into T225, that is, the cell seeding density is 5×10⁶. 4 Mix each sample per mL by gently blowing and mixing.

[0052] 2.3 Place the T225 culture flasks in a 37℃, 5% CO2 incubator for incubation.

[0053] 3. Amplification and inoculation in cell culture bags 3.1 Repeat the cell observation and counting steps described above.

[0054] 3.2 Add 900 mL of RPMI 1640 medium containing 10% fetal bovine serum to a 1 L disposable sterile PET bottle, and preheat the PET bottle in a 37 °C incubator.

[0055] 3.3 Dilute the cell suspension in the T225 cell flask to 5 × 10⁻⁶. 5 Transfer 100 mL of the solution to a PET bottle, resulting in a cell seeding density of 5 x 10⁶ cells / mL. 4 Mix each sample per mL by gently blowing and mixing.

[0056] 3.4 Transfer all the cell suspension from the PET bottle into the cell culture bag.

[0057] 3.5 Place the cell culture bags in a 37℃, 5% CO2 incubator for incubation.

[0058] Step 3: Large-scale culture of Jurkat cells 1. Preparation of bioreactors 1.1 Reactor Body Installation 1.1.1 Place the reactor tank on a stable platform, connect the base support, and use a level to ensure it is level.

[0059] 1.1.2 Install the agitator and ensure that the distance between the agitator blade and the bottom of the tank is 1 / 3 of the tank diameter.

[0060] 1.1.3 Connect the motor drive shaft and manually rotate it to test whether it is smooth and there should be no metallic friction sound.

[0061] 1.2 Piping System Connection 1.2.1 Connect the air intake system. Connect two 0.22μm air sterilization filters connected in series to the air intake. Test whether the air vent at the bottom of the tank is unobstructed.

[0062] 1.2.2 Connect the air intake system, install the condenser, install a 0.22μm air sterilization filter at the exhaust port on the top of the tank, and test whether the exhaust is unobstructed.

[0063] 1.2.3 Use silicone tubing to connect the feeding / sampling tubing, connect the feeding bottle and the sampling bottle, ensure that all interfaces use CPC quick connectors, and seal them with aluminum foil.

[0064] 1.3 Sensor Calibration 1.3.1 pH electrode: Calibrate with standard buffer solutions at pH 4.0 and 7.0. A slope > 95% is acceptable.

[0065] 1.3.2 DO (dissolved oxygen) electrode: calibrated to 100% in saturated air and 0% in nitrogen.

[0066] 1.3.3 Temperature probe: Compared with the calibrated thermometer, the error is <0.1℃.

[0067] 2. Sterilization of bioreactors 2.1 Fill the bioreactor tank with 5L of purified water.

[0068] 2.2 Connect the piping system, install the pH electrode and DO electrode, and close all valves.

[0069] 2.3 Place the bioreactor into the sterilization container and start the sterilization program: 121°C, 15 psi, 30 minutes, moist heat sterilization.

[0070] 3. Bioreactor Installation 3.1 After cooling, remove the main body of the bioreactor.

[0071] 3.2 Wrap a temperature-controlled electric heating blanket around the tank and insert a temperature probe.

[0072] 3.3 Connect the motor and the central control device.

[0073] 3.4 Connect compressed air, oxygen, and carbon dioxide to the central control input port in sequence, and connect the central output port to the air inlet of the bioreactor. Turn on the gas, maintain positive pressure, and prevent contamination.

[0074] 3.5 Connect 0.5M NaOH to the bioreactor piping system via the CPC interface, and adjust the input rate using an alkali pump.

[0075] 4. Setting Key Parameters for Bioreactors 4.1 Set the temperature parameter to 37℃.

[0076] 4.2 Set the pH parameter to 7.4.

[0077] 4.3 Set the DO parameter to 60% air saturation.

[0078] 4.4 Set the stirring speed to 70 rpm.

[0079] 5. Cell seeding and culture 5.1 Drain the sterilized water from the bioreactor.

