Serum-free suspension cell domestication method

Abstract

The invention provides a serum-free suspension domestication method for adherent culture cells, which provides a mode of combining rapid serum reduction with flow sorting and clone screening to screen high-performance clone cell clusters so as to obtain suspension cells with high transfection efficacy. The suspension cell obtained by the method can avoid the risk caused by using serum in adherent cell culture, and has the characteristics of high-density transfection tolerance and obviously higher transfection efficiency. The method can be applied to suspension domestication of all adherent cells including but not limited to HEK293T cells.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for serum-free domestication of suspension cells including HEK293T cells and the like with high transfection efficiency. Background Art

[0002] HEK293 cells are derived from human embryonic kidney cells, and HEK293T cells are high-transfection-efficiency derivatives of HEK293 cells after the SV40 large T antigen gene is inserted. HEK293T cells carry the SV40 replication sequence, have high transfection efficiency and protein expression levels, and are therefore widely used in retrovirus production, gene expression, and protein expression.

[0003] Traditionally, HEK293T cells are cultured in monolayer adherent culture. A certain proportion of bovine serum (fetal bovine serum or newborn bovine serum) needs to be added to the culture medium for normal growth of cells. Because bovine serum comes from animals, it may carry infectious sources, including viruses and toxic substances. Any infectious source may bring risks to growing cells or later biological products. Bovine serum is also a natural mixture, and its specific components have not yet been fully analyzed. Therefore, the use of bovine serum may cause difficulties in downstream purification processes. If there is any residue, it will usually cause side effects in the vaccine recipient and affect product quality. In addition, adherent cells need to be digested with trypsin during passage and amplification, and trypsin has the risk of introducing porcine-related viruses. For the above reasons, in the field of pharmaceutical industrial production, whether HEK293T cells are used for retrovirus production or protein expression, for safety reasons, intermediates and finished products need to be tested for bovine-related viruses and porcine-related viruses, which not only increases production costs, but also increases safety risks. Therefore, the suspension domestication of HEK293T cells has become an important means to reduce safety risks, increase virus yields and product quality.

[0004] CN103205396B discloses a suspension acclimation method, which adopts the method of first suspending and then reducing serum, and uses pancreatic enzyme to control cell clusters; CN110713971B adopts the method of gradient serum reduction, uses screens and anticoagulants to control agglomeration, and selects better cells by monoclonal method; CN113717927B adopts the method of gradient serum reduction, and uses external force to suspend cells when it drops to 0.2%, and culture on a shaking table; CN113981007A adopts the method of first cloning and screening cells with higher transfection efficiency and then quickly reducing serum for suspension; CN109280636A uses the method of gradient serum reduction to suspend 293T cells; CN102604889B uses the method of gradient serum reduction to suspend 293T cells and uses the presence or absence of poly-lysine to control the attachment or suspension culture of 293T cells. In summary, currently reducing serum is a step that cannot be skipped in suspension culture, but the method adopted by the above patents has the disadvantages of low efficiency and great damage to cells. Summary of the invention

[0005] The present invention provides a method for domesticating a serum-free suspension cell line capable of efficiently packaging lentiviruses, and can provide a serum-free suspension cell line capable of efficiently packaging lentiviruses. HEK293T cells are used as the original material, a cell line adapted to serum-free culture is domesticated and screened, and the transfection efficiency of the lentivirus is studied.

[0006] In view of a series of technical problems caused by serum, the present invention provides a method for suspension domestication of adherent cultured cells, which can be applied to suspension domestication of all adherent cells including but not limited to HEK293T cells.

