Cell cryopreservation solution for high-throughput cryopreservation of chinese hamster ovary cells

Abstract

Provided are a serum-free cryopreservation solution for high-throughput cryopreservation of Chinese hamster ovary cell samples and a method for using the cryopreservation solution for cryopreservation. The cryopreservation solution can ensure that recovered Chinese hamster ovary cells still have good biological activity after cryopreservation, and expression products thereof still have stable expression levels and good biological functions.

Description

Cell freezing medium for high-throughput cryopreservation of Chinese hamster ovary cells Technical Field

[0001] The present invention relates to the field of cell preservation, and in particular to a cryopreservation solution for high-throughput deep-low temperature preservation of Chinese hamster ovary cells and a cryopreservation method using the cryopreservation solution. Background Art

[0002] Cryopreservation is one of the primary methods for cell preservation. Using cryopreservation technology to store cells at deep temperatures in liquid nitrogen allows for long-term storage. Furthermore, high-quality cryopreservation ensures that cells retain the same pre-cryopreservation characteristics upon thawing, such as highly stable and consistent cell doubling times and the quantity and quality of expressed products. This provides the necessary support for the long-term stability of production and experimental work.

[0003] In the field of biopharmaceuticals, engineered cell lines are important raw materials for production and experiments. Chinese Hamster Ovary (CHO) cells are currently an important host cell line for protein production and have many advantages over other expression systems. An increasing number of pharmaceutical proteins have been efficiently expressed in CHO cells and commercialized. The effective preservation and culture of CHO cells play a vital role in the stability, continuity, reproducibility and controllability of production. Large-scale, standardized CHO cell banks with long-term storage stability can continuously provide companies with CHO cells that stably express target proteins. The storage stability of such cell banks mainly depends on the quality of cell freezing, among which the cell freezing medium is an important factor affecting the quality of cell freezing.

[0004] Currently, cell cryopreservation often uses cryopreservation fluid containing serum components for cell cryopreservation, and the number of cells frozen is generally around 20-50. For biopharmaceutical companies, the scale of a cell bank of 20-50 cells cannot meet their long-term stable and continuous production needs. At the same time, the cost of cell bank preparation and testing is high, especially the time period for external testing of cell banks is long. Therefore, there is still a demand in this field for high-throughput (for example, more than 200 cells) and high-quality cell cryopreservation technology.

[0005] The inventors of the present invention have conducted research on establishing a high-throughput standardized cell bank for Chinese hamster ovary cells. The core of the research is to provide a cell freezing solution for high-throughput cell freezing, and a method for freezing cells using this cell freezing solution.

[0006] Summary of the Invention

[0007] One aspect of the present application provides a serum-free freezing solution for freezing Chinese hamster ovary cells, comprising a serum-free culture medium, 5%-10% (v / v) DMSO, 0.05-0.1M trehalose, 5-10g / L human albumin, and 2-5g / L methylcellulose, wherein the serum-free culture medium is selected from CD CHO culture medium, CD OptiCHO culture medium, TM medium, Hycell CHO medium, S3CD CHO medium, and S4CHO medium.

[0008] In certain embodiments, the serum-free freezing solution comprises 7.5% (v / v) DMSO. In certain embodiments, the serum-free freezing solution comprises 10% (v / v) DMSO.

[0009] In certain embodiments, the serum-free freezing solution comprises 0.05 M trehalose. In certain embodiments, the concentration of trehalose is 0.1 M.

[0010] In certain embodiments, the serum-free freezing solution comprises 5 g / L human albumin. In certain embodiments, the serum-free freezing solution comprises 10 g / L human albumin. In certain embodiments, the human albumin contained in the serum-free freezing solution is obtained by recombinant expression. In certain embodiments, the human albumin contained in the serum-free freezing solution comprises a native human albumin sequence or a mutated human albumin sequence.

[0011] In certain embodiments, the serum-free freezing solution comprises 2 g / L methylcellulose. In certain embodiments, the serum-free freezing solution comprises 5 g / L methylcellulose.

[0012] In certain embodiments, the serum-free freezing medium comprises serum-free culture medium, 10% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin and 2 g / L methylcellulose.

[0013] In certain embodiments, the serum-free freezing medium comprises serum-free culture medium, 7.5% (v / v) DMSO, 0.1 M trehalose, 5 g / L human albumin and 2 g / L methylcellulose.

[0014] In certain embodiments, the Chinese hamster ovary cells of the present application are recombinant Chinese hamster ovary cells. In certain embodiments, the recombinant Chinese hamster ovary cells have been introduced with a nucleic acid encoding a target protein. In certain embodiments, the recombinant Chinese hamster ovary cells contain a vector with stably integrated nucleic acid encoding a target protein. In certain embodiments, the recombinant Chinese hamster ovary cells stably express the target protein.

[0015] In certain embodiments, the recombinant Chinese hamster ovary cells contain a nucleic acid encoding an anti-CD147 monoclonal antibody. In certain embodiments, the recombinant Chinese hamster ovary cells stably express the anti-CD147 monoclonal antibody. In certain embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:1 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:2, or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:11 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:12. In certain embodiments, the anti-CD147 monoclonal antibody is expressed in the Chinese hamster ovary cells at a level of 2.0 g / L.

[0016] Another aspect of the present application provides a method for cryopreserving Chinese hamster ovary cells, comprising cryopreserving the Chinese hamster ovary cells using the serum-free cryopreservation solution disclosed in the present application, mixing the Chinese hamster ovary cells with the cryopreservation solution to form a cell suspension, cooling the cell suspension to below -80°C within 90 minutes, and then transferring the cell suspension to liquid nitrogen for storage.