[0080] 5.2 Add 5.4 L of RPMI 1640 medium containing 10% fetal bovine serum to the bioreactor.

[0081] 5.3 Transfer the cell suspension from the cell culture bag to a PET bottle and dilute to 5 × 10⁻⁶. 5 The sample was collected at a rate of 1 / mL and transferred to the pre-added feed bottle.

[0082] 5.4 Connect the feed bottle to the bioreactor via CPC.

[0083] 5.5 The cell suspension was pumped into the bioreactor at a rate of 600 mL, which corresponds to a cell seeding concentration of 5 × 10⁻⁶ cells / mL. 4 per mL.

[0084] 5.6 Maintain the set bioreactor parameters for cultivation.

[0085] Step 4: Jurkat cell harvesting 1. Cell monitoring in bioreactors 1.1 Monitor the bioreactor parameters daily and take samples through the sampling port.

[0086] 1.2 Repeat the cell observation and counting steps in step two.

[0087] 1.3 Cell density reached 1.0 × 10⁻⁶ 6 Stop culturing when the number of cells / mL exceeds a certain level.

[0088] 2. Cell harvesting 2.1 Collect the cell suspension through the bioreactor outlet into a 10L sterile Schott bottle.

[0089] 2.2 Pour the cell suspension from the sterilized Schott flask into a sterilized 1L centrifuge container.

[0090] 2.3 Place the centrifuge container containing the cell suspension on an electronic balance and use a pipette to aspirate the liquid to balance the weight.

[0091] 2.4 Place the centrifuge bucket into a vertical centrifuge and centrifuge at 1000 rpm for 15 minutes.

[0092] 2.5 Discard the supernatant after centrifugation, resuspend the cell pellet in PBS, and harvest the cells into a PET bottle.

[0093] Comparative Example 1: Following the preparation method of Example 1, the main difference lies in replacing the cell culture bag in the "Jurkat cell expansion and culture" step with a 10-layer cell factory (purchased from Corning Incorporated, catalog number 3271) to prepare Jurkat control cells. Details are as follows: Jurkat's expansion in a 10-layer cell factory 1. Add 900 mL of RPMI 1640 medium containing 10% fetal bovine serum to a 1 L disposable sterile PET bottle, and preheat the PET bottle in a 37 °C incubator.

[0094] 2. Dilute the cell suspension in the T225 cell flask to 5 × 10⁻⁶. 5 Transfer 100 mL of the solution to a PET bottle, resulting in a cell seeding density of 5 × 10⁶ cells / mL. 4 Mix each sample per mL by gently blowing and mixing.

[0095] 3. Transfer all the cell suspension in the PET bottle into the 10-layer cell factory.

[0096] 4. Place the 10-layer cell factory in a 37℃, 5% CO2 incubator for incubation.

[0097] Comparative Example 2: Following the preparation method of Example 1, the difference lies in replacing the bioreactor in the "Jurkat cell large-scale culture" step with a 40-layer cell factory (purchased from Corning Incorporated, catalog number 3272) to prepare Jurkat control cells. Details are as follows: Large-scale culture of Jurkat in a 40-layer cell factory 1. Add 5L of RPMI 1640 medium containing 10% fetal bovine serum to a sterilized 10L Schott flask and preheat the flask in a 37℃ incubator.

[0098] 2. Transfer all 1L of cell suspension from the cell culture bag into a Schott flask, shake the Schott flask to disperse the cells.

[0099] 3. Transfer 600 mL of the cell suspension from the Schott flask into a 40-layer cell factory.

[0100] 4. Place the 40-layer cell factory into a large incubator at 37°C and 5% CO2 for incubation.

[0101] The technical solution of the present invention will be further explained through experiments below.

[0102] Comparison of cell proliferation in Experiment Example 1 I. Experimental Methods In the "Jurkat Cell Expansion Culture" step, the number of cells cultured in the cell culture bag according to the method of Example 1 and in the 10-layer cell factory according to the method of Comparative Example 1 were measured on days 1-4. The cell proliferation curves are shown below. Figure 1 As shown.