[0007] The first aspect of the present invention provides a method for screening high-performance cloned cell clusters by combining serum reduction with flow sorting, comprising the following steps:

[0008] 1) reviving adherent cells and subculturing them; taking cells in logarithmic growth phase and using serum-free medium and adding serum to culture for 2 to 3 days, collecting non-adherent cells into culture bottles, staining the cells with non-toxic live and dead dyes, and sorting live cells by flow cytometry; using serum-free medium and adding decreasing concentrations of serum to culture the sorted live cells for 2 to 3 days, and continuing to sort the non-adherent cells by flow cytometry according to the above sorting method; the concentration of the serum does not exceed 2.5% by volume;

[0009] 2) The living cells isolated above were suspended and cultured in a serum-free medium containing 0.5% dextran sulfate until the cell density reached 1×10 6 ~1.5×10 6When the cell density is 2.1447 W / mL, continue to perform flow cytometry sorting on the cells according to the method in step 1) to sort out the living cells, and culture the sorted cells on a shaker and perform adaptive subculture;

[0010] 3) When the specific growth rate of the cells reaches above 0.5 in a serum-free medium containing 0.5% dextran sulfate and the cell viability is greater than 90%, take the cells in the logarithmic growth phase and spread them on a 96-well plate, select the cells with good growth status and morphology and gradually expand them to shake flask culture; when the viability of the selected cells reaches 95-100%, the cells are well dispersed and there is no obvious aggregation, they are the cells that have been successfully suspended.

[0011] The present invention particularly provides a method for serum-free acclimation of suspended HEK293T cells with high transfection efficiency, comprising the following steps:

[0012] 1. Resuscitate adherent HEK293T cells and use DMEM high-glucose medium containing 10% newborn calf serum for adherent culture. When the cell confluence reaches 80% to 90%, subculture at a ratio of 1:3 to 1:15.

[0013] 2. Acclimation Steps

[0014] (1) The adherent HEK293T cells were subcultured for 2 to 3 generations using DMEM high-glucose medium containing 10% newborn calf serum.

[0015] (2) Take the HEK293T cells in the logarithmic growth phase in step (1) and culture them statically for 2 to 3 days in Free style medium containing 2% newborn calf serum. Collect the HEK293T cells that are not attached to the wall into a culture flask, stain the cells with a non-toxic live or dead dye, and sort the live cells using a flow cytometer.

[0016] (3) The living cells separated in step (2) were cultured in a Free style medium containing 0.5% newborn calf serum for 2 to 3 days, and the non-adherent HEK293T cells were further separated by flow cytometry according to the separation method in step (2).

[0017] (4) The living cells separated in step (3) were suspended and cultured in Free style medium containing 0.5% dextran sulfate until the cell density reached 1×10 6 ~1.5×10 6 When the concentration of 404 cells / mL is 2.14, the cells are flow sorted according to the method in step (2) to separate the living cells, and the separated cells are cultured on a shaker.

[0018] (5) Adaptively subculture the living cells separated in step (4) using Free style medium containing 0.5% dextran sulfate.

[0019] (6) When the specific growth rate of cells in Free style medium containing 0.5% dextran sulfate reaches above 0.5 and the cell viability is greater than 90%, cells in the logarithmic growth phase are plated on 96-well plates for monoclonal screening and culture. Cells with good growth status and morphology are selected and gradually expanded to shake flask culture.

[0020] (7) When the viability of the cells selected in step (6) reaches 95-100%, and the cells are well dispersed without obvious aggregation, and the cells can tolerate high-density electroporation, and the cells maintain a high viability and recover quickly after electroporation, the HEK293T cells are successfully suspended and domesticated.

[0021] As a further improvement of the above-mentioned serum-free acclimation method for HEK293T cells with high transfection efficiency, step 3 is also included as follows:

[0022] 3. Expand and freeze the successfully suspended HEK293T cells to build a library.

[0023] As a further improvement of the above-mentioned serum-free acclimation method for HEK293T cells with high transfection efficiency, in step 1, the subculture conditions are 37° C., 5% CO 2 , and the culture is carried out for 48-72 hours.

[0024] As a further improvement of the above-mentioned serum-free acclimation method for HEK293T cells with high transfection efficiency, in acclimation step 2 (4), the suspension culture conditions and shaking culture conditions are 36.5°C, 8% CO2, 80% humidity, and a rotation speed of 120 rpm.

