Cell cryopreservation method

Abstract

A cell cryopreservation method. By means of controlling the temperature gradient in a temperature lowering process and the temperature change rates at different stages, the cells are prevented from being injured in the cryopreservation process and still have good biological activity after thawing, and products expressed thereby still have stable expression quantity and good biological function.

Description

Cell cryopreservation methods Technical Field

[0001] The present invention relates to the field of cell preservation, and in particular to a cell freezing method. 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] Cell cryopreservation generally follows the principle of slow freezing and rapid thawing, with the typical cooling rate being 1°C / minute to below -80°C. Traditional cryopreservation methods generally include the following: The first involves wrapping the cell sample in cotton and placing it directly in a -80°C freezer. The second involves placing the cell sample in a 4°C environment for half an hour, a -20°C environment for an hour, and then a -80°C environment overnight. The third method involves using a programmed cooling box, where the cell sample is placed directly in a -80°C freezer overnight.

[0004] Although the above three methods can guarantee the cooling rate to a certain extent, the operation is cumbersome, the operation time is long, and large quantities of frozen food cannot be stored, which to a certain extent limits the application of these methods.

[0005] The inventors of the present invention have designed a cooling program suitable for cell cryopreservation, which sets multiple temperature change stages and controls the temperature gradient and temperature change rate in different stages during the temperature change process, thereby achieving strong controllability, avoiding damage to cells during the cryopreservation process, and being unaffected by the cryopreservation scale. It can also enable cells to still have good biological activity and biological functions after recovery.

[0006] Summary of the Invention

[0007] One aspect of the present application provides a cell freezing method, comprising setting a temperature-changing step to programmatically cool a cell sample, wherein the cell sample comprises the cells and a freezing solution, wherein the temperature-changing step comprises:

[0008] (a) placing the cell sample in an environment at a first temperature, wherein the first temperature is 2°C to 8°C;

[0009] (b) lowering the temperature of the cell sample to a second temperature at a temperature change rate of 2°C-10°C / min, wherein the second temperature is 0°C to -18°C;

[0010] (c) The ambient temperature of the cell sample is lowered to a third temperature at a temperature change rate of 30 ℃ -80 ℃ / minute, wherein the third temperature is -60 ℃ to -90 ℃;

[0011] (d) The ambient temperature of the cell sample is raised to a fourth temperature at a temperature change rate of 15°C-40°C / min, wherein the fourth temperature is -10°C to -30°C;

[0012] (e) The ambient temperature of the cell sample is lowered to a fifth temperature at a temperature change rate of 0.2°C-10°C / min, wherein the fifth temperature is -35°C to -50°C;

[0013] (f) Lowering the ambient temperature of the cell sample to a sixth temperature at a temperature change rate of more than 5°C / min, wherein the sixth temperature is below -80°C.

[0014] In some embodiments, the cell sample in step (a) is maintained at the first temperature for 1 to 60 minutes. In some embodiments, the cell sample in step (a) is maintained at the first temperature for 30 to 60 minutes. In some embodiments, the first temperature is 4°C.

[0015] In certain embodiments, the temperature change rate in step (b) is 2°C-3°C / minute. In certain embodiments, the temperature change rate in step (b) is 2°C / minute. In certain embodiments, the temperature change rate in step (b) is 3°C / minute. In certain embodiments, the second temperature is -4°C to -10°C. In certain embodiments, the second temperature is -7°C to -10°C. In certain embodiments, the second temperature is -7°C. In certain embodiments, the second temperature is -10°C.

[0016] In certain embodiments, the temperature change rate in the step (c) is 35°C-62°C / minute. In certain embodiments, the temperature change rate in the step (c) is 45°C-62°C / minute. In certain embodiments, the temperature change rate in the step (c) is 50°C-62°C / minute. In certain embodiments, the temperature change rate in the step (c) is 45°C / minute. In certain embodiments, the temperature change rate in the step (c) is 50°C / minute. In certain embodiments, the temperature change rate in the step (c) is 52°C / minute. In certain embodiments, the temperature change rate in the step (c) is 62°C / minute. In certain embodiments, the third temperature is -60°C to -80°C. In certain embodiments, the third temperature is -76°C to -80°C. In certain embodiments, the third temperature is -80°C.

