Reagent for reducing clotting rate of suspended HEK293F cells, culture method and culture medium

Abstract

The invention discloses a reagent for reducing the agglomeration rate of suspended HEK293F cells, a culture method and a culture medium. The reagent comprises the following components: 0.01-0.1% (v / v) of a nonionic surfactant, 0.05-0.2% (v / v) of polysaccharide or a derivative thereof, 1-5mM of L-glutamine or a derivative thereof, 0.001-0.01 mM of FeCl3, 14-16mM of HEPES and 50-100mu M of an RGD peptide inhibitor. According to the anti-caking agent, the nonionic surfactant, the polysaccharide or the polysaccharide derivative, the L-glutamine or the L-glutamine derivative, the FeCl3, the HEPES and the RGD peptide inhibitor are optimally proportioned, so that the HEK293F cell caking rate is remarkably reduced to 30% or below, after the anti-caking agent is added, cells are in a uniform single-cell suspension state, macroscopic cell agglomerates do not exist, and the anti-caking effect is good. The uptake efficiency of cells on nutrient substances is obviously improved. Compared with a commercially available PEG (Polyethylene Glycol)-containing anti-caking agent, the anti-caking agent disclosed by the invention has the advantages that the anti-caking rate is lower than 35% (the anti-caking rate is only 30% vs after being cultured for 48 hours, and the anti-caking rate is 40%) while the cell viability is maintained to be 95% or above, and no cytotoxic accumulation exists after long-term passage (more than or equal to 20 generations).

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a reagent, a culture method and a culture medium for reducing the agglomeration rate of suspended HEK293F cells. Background Art

[0002] HEK293F cells, also known as human embryonic kidney 293 cells, are a cell line derived from human embryonic kidney cells. Due to their ease of transfection, high protein expression, and suitability for suspension culture, they are widely used in recombinant protein production, viral vector packaging (such as adenovirus and lentivirus), and gene function research. However, during suspension culture, 293F cell clumping seriously limits their effectiveness. The resulting cell clumps hinder nutrient and oxygen transfer, reduce cell viability, and increase downstream harvesting difficulties, resulting in reduced product yield and quality.

[0003] There are three traditional methods to reduce the aggregation rate of HEK293F cells. The first is enzymatic digestion, but enzymatic digestion can easily damage the cell membrane and affect cell activity. The second is physical dispersion, such as filtering cells through a sieve, but this requires repeated and tedious operations on the cells and can easily cause contamination of the cells. The third is to add some chemical additives, such as EDTA, PEG, and some surfactants, but these substances can inhibit cell growth and virus packaging, and serious residual substances may be challenged by some regulations or guidelines of regulatory agencies.

[0004] Therefore, there is an urgent need to develop reagents that can maintain cell growth viability and effectively inhibit HEK293F cell aggregation. Summary of the Invention

[0005] The main purpose of the present invention is to propose a reagent, culture method and culture medium for reducing the clumping rate of suspended HEK293F cells, aiming to provide a low-toxic, low-cost reagent that does not affect cell activity and significantly reduces the clumping rate of HEK293 suspension culture.

[0006] To achieve the above objectives, the present invention provides a reagent for reducing the clumping rate of suspended HEK293F cells, comprising the following components: 0.01-0.1% (v / v) nonionic surfactant, 0.05-0.2% (v / v) polysaccharide or its derivative, 1-5 mM L-glutamine or its derivative, 0.001-0.01 mM FeCl3, 14-16 mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), and 50-100 μM RGD peptide inhibitor, wherein v / v refers to volume percentage, that is, the ratio of the component volume to the total volume of the reaction system.

[0007] Preferably, the reagent further comprises hyaluronidase, and the concentration of the hyaluronidase is 0.4 to 0.6 U / mL.

[0008] Preferably, the RGD peptide inhibitor comprises cyclic RGDfK (arginine-glycine-aspartic acid-D-phenylalanine-lysine).

[0009] Preferably, the nonionic surfactant includes: any one of a block copolymer surfactant and a polyoxyethylene surfactant;

[0010] The block copolymer surfactant includes any one of the Pluronic series and the Tetronic series; the polyoxyethylene surfactant includes any one of polyethylene glycol derivatives, nonylphenol polyoxyethylene ether, and octylphenol polyoxyethylene ether;

[0011] The polysaccharide or its derivatives include: any one of cellulose and its derivatives (including hydroxyethyl cellulose, carboxymethyl cellulose), heparin, dextran, and hyaluronic acid.