[0017] In some embodiments, the initial concentration of the cell suspension after the Chinese hamster ovary cells are mixed with the freezing solution is 1×10 7 –5×10 8 In some embodiments, the initial concentration of the cell suspension after the Chinese hamster ovary cells are mixed with the freezing solution is 5×10 7 cells / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG1 shows the results of the storage stability study of cells cryopreserved using the cryopreservation solutions of Experimental Examples 1-11 and 1-14. DETAILED DESCRIPTION

[0019] Although the present application will disclose various aspects and embodiments, it is obvious that, without departing from the subject matter and scope of the present application, those skilled in the art may make various equivalent changes and modifications to these aspects and embodiments. The various aspects and embodiments disclosed in this application are for illustrative purposes only and are not intended to be limiting. The true scope is represented by the appended claims. All publications, patents or patent applications cited in this application are incorporated herein by reference in their entirety. Unless otherwise stated, all scientific and technological terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs.

[0020] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "one or more" and "at least one" are used interchangeably in this application. It should also be noted that the terms "including," "comprising," and "having" are used interchangeably.

[0021] One aspect of the present application discloses a serum-free freezing solution for freezing Chinese hamster ovary cells, which comprises serum-free culture medium, 5%-10% (v / v) DMSO, 0.05-0.1M trehalose, 5-10g / L human albumin and 2-5g / L methylcellulose.

[0022] The "serum-free medium" used in this article is selected from CD CHO medium, CD OptiCHO TM CHO medium, Hycell CHO medium, S3CD CHO medium and S4CHO medium. The "serum-free medium" used in this article is a medium without animal-derived components, serum-free and chemically defined, suitable for the culture of Chinese hamster ovary cells. Specifically, CD CHO medium is suitable for the growth of Chinese hamster ovary cells and the expression of recombinant proteins in suspension culture. CD CHO medium can be purchased from Thermo Fisher Scientific (catalog number 12490003) or selected from other culture media with the same components. CD OptiCHO TM The medium provides high performance and yield of recombinant CHO cells. TM The culture medium can be purchased from Thermo Fisher Scientific (Cat. No. 12681011) or selected from other culture media with the same components. Hycell CHO medium is suitable for high-yield Chinese hamster ovary cell culture and recombinant protein production. Hycell CHO medium can be purchased from Hyclone (Cat. No. SH30933.04) or selected from other culture media with the same components. S3CD CHO medium and S4CHO medium are suitable for batch culture, fed-batch culture and perfusion culture of Chinese hamster ovary cells. S3CD CHO medium and S4CHO medium can both be purchased from Shanghai Duoning Biotechnology Co., Ltd., with product numbers M014-001 (or MNP014) and MT022-1L (or MNP022-001), respectively; they can also be selected from other culture media with the same components.

[0023] "DMSO" in this article refers to "dimethyl sulfoxide", whose molecular formula is (CH3)2SO. Trehalose is a non-reducing sugar composed of two glucose molecules with a 1,1-glycosidic bond.

[0024] "Human albumin" herein refers to human serum albumin (HSA). Human serum albumin is a soluble, single-chain protein that is the major protein in human blood. Human serum albumin is responsible for many cellular functions in the human body. In certain embodiments, the human albumin has a native human albumin sequence. In certain embodiments, the human albumin has a mutated human albumin sequence, such as a truncation (at the N-terminus, C-terminus, or both), a deletion, an addition, or a substitution. In certain embodiments, the serum-free cryopreservative herein comprises a functional fragment of human albumin. An exemplary amino acid sequence of human albumin is set forth in SEQ ID NO:19. In certain embodiments, the human albumin comprises an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% sequence identity to the amino acid sequence set forth in SEQ ID NO:19. In certain embodiments, human albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-23. In certain embodiments, human albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-23. In certain embodiments, human albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20. In certain embodiments, human albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21. In certain embodiments, human albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22. In certain embodiments, human albumin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23. In certain embodiments, the human albumin is obtained by recombinant expression. In certain embodiments, the human albumin is obtained by expression in plant cells. In certain embodiments, the human albumin is obtained by expression in transgenic rice cells. Human albumin can be purchased from Sigma-Aldrich, catalog number A9731.

[0025] Methylcellulose, also known as methyl ether of cellulose (MC), is a long-chain substituted cellulose with a molecular weight (average) ranging from 10,000 to 220,000 Da. Methylcellulose of different molecular weights has different viscosities. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 12-2,000 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 12-1,200 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 1,200-1,800 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 12-30 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 30-100 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 100-300 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 300-600 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 600-900 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 900-1,200 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 1,200-1,400 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 1,400-1,600 cp. In some embodiments, the viscosity of the methylcellulose added to the serum-free freezing solution is 1,600-1,800 cp. In some embodiments, methylcellulose can be purchased from Sigma-Aldrich, catalog number M0387.

[0026] In certain embodiments, the serum-free freezing solution of the present invention comprises serum-free culture medium, 5%-10% DMSO (v / v), 0.05-0.1 M trehalose, 5-10 g / L human albumin, and 2-5 g / L methylcellulose. Specifically, the serum-free freezing solution comprises:

[0027] (1) Serum-free culture medium solution selected from CD CHO culture medium, CD OptiCHO TM culture medium, Hycell CHO culture medium, S3CD CHO culture medium and S4CHO culture medium,

[0028] (2) 5%-10% (v / v) DMSO, wherein 5%-10% (v / v) means that the volume of DMSO used is 5%-10% of the total volume of the serum-free cryopreservation solution.

[0029] (3) 0.05-0.1M trehalose, that is, the concentration of trehalose in the serum-free freezing solution is 0.05-0.1mol / L,

[0030] (4) 5-10 g / L human albumin, i.e., the concentration of human albumin in serum-free cryopreservative solution is 5-10 g / L, and

[0031] (5) 2-5 g / L methylcellulose, i.e., the concentration of methylcellulose in the serum-free cryopreservation solution is 2-5 g / L. The total volume of the serum-free cryopreservation solution is the sum of the volumes of the components after addition.