[0017] In certain embodiments, the temperature change rate in the step (d) is 17°C-33°C / minute. In certain embodiments, the temperature change rate in the step (d) is 23°C-33°C / minute. In certain embodiments, the temperature change rate in the step (d) is 23°C-30°C / minute. In certain embodiments, the temperature change rate in the step (d) is 23°C-26°C / minute. In certain embodiments, the temperature change rate in the step (d) is 23°C / minute. In certain embodiments, the temperature change rate in the step (d) is 24°C / minute. In certain embodiments, the temperature change rate in the step (d) is 25°C / minute. In certain embodiments, the temperature change rate in the step (d) is 26°C / minute. In certain embodiments, the temperature change rate in the step (d) is 30°C / minute. In certain embodiments, the temperature change rate in the step (d) is 33°C / minute. In certain embodiments, the fourth temperature is -12°C to -26°C. In certain embodiments, the fourth temperature is between -20°C and -26°C. In certain embodiments, the fourth temperature is -20°C. In certain embodiments, the fourth temperature is -23°C. In certain embodiments, the fourth temperature is -26°C.

[0018] In certain embodiments, the temperature ramp rate in step (e) is 1°C / min. In certain embodiments, the fifth temperature is -40°C.

[0019] In some embodiments, the temperature ramp rate in step (f) is 10°C / min. In some embodiments, the sixth temperature is -80°C. In some embodiments, the sixth temperature is -90°C.

[0020] In certain embodiments, the freezing method of the present application cools the cell sample to -80°C or below within 90 minutes. In certain embodiments, the freezing method of the present application cools the cell sample to -80°C or below within 60-75 minutes.

[0021] In some embodiments, the cell sample is placed in a cryovial. In some embodiments, the temperature of the cell sample is controlled at the first temperature before the programmed cooling. In some embodiments, after the programmed cooling, the cooled cell sample is transferred to liquid nitrogen for storage.

[0022] In some embodiments, the cells are Chinese hamster ovary cells. In some embodiments, the starting concentration of cells in the cell sample is 1×10 7 -5×10 8 In some embodiments, the initial cell concentration in the cell sample is 5×10 7 cells / mL.

[0023] In certain embodiments, the cryopreservation solution is a serum-free cryopreservation solution comprising serum-free culture medium, DMSO, trehalose, human albumin, and methylcellulose. In certain embodiments, the cryopreservation solution is a serum-free cryopreservation solution comprising serum-free culture medium, 5%-10% (v / v) DMSO, 0.05-0.1M trehalose, 5-10g / L human albumin, and 2-5g / L methylcellulose. In certain embodiments, 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.

[0024] 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.

[0025] In certain embodiments, the serum-free freezing solution comprises 0.05 M trehalose. In certain embodiments, the concentration of trehalose is 0.1 M.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] In certain embodiments, the serum-free freezing medium comprises serum-free medium, 7.5% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin and 2 g / L methylcellulose. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG1 shows the storage stability test results of cells frozen using the cooling program of Program 4 and the freezing solutions of Formula A3 and Formula A6, respectively. DETAILED DESCRIPTION

[0032] 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.

[0033] As used in this application and 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.

[0034] One aspect of the present application discloses a cell freezing method, comprising setting a temperature changing step to programmatically cool a cell sample, wherein the cell sample comprises the cells and a freezing solution, wherein the temperature changing step comprises:

[0035] (a) placing the cell sample in an environment at a first temperature, wherein the first temperature is 2°C to 8°C;

[0036] (b) lowering the temperature of the cell sample to a second temperature at a temperature change rate of 2°C-10°C / min, wherein the second temperature is 0°C to -18°C;

[0037] (c) The ambient temperature of the cell sample is lowered to a third temperature at a temperature change rate of 30 ℃ -80 ℃ / minute, wherein the third temperature is -60 ℃ to -90 ℃;

[0038] (d) The ambient temperature of the cell sample is raised to a fourth temperature at a temperature change rate of 15°C-40°C / min, wherein the fourth temperature is -10°C to -30°C;

[0039] (e) The ambient temperature of the cell sample is lowered to a fifth temperature at a temperature change rate of 0.2°C-10°C / min, wherein the fifth temperature is -35°C to -50°C;

[0040] (f) Lowering the ambient temperature of the cell sample to a sixth temperature at a temperature change rate of more than 5°C / min, wherein the sixth temperature is below -80°C.