[0012] Preferably, the Pluronic series includes any one of Pluronic F-68 and F127.

[0013] Preferably, the L-glutamine derivative includes any one of N-acetyl-L-glutamine, L-glutamine dipeptide, γ-glutamyl derivative, polyglutamine, and L-glutamine analogs.

[0014] The present invention also provides a culture method for reducing the aggregation rate of suspended HEK293F cells, wherein the reagent as described above is added when the suspended HEK293F cells are in the recovery period and the passage period.

[0015] Preferably, the reagents added during the recovery period are: 0.05% Pluronic F-68+0.001 mM FeCl3+0.1% hydroxyethyl cellulose+4 mM glutamine+15 mM HEPES+75 μM RGD peptide inhibitor.

[0016] Preferably, the reagents added during the passage period are: 0.05% Pluronic F-68 + 0.001 mM FeCl3 + 0.1% hydroxyethyl cellulose + 4 mM glutamine + 15 mM HEPES + 75 μM RGD peptide inhibitor.

[0017] Preferably, the culture method specifically comprises the following steps:

[0018] S1. Cell recovery: Thaw the frozen HEK293F cells, centrifuge, collect the precipitated cells, resuspend them for culture, transfer them to a shake flask, add the above reagents and hyaluronidase, treat for 12-18 minutes, then replace the medium to remove the hyaluronidase;

[0019] S2. Cell passaging: Take out the HEK293F cell suspension and count the cells. Calculate the volume of cell suspension and the volume of supplementary culture medium required for passaging. Add the reagents described above to the passaged cells and culture them. Take samples and count them every 24 hours, and calculate the clumping rate.

[0020] The present invention also provides a culture medium for reducing the aggregation rate of suspended HEK293F cells, wherein the culture medium comprises the reagent as described above.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The anti-agglomeration agent provided by the present invention significantly reduces the HEK293F cell agglomeration rate to less than 30% by optimizing the ratio of non-ionic surfactant, polysaccharide or its derivative, L-glutamine or its derivative, FeCl3, HEPES, and RGD peptide inhibitor. After adding the anti-agglomeration agent of the present invention, the cells are in a uniform single-cell suspension state, without visible cell clumps, and significantly improve the cell's efficiency in nutrient uptake. The formula first reduces the surface tension of the culture medium by a non-ionic surfactant, and the polysaccharide derivative forms a protective hydration film, jointly constructing a physical barrier from two aspects to inhibit cell aggregation; the RGD peptide inhibitor biochemically blocks integrin-mediated cell adhesion, and hyaluronidase degrades the extracellular matrix, inhibiting agglomeration from two independent targets. The physical barrier and biochemical blocking mechanisms formed complement each other to improve the anti-agglomeration effect; finally, glutamine or its derivative, HEPES and FeCl3 jointly maintain the stability of cell metabolism, pH and trace element environment, ensure that the cells are in good condition, and reduce abnormal adhesion of cells due to metabolic disorders or environmental pressure. Compared with commercially available PEG-containing anti-agglomerating agents, the present invention maintains a cell viability of over 95% while achieving an agglomeration rate of less than 35% (the agglomeration rate after 48 hours of culture is only 30% vs. 40% for commercial products), and exhibits no cytotoxicity accumulation during long-term passage (≥20 generations), providing a more stable cell state for viral vector production (such as adenovirus and lentivirus) and recombinant protein expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is a microscope image of HEK293F cells after 48 hours of culture.

[0025] Figure 2This is a comparison chart of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 2 after culturing for 24 and 48 hours.

[0026] Figure 3 This is a comparison chart of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 3 after culturing for 24 and 48 hours in the present invention.

[0027] Figure 4 This is a comparison chart of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 4 after culturing for 24 and 48 hours.

[0028] Figure 5 This is a comparison chart of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 5 after culturing for 24 and 48 hours in the present invention.

[0029] Figure 6 This is a comparison chart of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 6 after culturing for 24 and 48 hours in the present invention.

[0030] Figure 7 This is a comparison chart of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 7 after culturing for 24 and 48 hours in the present invention.