[0025] As a further improvement of the above serum-free acclimation method for HEK293T cells with high transfection efficiency, the non-toxic live and dead dye is used at a concentration of 1×10 6 cells were diluted with the fluorescent dye ZombieAqua at a ratio of 1:1000.

[0026] As a further improvement of the above serum-free acclimation method for highly efficient transfection of HEK293T cells, in acclimation step 2 (5), adaptive subculture was performed for 2 to 3 generations, and the inoculation density was 6×10 5 ~8×10 5 cells / mL, and wait until the cell density reaches 2×10 6 ~3×10 6 cells / mL for subculture.

[0027] As a further improvement of the above-mentioned serum-free acclimation method for highly efficient transfection of HEK293T cells, in acclimation step 2 (6), monoclonal screening was performed by limiting dilution method at 5-20 cells / mL inoculation in 96-well cell culture plates, with 100 μl of cell suspension per well, and then 100 μl of Free style culture medium was added to each well, and clone observation was performed after 5-7 days of culture.

[0028] As a further improvement of the above serum-free acclimation method for highly efficient transfection of HEK293T cells, in step 3, the cell passage expansion method is to wait until the cell density reaches 1.5×10 6 ~3.0×10 6 cells / mL, and the density was maintained at 2×10 5 ~8×10 5 cells / mL, subcultured every 3 to 4 days.

[0029] As a further improvement of the above-mentioned serum-free acclimation method for HEK293T cells with high transfection efficiency, in step 3, the culture conditions for cell subculture and expansion are temperature 36.5° C., humidity 80%, carbon dioxide 8%, and rotation speed 120 rpm.

[0030] As a further improvement of the above serum-free acclimation method for highly efficient transfection of HEK293T cells, the cryopreservation and library construction method in step 3 is as follows: When the cell density reaches 1.5×10 6 ~3.0×10 6 When the cell density was 2 × 10 cells / mL, the cells were collected by centrifugation at 1500 rpm for 10 min, resuspended in freezing solution, sampled, and counted. The cell density was adjusted to 2 × 10 cells / mL using freezing solution. 7 cells / mL, aliquoted, 1 mL per cryovial, placed in a program cooling box, -80°C overnight, and transferred to liquid nitrogen for long-term storage the next day. The proliferation and expansion library construction medium used was Free style medium containing 0.5% dextran sulfate, without serum.

[0031] Preferably, a method for suspension acclimation of adherent cultured cells of the present invention comprises:

[0032] Phase 1: Domestication steps:

[0033] 1. Resuscitate the HEK293T cells that have been cultured adherently, and use DMEM medium containing 10% newborn calf serum for adherent culture. When the cell adhesion rate reaches about 80% to 90%, subculture at a ratio of 1:3 to 1:15. Culture conditions are 37°C, 5% CO2. Subculture the HEK293T cells that have been cultured adherently for 2 to 3 generations using DMEM medium containing 10% newborn calf serum.

[0034] 2. Take the adherent HEK293T cells in the logarithmic growth phase and culture them statically in Free style medium containing 2% newborn calf serum for 2 to 3 days. Collect the non-adherent HEK293T cells into a culture flask, stain the cells with a non-toxic live or dead dye, and sort out the live cells using a flow cytometer.

[0035] 3. The living cells separated in step 2 were cultured in Free style medium containing 0.5% newborn calf serum for 2 to 3 days, the non-adherent HEK293T cells were collected into a culture flask, the cells were stained with a non-toxic live or dead dye, and the living cells were separated by flow cytometry.

[0036] 4. The living cells sorted in step 3 were suspended and cultured in Free style medium containing 0.5% dextran sulfate until the cell density reached 1×10 6 ~1.5×10 6 When the concentration of 100 cells / mL was 400 μg / mL, the cells were stained with a non-toxic live or dead dye, live cells were sorted out using a flow cytometer, and the suspended cells were cultured using a cell culture shaker. The culture conditions were: 36.5°C, 8% CO2, 80% humidity, and 120 rpm.