[0032] In certain embodiments of the above embodiments, the serum-free cryopreservative comprises 7.5%-10% (v / v) DMSO. In certain embodiments of the above embodiments, the serum-free cryopreservative comprises 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% (v / v) DMSO. In certain embodiments, the serum-free cryopreservative comprises 7.5% (v / v) DMSO. In certain embodiments, the serum-free cryopreservative comprises 10% (v / v) DMSO.

[0033] In certain embodiments of the above embodiments, the serum-free freezing solution comprises 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, or 0.1 M trehalose. In certain embodiments, the serum-free freezing solution comprises 0.05 M trehalose. In certain embodiments, the serum-free freezing solution comprises 0.1 M trehalose.

[0034] In certain embodiments of the above embodiments, the serum-free cryopreservative comprises 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, or 10 g / L human albumin. In certain embodiments, the serum-free cryopreservative comprises 5 g / L human albumin. In certain embodiments, the serum-free cryopreservative comprises 10 g / L human albumin.

[0035] In certain embodiments of the above embodiments, the serum-free freezing solution comprises 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, or 5 g / L methylcellulose. In certain embodiments, the serum-free freezing solution comprises 2 g / L methylcellulose. In certain embodiments, the serum-free freezing solution comprises 5 g / L methylcellulose.

[0036] In certain embodiments of the aforementioned embodiments, the serum-free freezing solution comprises serum-free culture medium solution, 10% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin, and 2 g / L methylcellulose.

[0037] In certain embodiments of the aforementioned embodiments, the serum-free freezing solution comprises serum-free culture medium solution, 7.5% (v / v) DMSO, 0.1 M trehalose, 5 g / L recombinant human albumin, and 2 g / L methylcellulose.

[0038] Chinese hamster ovary cells are a type of mammalian cell that is easy to genetically manipulate and easy to culture and produce target proteins. In certain embodiments, the Chinese hamster ovary cells are CHO-K1 cells, CHO-S cells, or CHO-DG44 cells (CHO-DHFR cells). Recombinant Chinese hamster ovary cells expressing target proteins can be provided by genetic engineering methods. For example, they can be provided by the following steps: (1) constructing a vector comprising a nucleic acid sequence encoding the target protein; (2) introducing the aforementioned vector comprising a nucleic acid sequence encoding the target protein into Chinese hamster ovary cells (e.g., dihydrofolate reductase-deficient Chinese hamster ovary cells, i.e., CHO-DHFR cells) to produce recombinant Chinese hamster ovary cells; (3) screening one or more recombinant Chinese hamster ovary cells that stably express the target protein; (4) culturing the recombinant Chinese hamster ovary cells that can stably express the target protein obtained by screening to produce the target protein. The methods involved in the above steps of constructing and introducing vectors, screening and culturing recombinant cells or cell lines that stably express the target protein, and the vector elements used therein are well known to those skilled in the art, for example, various techniques described in molecular biology laboratory manuals, including but not limited to Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Laboratory Press) by Michael R. Green and Joseph Sambrook.

[0039] In certain embodiments, the serum-free freezing solution of the present invention is used to freeze recombinant Chinese hamster ovary cells. In certain embodiments, a nucleic acid encoding a target protein is introduced into the recombinant Chinese hamster ovary cells. In certain embodiments, the recombinant Chinese hamster ovary cells contain a vector with a stably integrated nucleic acid encoding a target protein. In certain embodiments, the recombinant Chinese hamster ovary cells stably express the target protein. In certain embodiments, the target protein is an antibody, such as a human monoclonal antibody, a humanized monoclonal antibody, a chimeric antibody, or an antigen-binding fragment of an antibody. The "antigen-binding fragment" of the antibody refers to an antibody fragment that retains the ability to specifically bind to an antigen, including, for example, Fab fragments, F(ab')2, Fd fragments (composed of VH and CH1 regions), and Fv fragments.

[0040] In certain embodiments, the recombinant Chinese hamster ovary cells are introduced with a nucleic acid encoding an anti-CD147 monoclonal antibody. In certain embodiments, the recombinant Chinese hamster ovary cells contain a stably integrated nucleic acid encoding an anti-CD147 monoclonal antibody. In certain embodiments, the recombinant Chinese hamster ovary cells express an anti-CD147 monoclonal antibody. The CD147 molecule is a family of transmembrane glycoproteins that are highly expressed on the surface of epithelial tumor cells, such as lung cancer, breast cancer, and liver cancer. Studies have shown that CD147 is also associated with malarial parasite invasion and viral infection. Anti-CD147 antibodies play an important role in the treatment of diseases such as tumors, malaria, and inflammation.

[0041] In certain of the aforementioned embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain variable region and a light chain variable region.

[0042] In certain embodiments of the above embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain CDR1 as shown in SEQ ID NO:3, a heavy chain CDR2 as shown in SEQ ID NO:4, a heavy chain CDR3 as shown in SEQ ID NO:5, a light chain CDR1 as shown in SEQ ID NO:6, a light chain CDR2 as shown in SEQ ID NO:7, and a light chain CDR3 as shown in SEQ ID NO:8.

[0043] In certain embodiments of the above embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.

[0044] In certain embodiments of the above embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO:9 and a light chain comprising the amino acid sequence shown in SEQ ID NO:10.

[0045] In certain embodiments of the above embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain CDR1 as shown in SEQ ID NO: 13, a heavy chain CDR2 as shown in SEQ ID NO: 14, a heavy chain CDR3 as shown in SEQ ID NO: 15, a light chain CDR1 as shown in SEQ ID NO: 16, a light chain CDR2 as shown in SEQ ID NO: 17, and a light chain CDR3 as shown in SEQ ID NO: 18.

[0046] In certain embodiments of the above embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:11 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:12.

[0047] Table 1 Amino acid sequence

[0048] In certain embodiments of the above embodiments, the expression level of the anti-CD147 antibody in the Chinese hamster ovary cells can reach 2.0 g / L to 3.8 g / L. In certain embodiments, the expression level of the anti-CD147 antibody in the Chinese hamster ovary cells can reach 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L or 3.8 g / L.