[0031] Figure 8 This is a comparison chart of the HEK293F cell viability in Application Example 1 and Application Example 7 after culturing for 24 and 48 hours in the present invention.

[0032] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0035] Example 1

[0036] A reagent for reducing the clumping rate of suspended HEK293F cells, comprising the following components:

[0037] Poloxamer 188 0.05% (v / v), hydroxyethylcellulose 0.1% (v / v), L-glutamine 4 mM, 0.001 mM FeCl 3 , HEPES 15 mM, cyclic RGDfK 75 μM.

[0038] Example 2

[0039] A reagent for reducing the clumping rate of suspended HEK293F cells, comprising the following components:

[0040] Poloxamer 188 0.01% (v / v), hydroxyethylcellulose 0.05% (v / v), L-glutamine 1 mM, 0.005 mM FeCl 3 , HEPES 14 mM, cyclo-RGDfK 50 μM.

[0041] Example 3

[0042] A reagent for reducing the clumping rate of suspended HEK293F cells, comprising the following components:

[0043] Poloxamer 188 0.1% (v / v), hydroxyethylcellulose 0.2% (v / v), L-glutamine 5 mM, 0.01 mM FeCl 3 , HEPES 15 mM, cyclo-RGDfK 100 μM.

[0044] Comparative Example 1

[0045] A reagent for reducing the aggregation rate of suspended HEK293F cells, comprising the following components: 0.1% (v / v) hydroxyethyl cellulose, 4 mM L-glutamine, 0.001 mM FeCl3, 15 mM HEPES, and 75 μM cyclic RGDfK.

[0046] Comparative Example 2

[0047] A reagent for reducing the aggregation rate of suspended HEK293F cells, comprising the following components: poloxamer 188 0.05% (v / v), L-glutamine 4mM, 0.001mM FeCl3, HEPES 15mM, and cyclic RGDfK 75μM.

[0048] Comparative Example 3

[0049] A reagent for reducing the clumping rate of suspended HEK293F cells comprises the following components: poloxamer 188 0.05% (v / v), hydroxyethyl cellulose 0.1% (v / v), L-glutamine 4mM, 0.001mM FeCl3, and HEPES 15mM.

[0050] Comparative Example 4

[0051] A reagent for reducing the aggregation rate of suspended HEK293F cells, comprising the following components: poloxamer 188 0.05% (v / v), hydroxyethyl cellulose 0.1% (v / v), L-glutamine 4mM, 0.001mM FeCl3, and cyclo-RGDfK 75μM.

[0052] Comparative Example 5

[0053] A reagent for reducing the aggregation rate of suspended HEK293F cells, comprising the following components: poloxamer 188 0.05% (v / v), hydroxyethyl cellulose 0.1% (v / v), L-glutamine 4mM, HEPES 15mM, and cyclic RGDfK 75μM.

[0054] Application Example 1

[0055] 1. Experimental Materials

[0056] Basal medium: transpro CD1 plus + 4mM L-glutamine

[0057] 2. A method for culturing suspension HEK293F cells, comprising the following steps:

[0058] S1. Cell recovery

[0059] (1) Remove the cryovial containing HEK293F cells and immediately thaw it in a 37°C water bath. Gently shake the cryovial to accelerate thawing until the cryosol is almost completely melted.

[0060] (2) transferring the cell suspension in the cryopreservation tube into a centrifuge tube containing basal culture medium, then centrifuging and discarding the supernatant to obtain a cell pellet; wherein the volume of the culture medium is 5 times the volume of the cell suspension;

[0061] (3) Add 8 mL of preheated basal medium to the centrifuge tube, gently pipette to resuspend the cells, and use an automatic cell counter to measure the cell density and viability. Adjust the cell density to the appropriate inoculation concentration (0.4-1×10 6 cells / mL);

[0062] (4) The cell suspension was transferred to a shake flask, preheated to 37°C, and basal medium was added. At the same time, the reagent for reducing the aggregation rate of suspended HEK293F cells described in Example 1 was added. The suspension was cultured at 120 rpm, 8% CO2, and 37°C.