[0037] 5. Use Free style medium containing 0.5% dextran sulfate to adaptively subculture the living cells sorted in step 4 for 2 to 3 generations, with a seeding density of 6×10 5 ~8×10 5 cells / mL, and wait until the cell density reaches 2×10 6 ~3×10 6 cells / mL for subculture.

[0038] 6. When the specific growth rate of cells in Free style medium containing 0.5% dextran sulfate reaches above 0.5 and the cell viability is greater than 90%, take the cells in the logarithmic growth phase and spread them on 96-well plates. After culturing for 5 to 7 days, observe the clones and select cells with good growth status and morphology to gradually expand them to shake flask culture. When the cell viability reaches 95-100%, the cells are well dispersed and have no obvious aggregation. The cells can tolerate high-density electroporation. After electroporation, the cells maintain a high viability and recover quickly. These are HEK293T cells that have been successfully domesticated in suspension and are named 293TF cells.

[0039] Phase II: Proliferation and expansion of suspension 293T cells and library construction

[0040] 1. The standard for successful suspension culture of HEK293TF cells: the cell density reaches 1.5×10 6~3.0×10 6 cells / mL, and then subculture and amplification, and the seeding density was maintained at 2×10 5 ~8×10 5 cells / mL, subcultured every 3-4 days.

[0041] 2. Culture conditions: temperature 36.5℃, humidity 80%, carbon dioxide 8%, rotation speed 120rpm.

[0042] 3. When the cell density reaches 1.5×10 6 ~3.0×10 6 When the cell density was 2 × 10 cells / mL, the cells were collected by centrifugation at 1500 rpm for 10 min, resuspended in freezing solution, sampled, and counted. The cell density was adjusted to 2 × 10 cells / mL using freezing solution. 7 cells / mL, and divided into 1 mL per cryovial. The cryovials were placed in a programmed cooling box at -80°C overnight and transferred to liquid nitrogen for long-term storage the next day.

[0043] The culture medium used for proliferation and expansion was Free style medium containing 0.5% dextran sulfate and no serum.

[0044] The second aspect of the present invention provides 293TF cells that have been successfully acclimated in serum-free suspension and have high transfection efficiency, and the main characteristics thereof are:

[0045] 1. The cell viability is 95-100%, and the cells are well dispersed without obvious aggregation. The selected 293TF cells with high transfection efficiency are expanded and cultured and a library is established.

[0046] 2. When the CO2 concentration is 6% to 8%, the temperature is 36.5℃ to 37℃, the humidity is 70% to 80%, and the shaking speed is 100 to 120rpm, the cells can grow at a rate higher than 1.3×10 7 cells / mL, thus having a higher protein expression level.

[0047] 3. The cells can tolerate high-density electroporation. After electroporation, the cells maintain a high viability and recover quickly.

[0048] 4. Compared with adherent HEK293T cells, the positive rate of CAR-T cells obtained by lentivirus-transfected T cells prepared by suspended 293TF cells was significantly improved.

[0049] In the third aspect of the present invention, the 293TF cells provided in this study can be widely used in the fields of virus transfection, protein expression, etc.:

[0050] 1. Resuscitate the frozen suspended 293TF cells, resuspend them in Free style medium containing 0.5% dextran sulfate, and transfer them to a 125 mL shake flask for culture. Culture conditions: temperature 36.5°C, humidity 80%, carbon dioxide 8%, rotation speed 120 rpm.

[0051] 2. When the cell density reaches 1.5×10 6 ~3.0×10 6 cells / mL, the lentiviral transfer plasmid and packaging plasmid were co-transfected, and after suspension culture for 96 hours, the solution was collected and the virus was concentrated. After testing, the lentiviral titer was 1×10 8 TU / mL, which was significantly higher than the lentivirus titer prepared by adherent HEK293T cells.