[0049] In certain embodiments, the expression level of the antibody in the Chinese hamster ovary cells does not decrease in the cells after freezing, or decreases by no more than 5%, 10%, 15%, or 20%. In certain embodiments, the expression level of the anti-CD147 antibody in the Chinese hamster ovary cells does not decrease in the cells after freezing. In certain embodiments, the expression level of the anti-CD147 antibody in the Chinese hamster ovary cells decreases by no more than 5% in the cells after freezing. In certain embodiments, the expression level of the anti-CD147 antibody in the Chinese hamster ovary cells decreases by no more than 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4.0%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3.0%, 2%, or 1% in the cells after freezing.

[0050] In certain embodiments, the expression level of the anti-CD147 antibody in the Chinese hamster ovary cells after cell cryopreservation can reach 2.0 g / L to 3.8 g / L. In certain embodiments, the expression level of the anti-CD147 antibody in the Chinese hamster ovary cells after cell cryopreservation can reach 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L or 3.8 g / L.

[0051] In some embodiments, the serum-free cryopreservation solution in the present application is particularly suitable for high-throughput deep-low temperature preservation of CHO cells. In the process of laboratory and industrial cell freezing, the sedimentation of cells in the cryopreservation solution limits the number of cryopreservation tubes that can be frozen at one time, making it difficult to obtain a large-scale, highly consistent and long-term storage stable CHO cell bank. The serum-free cryopreservation solution in the present application, based on the combination and content of its added components, unexpectedly enables cells to be stably suspended in the cryopreservation solution for a long time (e.g., 1, 1.5, 2, 2.5, 3 hours), so as to allow large-scale cell freezing at one time. Specifically, cryopreservation solution can be provided for a CHO cell bank containing 200, 300, 400, 450, or 500 or even more cryopreservation tubes (2 mL / tube) at one time, thereby achieving high-throughput freezing. At the same time, the cryopreservation solution of the present application allows the initial freezing concentration of the frozen cell suspension (i.e., the cell suspension after the Chinese hamster ovary cells and the cryopreservation solution are mixed) to be greater than 1×10 7 cells / mL, for example, 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, 1×10 8 cells / mL, 2×10 8 cells / mL, 3×10 8 cells / mL, 4×10 8 cells / mL or 5×10 8 The initial freezing concentration of the cell suspension containing Chinese hamster ovary cells and the freezing solution of the present application is much higher than the initial concentration of the cell suspension frozen with the general freezing solution in the art.

[0052] Another aspect of the present application provides a method for cryopreserving Chinese hamster ovary cells, comprising cryopreserving the Chinese hamster ovary cells using the serum-free cryopreservation solution disclosed herein, mixing the Chinese hamster ovary cells with the cryopreservation solution to form a cell suspension, cooling the cell suspension to below -80°C within 90 minutes, and then transferring the cell suspension to liquid nitrogen for storage. The cell cryopreservation method provided herein precisely controls the changes in the cell suspension temperature and the ambient temperature by setting multiple temperature change steps (including the starting and ending temperatures of the temperature change, as well as the cooling rate), achieving strong controllability and fully protecting the cell performance from damage during the cryopreservation process.

[0053] In certain embodiments, the cell suspension is cooled from 2°C, or 3°C, or 5°C, or 6°C, or 7°C, or 8°C to below -80°C within 90 minutes. In certain embodiments, the cell suspension is cooled from 4°C to -80°C within 90 minutes. In certain embodiments, the cell suspension is cooled from 4°C to -90°C within 90 minutes. In certain embodiments, the cell suspension is cooled from 4°C to -100°C within 90 minutes. In certain embodiments, the cooling to below -80°C is completed within 90 minutes. In certain embodiments, the cooling to below -80°C is completed in 65-75 minutes. In certain embodiments, the cooling to below -80°C is completed in about 90 minutes, about 89 minutes, about 88 minutes, about 87 minutes, about 86 minutes, about 85 minutes, about 84 minutes, about 83 minutes, about 82 minutes, about 81 minutes, about 80 minutes, about 79 minutes, about 78 minutes, about 77 minutes, about 76 minutes, about 75 minutes, about 74 minutes, about 73 minutes, about 72 minutes, about 71 minutes, about 70 minutes, about 69 minutes, about 68 minutes, about 67 minutes, about 66 minutes, about 65 minutes, about 64 minutes, about 63 minutes, about 62 minutes, about 61 minutes, about 60 minutes, about 59 minutes, about 58 minutes, about 57 minutes, about 56 minutes, about 55 minutes, about 54 minutes, about 53 minutes, about 52 minutes, about 51 minutes, or about 50 minutes. In certain embodiments, the cell suspension is cooled from 4°C to -80°C within 70 minutes. In certain embodiments, the cell suspension is cooled from 4°C to -90°C within 70 minutes.

[0054] In some embodiments, the cell suspension is cooled by setting a temperature change step. In some embodiments, the cell suspension is subjected to a staged uniform temperature change by setting a temperature change step, so as to ultimately achieve the purpose of cooling. In some embodiments, the staged uniform temperature change includes a uniform cooling step and a uniform temperature recovery step. In some embodiments, the uniform temperature recovery step occurs after a rapid cooling step, and the rapid cooling step refers to cooling at a cooling rate greater than 10°C / minute. In some embodiments, the uniform temperature recovery step occurs after a rapid cooling step of cooling at a cooling rate of 20°C-80°C / minute. In some embodiments, the uniform temperature recovery step occurs after a rapid cooling step of cooling at a cooling rate of 30°C-80°C / minute. In some embodiments, the uniform temperature recovery step occurs after a rapid cooling step of cooling at a cooling rate of 40°C-80°C / minute. In some embodiments, the uniform temperature recovery step occurs after a rapid cooling step at a cooling rate of 40°C-60°C / min. In some embodiments, the uniform temperature recovery step occurs after a rapid cooling step at a cooling rate of 50°C-55°C / min.