[0063] S2. Cell Passaging

[0064] (5) Take out 0.1 mL of HEK293F cell suspension from the above shake flask and count the cells. When the viable cell density is 2-4×10 6 cells / mL, the cells were passaged, and the cell seeding density during passage was 3-5×10 5 cells / mL, calculate the volume of cell suspension and the volume of supplementary basal medium required for subculture, and simultaneously add the reagent for reducing the clumping rate of suspended HEK293F cells described in Example 1 to the subcultured cells. Incubate at 120 rpm, 8% CO2, and 37°C, take samples and count them every 24 h, and calculate the clumping rate and cell viability.

[0065] Application Example 2

[0066] 1. Experimental Materials

[0067] Basal medium: transpro CD1 plus + 4mM L-glutamine

[0068] 2. A method for culturing suspension HEK293F cells, comprising the following steps:

[0069] S1. Cell recovery

[0070] (1) Remove the cryovial containing HEK293F cells and immediately thaw it in a 37°C water bath. Gently shake the cryovial to accelerate thawing until the cryosol is almost completely melted.

[0071] (2) transferring the cell suspension in the cryopreservation tube into a centrifuge tube containing basal culture medium, then centrifuging and discarding the supernatant to obtain a cell pellet; wherein the volume of the culture medium is 5 times the volume of the cell suspension;

[0072] (3) Add 8 mL of preheated basal medium to the centrifuge tube, gently pipette to resuspend the cells, and use an automatic cell counter to measure the cell density and viability. Adjust the cell density to the appropriate inoculation concentration (0.4-1×10 6 cells / mL);

[0073] (4) The cell suspension was transferred to a shake flask, preheated to 37°C, and basal culture medium was added. At the same time, the reagent for reducing the aggregation rate of suspended HEK293F cells in Comparative Example 1 was added, and cultured at 120 rpm, 8% CO2, and 37°C;

[0074] S2. Cell Passaging

[0075] (5) Take out 0.1 mL of HEK293F cell suspension from the above shake flask and count the cells. When the viable cell density is 2-4×10 6 cells / mL, the cells were passaged, and the cell seeding density during passage was 3-5×10 5 cells / mL, calculate the volume of cell suspension and the volume of supplementary culture medium required for subculture, and at the same time add the reagent for reducing the clumping rate of suspended HEK293F cells in Comparative Example 1 to the subcultured cells. Incubate at 120 rpm, 8% CO2, and 37°C, take samples and count them every 24 h, and calculate the clumping rate.

[0076] Application Example 3

[0077] 1. Experimental Materials

[0078] Basal medium: transpro CD1 plus + 4mM L-glutamine

[0079] 2. A method for culturing suspension HEK293F cells, comprising the following steps:

[0080] S1. Cell recovery

[0081] (1) Remove the cryovial containing HEK293F cells and immediately thaw it in a 37°C water bath. Gently shake the cryovial to accelerate thawing until the cryosol is almost completely melted.

[0082] (2) transferring the cell suspension in the cryopreservation tube into a centrifuge tube containing basal culture medium, then centrifuging and discarding the supernatant to obtain a cell pellet; wherein the volume of the culture medium is 5 times the volume of the cell suspension;

[0083] (3) Add 8 mL of preheated basal medium to the centrifuge tube, gently pipette to resuspend the cells, and use an automatic cell counter to measure the cell density and viability. Adjust the cell density to the appropriate inoculation concentration (0.4-1×10 6 cells / mL);

[0084] (4) The cell suspension was transferred to a shake flask, preheated to 37°C, and basal culture medium was added. At the same time, the reagent for reducing the aggregation rate of suspended HEK293F cells in Comparative Example 2 was added, and cultured at 120 rpm, 8% CO2, and 37°C;

[0085] S2. Cell Passaging

[0086] (5) Take out 0.1 mL of HEK293F cell suspension from the above shake flask and count the cells. When the viable cell density is 2-4×10 6cells / mL, the cells were passaged, and the cell seeding density during passage was 3-5×10 5 cells / mL, calculate the volume of cell suspension and the volume of supplementary culture medium required for subculture, and at the same time add the reagent for reducing the clumping rate of suspended HEK293F cells in Comparative Example 2 to the subcultured cells, culture at 120 rpm, 8% CO2, and 37°C, take samples and count them every 24 h, and calculate the clumping rate.