[0052] 3. Add the concentrated lentivirus to T cells, and after culturing for 5-6 days, test the positive rate of virus transfection in T cells, which can reach more than 95%, which is about 50% higher than the positive rate of lentivirus transfection prepared by adherent HEK293T cells.

[0053] The present invention adds monoclonal screening technology and more efficient and precise flow cytometry sorting technology during the suspension process, which can quickly and accurately screen out suspended cells with less damage to the cells, and can quickly obtain suspended cells with better activity and higher transfection efficiency.

[0054] In the preparation method provided by the present invention, in the steps of domestication and subculture, the serum content in the culture medium of each generation is scientifically and rationally controlled, and living cells are sorted out by flow cytometer. The advantages of cell sorting by flow cytometer are as follows: 1. High sorting accuracy (more than 99%) and fast speed; 2. Flow cytometer can separate cells according to any detectable luminescence or fluorescence scattering difference, and the sorted cells can also be cultured.

[0055] The serum-free culture medium can be a Free style expression medium, which is a non-animal-derived, chemically defined protein-free culture medium that supports the growth and transfection of 293 cells under suspension culture conditions, and can significantly save the time and cost used to adapt cells to serum-free conditions.

[0056] The anticoagulant dextran sulfate is a polyanionic derivative of dextran. Its negative charge will change the surface charge of the cell, thereby maintaining the single cell morphology culture. The addition of dextran sulfate can reduce cell aggregation, promote cell growth, and increase cell viability, so that the suspension culture characteristics of the cell are enhanced, and a stable serum-free suspension culture type 293TF cell is formed at a faster rate and the acclimation time of the entire preparation method is effectively shortened. The dextran sulfate is added to the serum-free culture medium at a volume ratio of 0.2 to 0.5:100. That is, 0.2 to 0.5 mL of dextran sulfate is added to every 100 mL of culture medium.

[0057] Through the monoclonal selection step, cells with better proliferation rate, cell morphology and shear resistance can be separated. Through selection, serum-free suspension culture 293TF cells with fast growth rate, good passage stability and high virus packaging efficiency can be obtained.

[0058] The present invention uses adherent culture type HEK293T cells as the initial raw material for domestication. The provided serum-free suspension culture type 293TF cells do not need to add serum with unclear components during the culture process, which greatly reduces the cell culture cost and the risk of clinical application. In particular, it has the characteristics of fast growth rate, good passage stability, high lentivirus packaging efficiency (up to 95%), etc., and is particularly suitable for large-scale culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is the viability of 293TF cells.

[0060] Figure 2 is the specific growth rate of 293TF cells.

[0061] Figure 3 293TF cells were transfected with green fluorescent protein.

[0062] Figure 4 is the CAR-T cell positivity rate (left: CAR-T positivity rate obtained by preparing virus-infected T cells with 293T cells; right: CAR-T positivity rate obtained by preparing virus-infected T cells with 293TF cells). DETAILED DESCRIPTION

[0063] The following examples are intended to further illustrate the present invention but should not be construed as limiting the present invention.

[0064] Example 1

[0065] The reagents and instruments involved in this patent are as follows:

[0066] HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-502, and were expanded and preserved by our laboratory. High-glucose DMEM, trypsin, and DPBS were purchased from Hyclone; newborn calf serum was purchased from Lanzhou Rongye; FreeStyle medium and GlutaMAX-I were purchased from Gibco; dextran sulfate was purchased from Sigma; and flow cytometry reagents were purchased from Biolegend.

[0067] The brand and model of the biological safety cabinet is Haier HR1200-ⅡA2; the brand and model of the carbon dioxide incubator is Thermo4111; the brand and model of the cell culture shaker is Conai ISF-1-W; the brand and model of the cell counter is Thermo COUNTESS; the brand and model of the flow sorter is Beckman CytoFLEX; the brand and model of the desktop refrigerated centrifuge is Thermo ST 16R; the brand and model of the inverted microscope is OLYMPUS CKX41; the brand and model of the fluorescence microscope is Thermo EVOS ® FL Auto.