[0055] In some embodiments, the staged uniform temperature change includes six stages: a first pre-cooling stage, a second cooling stage, a third cooling stage, a fourth warming stage, a fifth cooling stage, and a sixth cooling stage. Among them, in the first pre-cooling stage, the cryopreservation tube is placed at a first temperature of 2℃ to 8℃ for 30 to 60 minutes; in the second cooling stage, the cryopreservation tube is cooled from the first temperature to a second temperature of 0℃ to -18℃ at a cooling rate of 0.2℃-10℃ / minute; in the third cooling stage, the cryopreservation tube is quickly cooled from the second temperature to a third temperature of -60℃ to -90℃ at a cooling rate of 20℃-80℃ / minute; in the fourth warming stage, the cryopreservation tube is heated from the third temperature to a fourth temperature of -10℃ to -25℃ at a heating rate of 10℃-40℃ / minute; in the fifth cooling stage, the cryopreservation tube is cooled from the fourth temperature to a fifth temperature of -36℃ to -50℃ at a cooling rate of 0.2℃-10℃ / minute; in the sixth cooling stage, the cryopreservation tube is cooled from the fifth temperature to a sixth temperature below -80℃ at a cooling rate greater than 5℃ / minute.

[0056] In some embodiments, the first temperature is 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C. In some embodiments, the first temperature is 4°C. In some embodiments, the first pre-cooling phase lasts for 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. In some embodiments, the first pre-cooling phase lasts for 30 minutes.

[0057] In some embodiments, the second temperature is 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -8°C, -9°C, -10°C, -11°C, -12°C, -13°C, -14°C, -15°C, -16°C, -17°C, or -18°C. In some embodiments, the second temperature is -10°C. In some embodiments, the cooling rate of the second cooling stage is 0.2°C / min, 0.3°C / min, 0.4°C / min, 0.5°C / min, 0.6°C / min, 0.7°C / min, 0.8°C / min, 0.9°C / min, 1°C / min, 2°C / min, 3°C / min, 4°C / min, 5°C / min, 6°C / min, 7°C / min, 8°C / min, 9°C / min, or 10°C / min. In some embodiments, the cooling rate of the second cooling stage is 2°C / min.

[0058] In some embodiments, the third temperature is -60°C, -61°C, -62°C, -63°C, -64°C, -65°C, -66°C, -67°C, -68°C, -69°C, -70°C, -71°C, -72°C, -73°C, -74°C, -75°C, -76°C, -77°C, -78°C, -79°C, -80°C, -81°C, -82°C, -83°C, -84°C, -85°C, -86°C, -87°C, -88°C, -89°C or -90°C. In some embodiments, the third temperature is -80°C. In some embodiments, the cooling rate of the third cooling stage is 30°C-80°C / min. In some embodiments, the cooling rate of the third cooling stage is 40°C-80°C / min. In some embodiments, the cooling rate of the third cooling stage is 40°C-60°C / min. In some embodiments, the cooling rate in the third cooling stage is 41°C / min, 42°C / min, 43°C / min, 44°C / min, 45°C / min, 46°C / min, 47°C / min, 48°C / min, 49°C / min, 50°C / min, 51°C / min, 52°C / min, 53°C / min, 54°C / min, 55°C / min, 56°C / min, 57°C / min, 58°C / min, 59°C / min or 60°C / min. In some embodiments, the cooling rate in the third cooling stage is 52°C / min.

[0059] In some embodiments, the fourth temperature is -10°C, -11°C, -12°C, -13°C, -14°C, -15°C, -16°C, -17°C, -18°C, -19°C, -20°C, -21°C, -22°C, -23°C, -24°C, or -25°C. In some embodiments, the fourth temperature is -20°C. In some embodiments, the heating rate in the fourth temperature regeneration stage is 20°C-40°C / minute. In some embodiments, the heating rate in the fourth temperature regeneration stage is 25°C-35°C / minute. In some embodiments, the heating rate in the fourth temperature return stage is 25°C / min, 26°C / min, 27°C / min, 28°C / min, 29°C / min, 30°C / min, 31°C / min, 32°C / min, 33°C / min, 34°C / min, 35°C / min, 36°C / min, 37°C / min, 38°C / min, 39°C / min or 40°C / min. In some embodiments, the heating rate in the fourth temperature return stage is 30°C / min.

[0060] In some embodiments, the fifth temperature is -36°C, -37°C, -38°C, -39°C, -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C or -50°C. In some embodiments, the fifth temperature is -40°C. In some embodiments, the cooling rate of the fifth cooling stage is 0.2°C / min, 0.3°C / min, 0.4°C / min, 0.5°C / min, 0.6°C / min, 0.7°C / min, 0.8°C / min, 0.9°C / min, 1°C / min, 2°C / min, 3°C / min, 4°C / min, 5°C / min, 6°C / min, 7°C / min, 8°C / min, 9°C / min or 10°C / min. In some embodiments, the cooling rate of the fifth cooling stage is 1°C / min.

[0061] In some embodiments, the sixth temperature is -80°C, -81°C, -82°C, -83°C, -84°C, -85°C, -86°C, -87°C, -88°C, -89°C, -90°C, -91°C, -92°C, -93°C, -94°C, -95°C, -96°C, -97°C, -98°C, -99°C, -100°C or lower. In some embodiments, the sixth temperature is -80°C. In some embodiments, the sixth temperature is -90°C. In some embodiments, the cooling rate of the sixth cooling stage is 5°C-40°C / minute. In some embodiments, the cooling rate of the sixth cooling stage is 5°C-30°C / minute. In some embodiments, the cooling rate of the sixth cooling stage is 5°C / minute, 6°C / minute, 7°C / minute, 8°C / minute, 9°C / minute, 10°C / minute, 11°C / minute, 12°C / minute, 13°C / minute, 14°C / minute, 15°C / minute, 16°C / minute, 17°C / minute, 18°C / minute, 19°C / minute, 20°C / minute, 21°C / minute, 22°C / minute, 23°C / minute, 24°C / minute, 25°C / minute, 26°C / minute, 27°C / minute, 28°C / minute, 29°C / minute or 30°C / minute. In some embodiments, the cooling rate of the sixth cooling stage is 10°C / minute.