[0041] In certain embodiments, the cell sample in the step (a) is maintained in the environment of the first temperature for 1 to 60 minutes. In certain embodiments, the cell sample in the step (a) is maintained in the environment of the first temperature for 30 to 60 minutes. In certain embodiments, the cell sample in the step (a) is maintained in the environment of the first temperature for about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes or about 60 minutes. In certain embodiments, the cell sample in the step (a) is maintained in the environment of the first temperature for about 30 minutes.

[0042] In certain embodiments, the first temperature is 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., or 8° C. In certain embodiments, the first temperature is 4° C.

[0043] In certain embodiments, the temperature ramp rate in step (b) is 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 certain embodiments, the temperature ramp rate in step (b) is 2°C-3°C / min. In certain embodiments, the temperature ramp rate in step (b) is 2°C / min. In certain embodiments, the temperature ramp rate in step (b) is 3°C / min.

[0044] In certain 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 certain embodiments, the second temperature is between -2°C and -10°C, -4°C and -10°C, -7°C and -10°C, -8°C and -10°C, -2°C and -8°C, -4°C and -8°C, -7°C and -8°C, -2°C and -7°C, or -4°C and -7°C. In certain embodiments, the second temperature is between -7°C and -10°C. In certain embodiments, the second temperature is -4°C. In certain embodiments, the second temperature is -7°C. In certain embodiments, the second temperature is -8°C. In certain embodiments, the second temperature is -10°C.

[0045] In certain embodiments, the temperature ramp rate in step (c) is 30°C-80°C / min. In certain embodiments, the temperature ramp rate in step (c) is 30°C-65°C / min. In certain embodiments, the temperature ramp rate in step (c) is 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, 40°C / min, 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, 60°C / min, 61°C / min, 62°C / min, 63°C / min, 64°C / min, or 65°C / min. In certain embodiments, the ramp rate in step (c) is 30-80°C / min, 50-80°C / min, 60-80°C / min, 30-65°C / min, 35-65°C / min, 45-65°C / min, 50-65°C / min, 60-65°C / min, 30-62°C / min, 35-62°C / min, 45-62°C / min, 50-62°C / min, 35-52°C / min, 45-52°C / min, 50-52°C / min, 35-50°C / min, 45-50°C / min, or 52-62°C / min. In certain embodiments, the ramp rate in step (c) is 50-62°C / min. In certain embodiments, the ramp rate in step (c) is 35°C / min. In certain embodiments, the ramp rate in step (c) is 45°C / min. In certain embodiments, the ramp rate in step (c) is 50°C / min. In certain embodiments, the ramp rate in step (c) is 52°C / min. In certain embodiments, the ramp rate in step (c) is 62°C / min.

[0046] In certain 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 certain embodiments, the third temperature is between -60° C. and -80° C., between -60° C. and -90° C., between -70° C. and -80° C., or between -70° C. and -90° C. In certain embodiments, the third temperature is between -76°C and -80°C, between -78°C and -80°C, or between -76°C and -78°C. In certain embodiments, the third temperature is -60°C. In certain embodiments, the third temperature is -76°C. In certain embodiments, the third temperature is -78°C. In certain embodiments, the third temperature is -80°C.

[0047] In certain embodiments, the ramp rate in step (d) is 15-40°C / min. In certain embodiments, the ramp rate in step (d) is 15°C / min, 16°C / min, 17°C / min, 18°C / min, 19°C / min, 20°C / min, 21°C / min, 22°C / min, 23°C / min, 24°C / min, 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 certain embodiments, the ramp rate in step (d) is 17-33°C / min, 23-33°C / min, 26-33°C / min, or 30-33°C / min. In certain embodiments, the temperature change rate in step (d) is 17°C-30°C / minute, 23°C-30°C / minute, or 26°C-30°C / minute. In certain embodiments, the temperature change rate in step (d) is 17°C-23°C / minute or 23°C-26°C / minute. In certain embodiments, the temperature change rate in step (d) is 17°C / minute. In certain embodiments, the temperature change rate in step (d) is 23°C / minute. In certain embodiments, the temperature change rate in step (d) is 24°C / minute. In certain embodiments, the temperature change rate in step (d) is 25°C / minute. In certain embodiments, the temperature change rate in step (d) is 26°C / minute. In certain embodiments, the temperature change rate in step (d) is 30°C / minute. In certain embodiments, the temperature change rate in step (d) is 33°C / minute.