[0068] The preparation method of serum-free suspension culture type 293T cells provided in this embodiment comprises the following steps:

[0069] 1. Recovery and culture

[0070] HEK293T cells were frozen and revived in a 37°C water bath. 6 ~5×10 6 The cells were inoculated into a T75 cell culture flask containing DMEM medium containing 10% newborn calf serum at a density of 1:3 cells / mL. The cells were cultured at 37°C and 5% CO2 for 48-72 hours. When the cell confluence reached about 80% to 90% and the cell edges were clear and epithelial-like, the cells were subcultured at a ratio of 1:3 to 1:15.

[0071] 2. Acclimation and cultivation

[0072] (1) Normally growing adherent HEK293T cells were subcultured for 2 to 3 generations using DMEM medium containing 10% newborn calf serum.

[0073] (2) Take 3×10 HEK293T cells in the logarithmic growth phase in the acclimation step (1) 6 ~5×10 6 Inoculate 10-15 mL of Free style medium containing 2% newborn calf serum and culture for 2 to 3 days. Collect HEK293T cells that have not adhered to the wall, wash once with PBS, and add 1×10 6The cells were stained with the fluorescent dye Zombie Aqua at a dilution ratio of 1:1000, and live cells were sorted under the 405nm excitation of the flow cytometer.

[0074] (3) The living cells separated in step (2) were cultured in a Free style medium containing 0.5% newborn calf serum for 2 to 3 days, and the non-adherent HEK293T cells were further separated by flow cytometry according to the separation method in step (2).

[0075] (4) The living cells separated in step (3) were suspended and cultured in Free style medium containing 0.5% dextran sulfate. The culture conditions were: 36.5°C, 8% CO2, 80% humidity, and 120 rpm. The cells were grown to a density of 1×10 6 ~1.5×10 6 When the concentration of viable cells / mL is 1.0447 W / mL, continue to perform flow cytometry sorting to separate live cells according to the method in step (2), and culture the sorted cells on a shaker under the same culture conditions as described above.

[0076] (5) Adaptively subculture the living cells isolated in step (4) for 2 to 3 generations using Free style medium containing 0.5% dextran sulfate, with a seeding density of 6 × 10 5 ~8×10 5 cells / mL, and wait until the cell density reaches 2×10 6 ~3×10 6 cells / mL for subculture.

[0077] (6) When the specific growth rate of cells in Free style medium containing 0.5% dextran sulfate reaches above 0.5 and the cell viability is greater than 90%, cells in the logarithmic growth phase are selected for monoclonal screening. The monoclonal screening is carried out by limiting dilution method at 5-20 cells / mL inoculated in 96-well cell culture plates, with 100 μl of cell suspension per well. Then, 100 μl of Freestyle medium is added to each well. After culturing for 5-7 days, clones are observed and cells with good proliferation rate, cell morphology and shear resistance are selected and gradually expanded to shake flask culture.

[0078] (7) When the cell viability of the cells selected in step (6) reaches 95% to 100%, and the cells are well dispersed and have no obvious aggregation, they are HEK293T cells that have been successfully suspended and domesticated, and are named 293TF cells.

[0079] 3. Proliferation, expansion, culture and cryopreservation

[0080] The successfully suspended 293TF cells were further cultured, expanded, and cryopreserved to build a library.

[0081] (1) Standard for successful suspension culture of 293TF cells: cell density reaches 1.5×10 6 ~3.0×10 6 cells / mL, and then subculture and amplification, and the seeding density was maintained at 2×10 5 ~8×10 5 cells / mL, subcultured every 3 to 4 days.

[0082] (2) Culture conditions: temperature 36.5°C, humidity 80%, carbon dioxide 8%, rotation speed 120 rpm.