[0062] In certain embodiments, the staged uniform temperature change described in the present application includes the following 6 stages: a first pre-cooling stage, placing the cryotube at 4°C for 30 to 45 minutes; a second cooling stage, cooling the cryotube from 4°C to -10°C to -18°C at a cooling rate of 2°C / minute; a third cooling stage, rapidly cooling the cryotube from -10°C to -18°C in the previous stage to -80°C at a cooling rate of 40°C-60°C / minute; a fourth warming stage, heating the cryotube from -80°C to -20°C at a heating rate of 25°C-35°C / minute; a fifth cooling stage, cooling the cryotube from -20°C to -40°C at a cooling rate of 0.2°C-2°C / minute; and a sixth cooling stage, cooling the cryotube from -40°C to below -80°C at a cooling rate of 5°C-15°C / minute.

[0063] In certain embodiments, the staged uniform temperature change described in the present application includes the following 6 stages: a first pre-cooling stage, placing the cryotube at 4°C for 30 minutes; a second cooling stage, cooling the cryotube from 4°C to -10°C at a cooling rate of 2°C / minute; a third cooling stage, rapidly cooling the cryotube from -10°C to -80°C at a cooling rate of 52°C / minute; a fourth warming stage, heating the cryotube from -80°C to -20°C at a heating rate of 30°C / minute; a fifth cooling stage, cooling the cryotube from -20°C to -40°C at a cooling rate of 1°C / minute; and a sixth cooling stage, cooling the cryotube from -40°C to -90°C at a cooling rate of 10°C / minute.

[0064] The temperature change can be controlled using any method or instrument known in the art that can achieve the above-mentioned temperature change step. In certain embodiments, the temperature change is controlled using a programmed cooling device. The programmed cooling device can be purchased from Thermo Fisher Scientific, Model 7455.

[0065] In certain embodiments, the initial concentration of the cell suspension after Chinese hamster ovary cells are mixed with the freezing solution of the present application is 1×10 7 -1×10 8 cells / mL. In some embodiments, the initial concentration of the cell suspension is 5×10 7 cells / mL, 6×10 7 cells / mL, 7×10 7 cells / mL, 8×10 7 cells / mL, 9×10 7 cells / mL, 1×10 8 cells / mL, 2×10 8 cells / mL, 3×10 8 cells / mL, 4×10 8 cells / mL or 5×10 8 cells / mL. In some embodiments, the initial concentration of the cell suspension is 5×10 7 In some embodiments, the initial concentration of the cell suspension is 1×10 8 cells / mL.

[0066] The application will be described in more detail below by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily appreciate that multiple noncritical parameters can be changed or modified to produce substantially the same result.

[0067] Example

[0068] The following examples are intended to further illustrate the present application. Through description, the advantages and features of the present application will become clear. However, these descriptions are merely exemplary and should not be construed as limiting the scope of the present application.

[0069] Example 1

[0070] 1. Preparation of cryopreservation solution

[0071] (1) Preparation of trehalose mother solution (400g / L)

[0072] Weigh 1.9 g of CD medium powder (Thermo Fisher Scientific, Cat. No. 12681011) and dissolve it in 70 mL of purified water. Stir to dissolve. Once dissolved, add 40 g of trehalose and continue stirring to dissolve. Once dissolved, dilute to 100 mL, filter sterilize, and store in a refrigerator at 4°C.

[0073] (2) Preparation of human albumin stock solution (100 g / L)

[0074] Weigh 1.9 g of CD medium powder (Thermo Fisher Scientific, Catalog No. 12681011) and dissolve it in 100 mL of purified water. Stir to dissolve the solution. After filtration and sterilization, use a syringe to dissolve 10 g of recombinant human albumin (Sigma-Aldrich, Catalog No. A9731) in 100 mL of CD medium. Store in a refrigerator at 4°C.

[0075] (3) Preparation of methyl cellulose mother liquor (100 g / L)

[0076] Dissolve 10g of methylcellulose powder (Sigma-Aldrich, Catalog No. M0387) in 50mL of purified water (70-80°C), autoclave, and cool to a uniform aqueous solution. Then, weigh 1.9g of CD medium powder (Thermo Fisher Scientific, Catalog No. 12681011) and dissolve it in 50mL of purified water. Stir to dissolve, then filter and sterilize. Mix this with the autoclaved methylcellulose solution and store in a refrigerator at 4°C.

[0077] (4) Preparation of serum-free culture medium solution

[0078] a) Add 80% of the prepared volume of purified water (determine the prepared volume according to the supplier's standard preparation process), add 19.3g of CD medium powder (Thermo Fisher Scientific, Cat. No. 12681011) to the purified water and dissolve completely;

[0079] b) adjusting the pH of the medium obtained in step (a) to 7.00 ± 0.05 with 5M NaOH or 5M HCl;

[0080] c) Add purified water to the prepared volume (i.e., adjust the culture medium solution to the prepared volume). The default density of CD culture medium is 1 kg / L.

[0081] d) After the volume is fixed, mix for 15 minutes and test the pH and osmotic pressure of the culture medium. The test results are as follows: the pH value of the culture medium is 7±0.2 and the osmotic pressure is 275-320mOsm / Kg. If the test results are qualified, proceed;

[0082] e) Sterile filter the culture medium using a 0.2 μm filter; store the filtered culture medium in a 4°C refrigerator until use.