[0048] In certain embodiments, the fourth temperature is between -10°C and -30°C. In certain embodiments, the fourth temperature is between -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, -25°C, -26°C, -27°C, -28°C, -29°C, or -30°C. In certain embodiments, the fourth temperature is between -12°C and -26°C, -12°C and -23°C, or -12°C and -20°C. In certain embodiments, the fourth temperature is between -20°C and -26°C, or -20°C and -23°C. In certain embodiments, the fourth temperature is -20°C. In certain embodiments, the fourth temperature is -23°C. In certain embodiments, the fourth temperature is -26°C.

[0049] In certain embodiments, the ramp rate in step (e) is 0.2°C-10°C / min. In certain embodiments, the ramp rate in step (e) 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 certain embodiments, the ramp rate in step (e) is 1°C / min. In certain embodiments, the temperature change rate in step (e) is 0.5°C-1°C / min, 0.5°C-2°C / min, 0.5°C-3°C / min, 0.5°C-4°C / min, 0.5°C-5°C / min, 1°C-2°C / min, 1°C-3°C / min, 1°C-4°C / min or 1°C-5°C / min.

[0050] In certain embodiments, the fifth temperature is -35°C to -50°C. In certain embodiments, the fifth temperature is -35°C, -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 certain embodiments, the fifth temperature is -40°C. In certain embodiments, the fifth temperature is -35°C to -50°C, -35°C to -45°C, -35°C to -40°C, or -40°C to -50°C.

[0051] In certain embodiments, the temperature ramp rate in step (f) is 5°C-40°C / minute. In certain embodiments, the temperature ramp rate in step (f) is 5°C-30°C / minute. In certain embodiments, the temperature ramp rate in step (f) 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 certain embodiments, the temperature ramp rate in step (f) is 10°C / minute. In certain embodiments, the temperature change rate in step (f) is 5°C-10°C / min, 5°C-15°C / min, 5°C-20°C / min, 5°C-25°C / min, 5°C-30°C / min, 10°C-15°C / min, 10°C-20°C / min, 10°C-25°C / min or 10°C-30°C / min.

[0052] In certain embodiments, the sixth temperature is between -80° C. and -190° C. In certain 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 less. In certain embodiments, the sixth temperature is -80°C to -90°C, -80°C to -100°C, -80°C to -110°C, -80°C to -120°C, -80°C to -130°C, -80°C to -140°C, -80°C to -150°C, -80°C to -160°C, -80°C to -170°C, -80°C to -180°C, or -80°C to -190°C. In certain embodiments, the sixth temperature is -80°C. In certain embodiments, the sixth temperature is -90°C.

[0053] In certain embodiments, the freezing method of the present application cools the cell sample to below -80°C within 90 minutes. In certain embodiments, the freezing method of the present application cools the cell sample from 2°C, or 3°C, or 4°C, or 5°C, or 6°C, or 7°C, or 8°C to below -80°C within 90 minutes. In certain embodiments, the freezing method of the present application cools the cell sample to -80°C to -120°C, -80°C to -110°C, -80°C to -100°C, or -80°C to -90°C within 90 minutes. In certain embodiments, the cell sample is cooled from 4°C to -80°C within 90 minutes. In certain embodiments, the cell sample is cooled from 4°C to -90°C within 90 minutes. In certain embodiments, the cell sample is cooled from 4°C to -100°C within 90 minutes. In certain embodiments, the cell sample is cooled to below -80°C within 90 minutes. In certain embodiments, the cell sample is cooled to below -80°C within 60-75 minutes. In certain embodiments, the cell sample is cooled to below -80°C within 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 sample is cooled from 4° C. to −80° C. within 70 minutes. In certain embodiments, the cell sample is cooled from 4° C. to −90° C. within 70 minutes.

[0054] In certain embodiments, the cell sample is a cell suspension comprising cells to be frozen and a freezing solution. In certain embodiments, the cell sample is contained in a freezing tube.