[0103] In the "Jurkat Cell Scale-up Culture" step, the number of cells cultured for days 1-4 using the method of Example 1 in a bioreactor and the method of Comparative Example 2 in a 40-layer cell factory were measured. The cell proliferation curves are shown below. Figure 2 As shown.

[0104] II. Experimental Results 1. Compared with the expansion culture method of 10-layer cell factory The cell proliferation obtained by the method in Example 1 and Comparative Example 1 is as follows: Figure 1 As shown in Tables 1-2, the number of Jurkat cells gradually increased with prolonged culture time; the cell proliferation in Example 1 was significantly better than that of cells cultured using the method in Comparative Example 1.

[0105] Table 1. Results of cell count detection in cell culture bags (×10) 5 (pcs / mL) Table 2. Cell count results in a 10-layer cell factory (×10) 5 (pcs / mL) 2. Compared with the expansion culture method of 40-layer cell factory The cell proliferation obtained by the method in Example 1 and Comparative Example 2 is as follows: Figure 2 As shown in Tables 3-4, the number of Jurkat cells gradually increased with prolonged culture time; the cell proliferation in Example 1 was significantly better than that of cells cultured using the method in Comparative Example 2.

[0106] Table 3. Cell count results in the bioreactor (×10) 5 (pcs / mL) Table 4. Cell count results in a 40-layer cell factory (×10⁻¹⁰) 5 (pcs / mL) The above experimental results show that, compared with the expansion culture using a 10-layer cell factory and the large-scale culture using a 40-layer cell factory, the cell proliferation rate is faster when the cell culture bag expansion culture is used in Example 1 of this invention, followed by large-scale culture using a stirred bioreactor. Furthermore, it is possible to replace the bioreactor with a larger volume one, which is more conducive to industrial production.

[0107] Experiment Example 2: Detection of CD molecule expression in Jurkat cells The CD molecule expression detection antibody, Anti-CD3 epsilon / CD3e Antibody (PE), is a product of Sinocare, catalog number 10977-M001-P; the Anti-CD4 Antibody (APC) is also from Sinocare, catalog number 10400-MM08-A; and the flow cytometer is a product of Agilent Technologies, model Novocyte 2060R.

[0108] I. Experimental Methods CD molecule expression was detected in Jukat cells collected in Example 1 and Comparative Example 2.

[0109] Step 1: Jurkat cell suspension preparation. Take the required cell volume, centrifuge at 1500 rpm for 5 min, resuspend in 1× PBS (pH 7.4), and prepare a suspension with a density of (1-5)×10⁻⁶ cells / mL. 6 Cell suspensions of 1 cell per mL were prepared for flow cytometry antibody staining detection.

[0110] Step 2: Antibody incubation CD3 molecular expression experimental group: Take 100 μL of cell suspension from step 1 and add it to a 1.5 mL EP tube, then add 1 μL of anti-CD3 antibody (PE) detection reagent and mix by pipetting.

[0111] CD4 molecular expression experimental group: Take 100 μL of cell suspension from step 1 and add it to a 1.5 mL EP tube, then add 1 μL of Anti-CD4 antibody (APC) detection reagent and mix well by pipetting.

[0112] Blank cell experimental group: 100 μL of cells were added to each well, without adding staining antibodies.

[0113] Cells from each experimental group were incubated at room temperature in the dark for 30 minutes. After incubation, 500 μL of PBS (pH 7.4) was added to each experimental group and mixed well. The cells were centrifuged at 400g for 5 minutes, the supernatant was discarded, and the cell pellet was mixed with 100 μL of 1×PBS (pH 7.4). Cells from each experimental group were then added to 96-well plates for flow cytometry analysis.