[0083] (5) In a biosafety cabinet, the serum-free medium obtained in step (4) and DMSO were mixed according to a specific volume ratio (see Table 2 below), pipetted evenly, and the resulting mixture was placed on an ice box at 4°C or in a refrigerator for later use.

[0084] (6) In a biosafety cabinet, add trehalose, human albumin, methylcellulose and other additives according to the final freezing volume required, pipette and pipette to mix evenly, and place on a 4°C ice box or in a refrigerator for later use.

[0085] The volume ratio of serum-free medium and DMSO in the freezing solution of each experimental example and control example, as well as the final concentrations of trehalose, human albumin and methylcellulose are shown in Table 2 below.

[0086] Table 2 Components and concentrations of cell freezing solutions

[0087] Note: In Table 2, “CD” indicates the serum-free culture medium prepared in step (4) above; “MC” indicates methylcellulose; and “HSA” indicates human albumin, purchased from Sigma-Aldrich, catalog number A9731.

[0088] 2. Cryopreservation of cells

[0089] (1) Take about 1 mL of a cell suspension containing recombinant CHO-1 cells (the CHO cells express an anti-CD147 antibody, the CD147 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2) and count the cells.

[0090] (2) When the density of the cell suspension is about 5×106 When the cell activity was >95%, 5000 mL of cell suspension was taken and centrifuged using 10 500 mL centrifuge cups at a speed of 1500 rpm for 10 minutes.

[0091] (3) Resuspend the cell pellet obtained after centrifugation in step (2) using the freezing solution of each experimental example and control example, and adjust the cell density to 5×10 7 cells / mL.

[0092] (4) Place the resuspended cells on a 4°C ice box.

[0093] (5) Use a multichannel screw cap to open the cryovial, and then use a multichannel pipette to quickly dispense the obtained cell suspension into 2 mL cryovials at a microenvironment of 4°C, with 1 mL of cell suspension in each cryovial, for a total of 480 cryovials.

[0094] (6) Place the packaged cryovials in a transport ice box (placed in a 4°C cold storage 12 hours in advance).

[0095] (7) Set up a programmed cooling apparatus (purchased from Thermo Fisher Scientific, Model 7455) according to the following temperature change steps and temperature change rates. Then, place the cryovials in the transport ice box into the programmed cooling apparatus for cooling. The total cooling time is 65.4 minutes.

[0096] a). Place the cryotube in a programmed cooling apparatus and wait at 4°C for 30 minutes;

[0097] b) Cool down to -10°C at a rate of 2°C per minute;

[0098] c) Cool down to -80°C at a rate of 52°C per minute;

[0099] d). Raise the temperature to -20°C at a rate of 30°C per minute;

[0100] e). Cool down to -40°C at a rate of 1°C per minute;

[0101] f). Cool down to -90℃ at a rate of 10℃ per minute.

[0102] (8) Transfer the cryovials into a liquid nitrogen tank for storage.

[0103] 3. Cell activity detection

[0104] (1) On the second day, the frozen cells were taken out of the liquid nitrogen tank. Three cryopreserved tubes of cells were randomly taken out for thaw in each experimental case and control case.

[0105] (2) Cell recovery:

[0106] a) Slowly rotate the cryovial in a water bath (37°C) until the ice crystals in the cryovial melt. Remove the cryovial from the water bath (melting time is about 2 minutes) and place it in a biosafety cabinet.

[0107] b) Use a pipette to transfer the cell suspension in the cryovial into 40 mL of preheated serum-free culture medium.

[0108] c) Pipette about 1 mL of the cell suspension from step (b), mix the cell suspension with trypan blue in a 1:1 ratio, and count the cells.

[0109] (3) Observe the cell status under a microscope. At the same time, count the number of live and dead cells using a cell counter and calculate the cell activity (cell activity = (number of live cells / total number of cells) × 100%).

[0110] (4) Results and analysis

[0111] The cell activities after recovery of each control example and experimental example are shown in Table 3 below.

[0112] Table 3 Cell activity after thawing of frozen cells in control and experimental examples

[0113] Compared with Control Example 1 and Control Example 2, the addition of 0.05M trehalose and 0.1M trehalose, 2g / L methylcellulose and 5g / L methylcellulose in the experimental examples with single components (i.e., Experimental Examples 1-1 to 1-6) had a certain degree of effect on improving cell recovery activity, but the effect was limited.

[0114] In the experimental groups to which trehalose and HSA were added in combination (i.e., Experimental Examples 1-7 to 1-9), the cell recovery activity was improved to a certain extent. In the experimental groups to which trehalose, HSA and methylcellulose were added in combination (i.e., Experimental Examples 1-10 to 1-14), the cell recovery activity was significantly improved. Specifically, in the experimental groups to which trehalose, HSA and methylcellulose were added in combination (i.e., Experimental Examples 1-10 to 1-14), the cell recovery activity was higher than 95%, and even reached 97.8%. In addition to having a better freezing effect, the freezing solution of the present invention is very suitable for large-scale cell freezing in production due to its low cost and ease of operation.

[0115] Example 2

[0116] The preparation of the freezing solution, cell freezing, cell recovery, and activity testing procedures were consistent with those of Example 1. A recombinant CHO-2 cell line (the CHO cells expressing the CD147 antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12) was used to study the applicability of the freezing solution formulation. The volume ratio of serum-free medium and DMSO in the freezing solution, as well as the final concentrations of trehalose, human albumin, and methylcellulose, were shown in Table 4 below.

[0117] Table 4 Components and concentrations of cell freezing solution

[0118] Note: "S3CD" in Table 4 is a serum-free medium prepared using S3CD medium according to step (4) in Example 1 above, wherein the CD CHO medium of ThermoFisher Scientific in the serum-free medium in step (4) of "Preparation of Cryopreservative Solution" in Example 1 is replaced with S3CD medium of Shanghai Duoning Biotechnology Co., Ltd., product number MNP014.