[0087] Application Example 4

[0088] 1. Experimental Materials

[0089] Basal medium: transpro CD1 plus + 4mM L-glutamine

[0090] 2. A method for culturing suspension HEK293F cells, comprising the following steps:

[0091] S1. Cell recovery

[0092] (1) Remove the cryovial containing HEK293F cells and immediately thaw it in a 37°C water bath. Gently shake the cryovial to accelerate thawing until the cryosol is almost completely melted.

[0093] (2) transferring the cell suspension in the cryopreservation tube into a centrifuge tube containing basal culture medium, then centrifuging and discarding the supernatant to obtain a cell pellet; wherein the volume of the culture medium is 5 times the volume of the cell suspension;

[0094] (3) Add 8 mL of preheated basal medium to the centrifuge tube, gently pipette to resuspend the cells, and use an automatic cell counter to measure the cell density and viability. Adjust the cell density to the appropriate inoculation concentration (0.4-1×10 6 cells / mL);

[0095] (4) The cell suspension was transferred to a shake flask, preheated to 37°C, and basal culture medium was added. At the same time, the reagent for reducing the aggregation rate of suspended HEK293F cells in Comparative Example 3 was added, and cultured at 120 rpm, 8% CO2, and 37°C;

[0096] S2. Cell Passaging

[0097] (5) Take out 0.1 mL of HEK293F cell suspension from the above shake flask and count the cells. When the viable cell density is 2-4×10 6 cells / mL, the cells were passaged, and the cell seeding density during passage was 3-5×10 5cells / mL, calculate the volume of cell suspension and the volume of supplementary culture medium required for subculture, and at the same time add the reagent for reducing the clumping rate of suspended HEK293F cells in Comparative Example 3 to the subcultured cells. Incubate at 120 rpm, 8% CO2, and 37°C, take samples and count them every 24 h, and calculate the clumping rate.

[0098] Application Example 5

[0099] 1. Experimental Materials

[0100] Basal medium: transpro CD1 plus + 4mM L-glutamine

[0101] 2. A method for culturing suspension HEK293F cells, comprising the following steps:

[0102] S1. Cell recovery

[0103] (1) Remove the cryovial containing HEK293F cells and immediately thaw it in a 37°C water bath. Gently shake the cryovial to accelerate thawing until the cryosol is almost completely melted.

[0104] (2) transferring the cell suspension in the cryopreservation tube into a centrifuge tube containing basal culture medium, then centrifuging and discarding the supernatant to obtain a cell pellet; wherein the volume of the culture medium is 5 times the volume of the cell suspension;

[0105] (3) Add 8 mL of preheated basal medium to the centrifuge tube, gently pipette to resuspend the cells, and use an automatic cell counter to measure the cell density and viability. Adjust the cell density to the appropriate inoculation concentration (0.4-1×10 6 cells / mL);

[0106] (4) The cell suspension was transferred to a shake flask, preheated to 37°C, and basal culture medium was added. At the same time, the reagent for reducing the aggregation rate of suspended HEK293F cells in Comparative Example 4 was added, and cultured at 120 rpm, 8% CO2, and 37°C;

[0107] S2. Cell Passaging

[0108] (5) Take out 0.1 mL of HEK293F cell suspension from the above shake flask and count the cells. When the viable cell density is 2-4×10 6 cells / mL, the cells were passaged, and the cell seeding density during passage was 3-5×10 5 cells / mL, calculate the volume of cell suspension and the volume of supplementary culture medium required for subculture, and at the same time add the reagent for reducing the clumping rate of suspended HEK293F cells of Comparative Example 4 to the subcultured cells. Incubate at 120 rpm, 8% CO2, and 37°C, take samples and count them every 24 h, and calculate the clumping rate.

[0109] Application Example 6

[0110] 1. Experimental Materials

[0111] Basal medium: transpro CD1 plus + 4mM L-glutamine

[0112] 2. A method for culturing suspension HEK293F cells, comprising the following steps:

[0113] S1. Cell recovery

[0114] (1) Remove the cryovial containing HEK293F cells and immediately thaw it in a 37°C water bath. Gently shake the cryovial to accelerate thawing until the cryosol is almost completely melted.