[0114] Step 3: FACS on-machine testing 96-well plates were used for assaying. The PE and APC fluorescence channels were enabled. The current and voltage were automatically adjusted for the blank cell group. 10,000 cells were collected. The blank cell group, with gate P1 representing a normal viable cell population, was used to analyze CD molecule expression in cells within gate P1. Data processing: For the CD molecule expression blank cell group settings, the background positivity rate of each antibody staining was adjusted to <1.0%, with an optimal range of <0.7%-1.0%. The blank cell group settings were used for analyzing CD3 and CD4 positivity rates in each experimental group.

[0115] II. Experimental Results In the preparation method of Example 1, the detection results of the positive rate of CD molecule expression in cells harvested in the bioreactor are as follows: Figure 3 As shown in Table 5; in the preparation method of Comparative Example 2, the cell number detection results in the 40-layer cell factory are as follows. Figure 4 As shown in Table 6.

[0116] Table 5. Results of CD molecule expression positivity rate (%) in cells cultured in bioreactors Table 6. Results of CD molecule expression positivity rate (%) in 40-layer cell factory culture cells The above experimental results show that, compared with the large-scale culture using a 40-layer cell factory, the Jurkat cells cultured on a large scale using a stirred bioreactor in Example 1 of this invention have a higher positive rate of CD3 and CD4 molecule expression, making them more suitable for industrial production.

[0117] As can be seen from the above embodiments and experimental examples, the present invention provides a method for large-scale preparation of Jurkat cells using a bioreactor. The preparation method includes the following steps: (1) Cell resuscitation: resuscitating cells from liquid nitrogen; (2) Cell expansion culture: progressively expanding and culturing cells in T-flasks and cell culture bags of different specifications; (3) Large-scale cell culture: large-scale culture using a bioreactor; (4) Cell harvesting: collecting the cell suspension and harvesting the cells by centrifugation. The preparation method of the present invention overcomes the problem of requiring large quantities of thymocytes or human lymphocytes as immunogens when preparing anti-human T lymphocyte immunoglobulins. This preparation method can simply and rapidly prepare large quantities of Jurkat cells for the preparation of anti-human lymphocyte immunoglobulins, improving quality, reducing biosafety risks, and is beneficial for industrial production, thus possessing good economic benefits.

Claims

1. A method for large-scale preparation of Jurkat cells, characterized in that, It includes: taking Jurkat cells and sequentially performing expansion culture and large-scale culture; The amplification culture includes cell culture in a cell culture bag; the large-scale culture includes cell culture using a bioreactor.

2. The method for preparing Jurkat cells according to claim 1, characterized in that, The amplification and culture steps include: sequentially amplifying and culturing in T25 flasks, T225 flasks, and cell culture bags.

3. The method for preparing Jurkat cells according to claim 2, characterized in that: The cell seeding density during the amplification culture was 5 × 10⁶. 4 ~1.0×10 5 Cells / mL, culture volume in T225 flask is 80~100mL, culture volume in cell culture bag is 800~1000mL.

4. The method for preparing Jurkat cells according to claim 1, characterized in that, The steps of the scaled-up culture include: seeding Jurkat cells cultured in cell culture bags into a bioreactor for further culture.

5. The method for preparing Jurkat cells according to claim 4, characterized in that: The bioreactor is a stirred bioreactor.

6. The method for preparing Jurkat cells according to claim 5, characterized in that: The conditions for large-scale cultivation are: cultivation temperature 36-38℃, stirring speed 60-80 rpm / min, pH 7.2-7.

6.

7. The method for preparing Jurkat cells according to claim 6, characterized in that: The large-scale cultivation time is 3-5 days.

8. The method for preparing Jurkat cells according to claim 4, characterized in that: The effective volume for cultivation in the bioreactor is ≤6L.

9. The method for preparing Jurkat cells according to claim 4, characterized in that: During the large-scale cultivation process, the dissolved oxygen parameter of the bioreactor is 60% air saturation.

10. The method for preparing Jurkat cells according to claim 1, characterized in that: The culture medium was RPMI 1640 medium containing 10% fetal bovine serum.

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