[0119] Table 5 shows the cell viability of cells cryopreserved using the freezing solutions of Experimental Examples 2-1 to 2-5 after cell thawing. Table 5 shows that the cell thawing activities of cells cryopreserved using the five freezing solution formulations were all high. This indicates that these freezing solution formulations can achieve similarly good cryopreservation results for different CHO cell types.

[0120] Table 5 Cell activity after thawing of frozen cells in experimental example

[0121] Example 3

[0122] The freezing solutions of Experimental Examples 1-11 and 1-14 were used to freeze recombinant CHO-1 and recombinant CHO-2 cells, respectively, according to the steps and methods in Example 1. The frozen cells were revived and evaluated after 24 hours, 1 month, 3 months, 6 months, 1 year, and 3 years of freezing, respectively, to investigate the effect of the freezing formula on the long-term storage stability of the cells. Specifically, at 24 hours, 1 month, 3 months, 6 months, 1 year, and 3 years of freezing, 3 cryopreservation tubes were taken out and the frozen cells were revived, and indicators such as cell activity, cell morphology, doubling period, and the binding activity of the target gene (nucleic acid sequence encoding anti-CD-147 antibody) and the antibody produced were investigated. Tables 6 and 7 below list the stability investigation results of freezing recombinant CHO-1 cells using the freezing solution of Experimental Examples 1-11 and freezing recombinant CHO-2 cells using the freezing solution of Experimental Examples 1-14, respectively.

[0123] Table 6 Stability study results of recombinant CHO-1 cells frozen using the freezing solution of Experimental Examples 1-11

[0124] Note: The expression level of anti-CD147 antibody in recombinant CHO-1 cells before freezing is 2.5±0.2g / L.

[0125] Table 7 Stability study results of recombinant CHO-2 cells frozen in cryopreservation solution of Experimental Examples 1-14

[0126] Note: The expression level of anti-CD147 antibody in recombinant CHO-2 cells before freezing is 3.5±0.2g / L.

[0127] As shown in Tables 6 and 7, cells frozen using the freezing solution formulations of Experimental Examples 1-11 and 1-14 recovered 24 hours, 1 month, 3 months, 6 months, 1 year, and 3 years after freezing. The cells showed good morphology, stable doubling time, and the target gene sequence was consistent with the theoretical sequence. The antibody expression level and antibody binding activity were minimally affected by freezing. It can be seen that the cell freezing solution containing 10% (v / v) DMSO + 0.05M trehalose + 10g / L HSA + 2g / L MC and the cell freezing solution containing 7.5% (v / v) DMSO + 0.1M trehalose + 5g / L HSA + 2g / L MC played a better role in the long-term deep cryopreservation of CHO cells.

[0128] At the same time, the cell activity of cells frozen using the freezing solution formulas of Experimental Examples 1-11 and Experimental Examples 1-14 after thawing is shown in Figure 1. As can be seen from Figure 1, the cells frozen using the freezing solution formulas of Experimental Examples 1-11 and Experimental Examples 1-14 were revived after 24 hours, 1 month, 3 months, 6 months, 1 year, and 3 years of freezing, and the cell activity was greater than 90%, which is much higher than the requirement of cell thawing activity > 80% in the "Chinese Pharmacopoeia" (2020 Edition, Part III).

Claims

1. A serum-free cryopreservation solution for cryopreservation of Chinese hamster ovary cells, comprising a serum-free culture medium, 5%-10% (v / v) DMSO, 0.05-0.1 M trehalose, 5-10 g / L human albumin and 2-5 g / L methylcellulose, wherein the serum-free culture medium is selected from CD CHO culture medium, CD OptiCHO TM medium, Hycell CHO medium, S3 CD CHO medium and S4 CHO medium.

2. The serum-free freezing solution according to claim 1, comprising 7.5% (v / v) DMSO. The serum-free freezing solution according to claim 1 , comprising 10% (v / v) DMSO. The serum-free freezing solution according to claim 1 , comprising 0.05 M trehalose. The serum-free freezing solution according to claim 1 , comprising 0.1 M trehalose. The serum-free cryopreservation solution according to claim 1 , comprising 5 g / L human albumin.

7. The serum-free cryopreservation solution according to claim 1, comprising 10 g / L human albumin. The serum-free freezing solution according to claim 1 , comprising 2 g / L methylcellulose.

9. The serum-free freezing solution according to claim 1, comprising 5 g / L methylcellulose. 10 . The serum-free cryopreservation solution according to claim 1 , comprising serum-free culture medium, 10% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin and 2 g / L methylcellulose. The serum-free cryopreservation solution according to claim 1 , comprising serum-free culture medium, 7.5% (v / v) DMSO, 0.1 M trehalose, 5 g / L human albumin and 2 g / L methylcellulose.

12. The serum-free cryopreservation solution according to claim 1, wherein the Chinese hamster ovary cells express anti-CD147 monoclonal antibodies.

13. The serum-free cryopreservation solution according to claim 12, wherein the anti-CD147 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

12.

14. The serum-free cryopreservation solution according to claim 12, wherein the expression level of the anti-CD147 monoclonal antibody in the Chinese hamster ovary cells reaches 2 g / L.

15. A method for freezing Chinese hamster ovary cells, comprising freezing the Chinese hamster ovary cells using the serum-free freezing solution according to claim 1, wherein the freezing comprises mixing the Chinese hamster ovary cells with the freezing solution to form a cell suspension, cooling the cell suspension to below -80°C within 90 minutes, and then transferring the cell suspension to liquid nitrogen for storage.

16. The method according to claim 15, wherein the initial concentration of the cell suspension after the Chinese hamster ovary cells and the freezing solution are mixed is 1×10 7 -5×10 8 cells / mL.

17. The method according to claim 16, wherein the initial concentration of the cell suspension after the Chinese hamster ovary cells and the freezing solution are mixed is 5×10 7 cells / mL.

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