[0115] (2) transferring the cell suspension in the cryopreservation tube into a centrifuge tube containing basal culture medium, then centrifuging and discarding the supernatant to obtain a cell pellet; wherein the volume of the culture medium is 5 times the volume of the cell suspension;

[0116] (3) Add 8 mL of preheated basal medium to the centrifuge tube, gently pipette to resuspend the cells, and use an automatic cell counter to measure the cell density and viability. Adjust the cell density to the appropriate inoculation concentration (0.4-1×10 6 cells / mL);

[0117] (4) The cell suspension was transferred to a shake flask, preheated to 37°C, and basal culture medium was added. At the same time, the reagent for reducing the aggregation rate of suspended HEK293F cells in Comparative Example 5 was added, and the suspension was cultured at 120 rpm, 8% CO2, and 37°C.

[0118] S2. Cell Passaging

[0119] (5) Take out 0.1 mL of HEK293F cell suspension from the above shake flask and count the cells. When the viable cell density is 2-4×10 6 cells / mL, the cells were passaged, and the cell seeding density during passage was 3-5×10 5 cells / mL, calculate the volume of cell suspension and the volume of supplementary basal medium required for subculture, and at the same time add the reagent for reducing the clumping rate of suspended HEK293F cells of Comparative Example 5 to the subcultured cells. Incubate at 120 rpm, 8% CO2, and 37°C, take samples and count them every 24 h, and calculate the clumping rate.

[0120] Application Example 7

[0121] 1. Experimental Materials

[0122] Basal medium: transpro CD1 plus + 4mM L-glutamine

[0123] 2. A method for culturing suspension HEK293F cells, comprising the following steps:

[0124] S1. Cell recovery

[0125] (1) Remove the cryovial containing HEK293F cells and immediately thaw it in a 37°C water bath. Gently shake the cryovial to accelerate thawing until the cryosol is almost completely melted.

[0126] (2) transferring the cell suspension in the cryopreservation tube into a centrifuge tube containing basal culture medium, then centrifuging and discarding the supernatant to obtain a cell pellet; wherein the volume of the culture medium is 5 times the volume of the cell suspension;

[0127] (3) Add 8 mL of preheated basal medium to the centrifuge tube, gently pipette to resuspend the cells, and use an automatic cell counter to measure the cell density and viability. Adjust the cell density to the appropriate inoculation concentration (0.4-1×10 6 cells / mL);

[0128] (4) Transfer the cell suspension to a shake flask, preheat to 37°C, add basal medium, and add a commercial anti-agglomerating agent, and culture at 120 rpm, 8% CO2, and 37°C;

[0129] S2. Cell Passaging

[0130] (5) Take out 0.1 mL of HEK293F cell suspension from the above shake flask and count the cells. When the viable cell density is 2-4×10 6 cells / mL, the cells were passaged, and the cell seeding density during passage was 3-5×10 5 cells / mL, calculate the volume of cell suspension and supplementary culture medium required for subculturing, and add a commercial anti-agglomeration agent (gbico) to the subcultured cells. Incubate at 120 rpm, 8% CO2, and 37°C, take samples and count them every 24 h, and calculate the clumping rate and cell viability.

[0131] Figure 1 This is a microscope photo of the HEK293F cells in Application Example 1 of the present invention after 48 hours of culture. Figure 1 It can be seen that the cells are well dispersed and no clumping occurs.

[0132] Figure 2 This is a comparison of the HEK293F cell agglomeration rates in Application Example 1 and Application Example 2 after culturing for 24 and 48 hours. Figure 2It can be seen that after adding the reagent for reducing the clumping rate of suspended HEK293F cells of the present invention, the cell clumping rate was lower than 30%, while after adding the reagent in Comparative Example 1, the clumping rate was greater than 35%.

[0133] Figure 3 This is a comparison of the HEK293F cell agglomeration rates in Application Example 1 and Application Example 3 after culturing for 24 and 48 hours. Figure 3 It can be seen that after adding the reagent for reducing the clumping rate of suspended HEK293F cells of the present invention, the cell clumping rate is lower than 30%, while after adding the reagent in Comparative Example 2, the clumping rate is greater than 40%.

[0134] Figure 4 This is a comparison of the HEK293F cell agglomeration rates in Application Example 1 and Application Example 4 after culturing for 24 and 48 hours. Figure 4 It can be seen that after adding the reagent for reducing the clumping rate of suspended HEK293F cells of the present invention, the cell clumping rate is lower than 30%, while after adding the reagent in Comparative Example 3, the clumping rate is greater than 45%.