[0083] (3) When the cell density reaches 1.5×10 6 ~3.0×10 6 When the cell density was 2 × 10 cells / mL, the cells were collected by centrifugation at 1500 rpm for 10 min, resuspended in freezing solution, sampled, and counted. The cell density was adjusted to 2 × 10 cells / mL using freezing solution. 7 cells / mL, aliquoted, 1 mL per cryovial, placed in a program cooling box, -80°C overnight, and transferred to liquid nitrogen for long-term storage the next day. The proliferation and expansion library construction medium used was Free style medium containing 0.5% dextran sulfate, without serum.

[0084] Cell freezing medium for library construction: Free style medium containing 10% DMSO

[0085] Example 2

[0086] This example provides a method for investigating the stability of passage and lentiviral transfection rate of successfully acclimated serum-free suspension cultured 293TF cells, comprising the following steps:

[0087] 1. Stability of cell culture

[0088] The 293TF cells obtained in step 3 (1) of Example 1 were selected and cultured in Free style medium. Culture conditions: temperature 36.5°C, humidity 80%, carbon dioxide 8%, rotation speed 120 rpm. The cell density reached 1.5×10 6 ~3.0×10 6 cells / mL, and the density was maintained at 2×10 5 ~8×10 5 cells / mL, and subcultured every 3-4 days. The cell density and cell viability were tested at each subculture and the cell specific growth rate was calculated. The results showed that the cell viability was above 95% and the specific growth rate was above 0.5. Figure 1 and Figure 2 .

[0089] 2. Evaluation of the effect of electroporation of green fluorescent protein

[0090] The collection density is 1×10 7 ~2×10 7 At least 1 mL of 293TF cells were suspended at 100 cells / mL and electroporated with green fluorescent protein. After 24 to 48 hours of electroporation, the cells were observed under an inverted fluorescence microscope. 293TF cells showed that they could tolerate high-density electroporation, and the cells maintained a high viability after electroporation, and the cell state recovered quickly. The results are shown in Figure 3 .

[0091] 3. Investigation of the transfection effect of lentivirus in 293TF cells

[0092] (1) Thaw the frozen 293TF cells, resuspend them in Free style medium containing 0.5% dextran sulfate, and transfer them to a 125 mL shake flask for culture. Culture conditions: temperature 36.5°C, humidity 80%, carbon dioxide 8%, and rotation speed 120 rpm.

[0093] (2) Preparation of transfection solution:

[0094]

[0095] When the cell density reached 1.5×10 6 ~3.0×10 6 When the transfection solution was 1×10 cells / mL, a certain proportion of transfection solution was gently added to 293TF cells to co-transfect the lentiviral transfer plasmid and packaging plasmid. After suspension culture for 96 hours, the solution was collected and the virus was concentrated. After testing, the lentiviral titer was 1×10 8 TU / mL, which is higher than the lentiviral titer prepared by adherent HEK293T cells (1×10 7 TU / mL).

[0096] (3) The concentrated lentivirus was added to T cells. After 5-6 days of culture, the virus transfection positive rate of T cells was tested. The transfection positive rate was over 95%, which was about 30% higher than that of lentivirus transfection prepared by adherent HEK293T cells. The results are shown in Figure 4 .

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for acclimating suspension cells without serum, characterized in that: The following steps are involved: 1) reviving adherent cells and subculturing them; taking cells in logarithmic growth phase and using serum-free medium and adding serum for static culture, collecting non-adherent cells into culture bottles, staining the cells with a non-toxic live or dead dye, and sorting live cells by flow cytometry; using serum-free medium and adding serum of decreasing concentration for static culture, and continuing to sort non-adherent cells by flow cytometry according to the above sorting method; the concentration of the serum does not exceed 2.5% by volume; 2) The living cells isolated above were suspended and cultured in a serum-free medium containing 0.5% dextran sulfate until the cell density reached 1×10 6 ~1.5×10 6 cells / mL, continue to perform flow cytometry sorting on the cells according to the method in step 1) to sort out the living cells, culture the sorted cells on a shaker, and then perform adaptive subculture; 3) When the specific growth rate of the cells reaches above 0.5 in a serum-free medium containing 0.5% dextran sulfate and the cell viability is greater than 90%, take the cells in the logarithmic growth phase and spread them on a 96-well plate, select the cells with good growth status and morphology and gradually expand them to shake flask culture; when the viability of the selected cells reaches 95-100%, the cells are well dispersed and there is no obvious aggregation, they are the cells that have been successfully suspended.

2. The preparation method according to claim 1, characterized in that The following steps are involved: (1) Resuscitate adherent HEK293T cells and culture them in DMEM high-glucose medium containing 10% newborn calf serum. Subculture when the cell confluence reaches about 80% to 90%; (2) Acclimation steps 1) Subculture the adherent HEK293T cells for 2 to 3 generations using DMEM high-glucose medium containing 10% newborn calf serum; 2) The HEK293T cells in the logarithmic growth phase of step 1) were cultured statically for 2 to 3 days in a Free style medium containing 2% newborn calf serum, and the non-adherent HEK293T cells were collected into a culture flask, and the cells were stained with a non-toxic live or dead dye, and live cells were sorted out by flow cytometry; 3) The living cells separated in step 2) were cultured in a Free style medium containing 0.5% newborn calf serum for 2 to 3 days, and the non-adherent HEK293T cells were further separated by flow cytometry according to the separation method in step 2); 4) The living cells separated in step 3) were suspended and cultured in Free style medium containing 0.5% dextran sulfate until the cell density reached 1×10 6 ~1.5×10 6 cells / mL, continue to perform flow cytometry sorting on the cells according to the method in step 2) to sort out the living cells, and culture the sorted cells on a shaker; 5) Adaptively subculture the living cells sorted in step 4) using Free style medium containing 0.5% dextran sulfate; 6) When the specific growth rate of cells in Free style medium containing 0.5% dextran sulfate reaches above 0.5 and the cell viability is greater than 90%, take the cells in the logarithmic growth phase and spread them on 96-well plates, select cells with good growth status and morphology and gradually expand them to shake flask culture; 7) When the cell viability selected in step 6) reaches 95-100%, and the cells are well dispersed and have no obvious aggregation, the HEK293T cells are successfully suspended and domesticated.

3. The preparation method according to claim 2, characterized in that: The method also includes step (3) amplifying and freezing the successfully suspended HEK293T cells to build a library.

4. The preparation method according to claim 2, characterized in that: In step (1), the culture conditions are 37°C, 5% CO2, and the culture time is 48 to 72 hours.

5. The preparation method according to claim 2, characterized in that: In step (2) 4), the suspension culture conditions and the shaking culture conditions are 36.5°C, 8% CO2, 80% humidity, and 120 rpm.

6. The preparation method according to claim 2, characterized in that: The non-toxic live and dead dye was used at a rate of 1×10 6 cells were diluted with the fluorescent dye Zombie Aqua at a ratio of 1:1000.

7. The preparation method according to claim 2, characterized in that: In step (2) 5), adaptive subculture was performed for 2 to 3 generations with an inoculation density of 6 × 10 5 ~8×10 5 cells / mL, and wait until the cell density reaches 2×10 6 ~3×10 6 cells / mL for subculture.

8. The preparation method according to claim 3, characterized in that: In step (3), cell subculture expansion method: when the cell density reaches 1.5×10 6 ~3.0×10 6 cells / mL, and the density was maintained at 2×10 5 ~8×10 5 cells / mL, subcultured every 3 to 4 days.

9. The cell line obtained according to the preparation method according to any one of claims 1 to 8.

10. The use of the cell line according to claim 9, characterized in that: The applications include virus transfection and protein expression.

Images