[0135] Figure 5 This is a comparison of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 5 after culturing for 24 and 48 hours. Figure 5 It can be seen that after adding the reagent for reducing the clumping rate of suspended HEK293F cells of the present invention, the cell clumping rate was lower than 30%, while after adding the reagent in Comparative Example 4, the clumping rate was greater than 35%.

[0136] Figure 6 This is a comparison of the aggregation rates of HEK293F cells in Application Example 1 and Application Example 6 after culturing for 24 and 48 hours. Figure 6 It can be seen that after adding the reagent for reducing the clumping rate of suspended HEK293F cells of the present invention, the cell clumping rate is lower than 30%, while after adding the reagent in Comparative Example 5, the clumping rate is greater than 40%.

[0137] Figure 7 and 8 The following are comparisons of the aggregation rate and viability of HEK293F cells in Application Example 1 and Application Example 7 after culturing for 24 and 48 hours, respectively. Figure 7 It can be seen that the cell agglomeration rate of the present invention is lower; while the cell dispersion in the group with commercial anti-agglomeration agent is poor, the agglomeration rate is higher, and the cell viability of the present invention is greater than that of the commercial group ( Figure 8 ).

[0138] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A reagent for reducing the clumping rate of suspended HEK293F cells, characterized in that: The reagent includes the following components: 0.01-0.1% (v / v) nonionic surfactant, 0.05-0.2% (v / v) polysaccharide or its derivative, 1-5mM L-glutamine or its derivative, 0.001-0.01mM FeCl3, 14-16mM HEPES, and 50-100μM RGD peptide inhibitor.

2. The reagent according to claim 1, characterized in that The reagent further comprises hyaluronidase, and the concentration of the hyaluronidase is 0.4-0.6 U / mL.

3. The reagent according to claim 1, characterized in that The RGD peptide inhibitors include cyclic RGDfK.

4. The reagent according to claim 1, characterized in that The nonionic surfactant includes any one of a block copolymer surfactant and a polyoxyethylene surfactant; The block copolymer surfactant includes any one of the Pluronic series and the Tetronic series; The polyoxyethylene surfactant includes any one of polyethylene glycol derivatives, nonylphenol polyoxyethylene ether, and octylphenol polyoxyethylene ether; The polysaccharide or its derivative includes any one of cellulose and its derivatives, heparin, dextran, and hyaluronic acid.

5. The reagent according to claim 1, characterized in that The L-glutamine derivatives include any one of N-acetyl-L-glutamine, L-glutamine dipeptide, γ-glutamyl derivatives, polyglutamine, and L-glutamine analogs.

6. A method for reducing the clumping rate of suspended HEK293F cells, characterized in that: The reagent according to any one of claims 1 to 5 is added to the HEK293F suspension cells during the recovery phase and the passage phase.

7. The culture method according to claim 6, characterized in that The reagents added during the recovery period were: 0.05% Pluronic F-68 + 0.001 mM FeCl3 + 0.1% hydroxyethyl cellulose + 4 mM glutamine + 15 mM HEPES + 75 μM RGD peptide inhibitor.

8. The culture method according to claim 6, characterized in that The reagents added during the passage period were: 0.05% Pluronic F-68 + 0.001 mM FeCl3 + 0.1% hydroxyethyl cellulose + 4 mM glutamine + 15 mM HEPES + 75 μM RGD peptide inhibitor.

9. The culture method according to claim 6, characterized in that The culture method specifically comprises the following steps: S1. Cell recovery: Thaw the frozen HEK293F cells, centrifuge, collect the precipitated cells, resuspend and culture, transfer to a shake flask for culture, add the reagent as described in claim 1, and add hyaluronidase, treat for 12 to 18 minutes, then replace the medium to remove the hyaluronidase; S2. Cell passaging: Take out the HEK293F cell suspension and count the cells. Calculate the volume of cell suspension and the volume of supplementary culture medium required for passaging. Add the reagent as described in claim 1 to the passaged cells and culture them. Take samples and count them every 24 hours, and calculate the clumping rate.

10. A culture medium for reducing the clumping rate of suspended HEK293F cells, characterized in that: The culture medium comprises the reagent according to any one of claims 1 to 5.

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