[0055] In certain embodiments, the freezing method of the present invention is to cool and freeze the cell sample by using a cooling device. The cooling device used in the present invention can be any device or system that can realize the temperature change step disclosed in the present application. In certain embodiments, the cooling device comprises at least one cavity for placing the cell sample. In certain embodiments, the cooling device is a programmed cooling device (also known as a program-controlled cooling device). In certain embodiments, the cooling device is a programmed cooling device utilizing liquid nitrogen injection ring technology (see U.S. Patent No. 6,044,648A, the disclosure of which is incorporated herein by reference). The programmed cooling device can be purchased from ThermoFisherScientific, model 7455.

[0056] In some embodiments, the temperature of the cell sample is controlled at the first temperature before the programmed cooling is performed. In some embodiments, after the programmed cooling is performed, the cooled cell sample is transferred to liquid nitrogen for storage.

[0057] In certain embodiments, the freezing method provided by the present disclosure can be used to freeze Chinese hamster ovary cells. 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 compared to other expression systems. More and more 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. A large-scale, standardized CHO cell bank with long-term storage stability can continuously provide companies with CHO cells that stably express the target protein. The storage stability of such a cell bank mainly depends on the quality of cell freezing, among which the cell freezing method is an important factor affecting the quality of cell freezing.

[0058] 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.

[0059] In certain embodiments, the freezing method provided by the present disclosure can be 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, a Fab fragment, F(ab')2, an Fd fragment (composed of VH and CH1 regions), and an Fv fragment, etc.

[0060] 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.

[0061] In certain of the aforementioned embodiments, the anti-CD147 monoclonal antibody comprises a heavy chain variable region and a light chain variable region.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Table 1. Amino acid sequence

[0068] 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.

[0069] 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.

[0070] 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.

[0071] In certain embodiments, the initial cell concentration in the cell sample is 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 In some embodiments, the initial cell concentration in the cell sample is 1×10 7 -5×10 8 In some embodiments, the initial cell concentration in the cell sample is 5×10 7 cells / mL.

[0072] In certain embodiments, the cryopreservation solution used in the present application is a serum-free cryopreservation solution comprising serum-free culture medium, DMSO, trehalose, human albumin, and methylcellulose. In certain embodiments, the serum-free cryopreservation solution comprises 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.

[0073] The "serum-free medium" used in this application is a medium without animal-derived components, serum-free and chemically defined. The serum-free medium can be selected from CD CHO medium, CD OptiCHO medium, TMCHO medium, Hycell CHO medium, S3CD CHO medium, and S4CHO medium. 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 (Cat. No. 12490003) or selected from other medium with the same composition. CD OptiCHO TM The medium provides high performance and yield of recombinant CHO cells. TM Culture media can be purchased from Thermo Fisher Scientific (Cat. No. 12681011) or selected from other media with the same composition. 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 media with the same composition. S3CD CHO medium and S4CHO medium are suitable for batch, fed-batch, and perfusion cultures of Chinese hamster ovary cells. S3CD CHO medium and S4CHO medium can be purchased from Shanghai Duoning Biotechnology Co., Ltd., with product numbers M014-001 (or MNP014) and MT022-1L (or MNP022-001), respectively; other media with the same composition can also be selected.

[0074] "DMSO" in this application 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.

[0075] "Human albumin" as used 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 cryopreservatives of the present application comprise functional fragments 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.

[0076] 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.

[0077] In certain embodiments, the serum-free freezing solution 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:

[0078] (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,

[0079] (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.

[0080] (3) 0.05-0.1M trehalose, that is, the concentration of trehalose in the serum-free freezing solution is 0.05-0.1mol / L,

[0081] (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

[0082] (5) 2-5 g / L methylcellulose, that is, the concentration of methylcellulose in the serum-free freezing solution is 2-5 g / L.

[0083] The total volume of the serum-free freezing solution is the sum of the volumes of the components added to the freezing solution.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In certain embodiments of the aforementioned embodiments, the serum-free freezing solution comprises serum-free culture medium, 10% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin, and 2 g / L methylcellulose.

[0089] In certain embodiments of the aforementioned embodiments, the serum-free freezing solution comprises serum-free culture medium, 7.5% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin, and 2 g / L methylcellulose.

[0090] In certain embodiments of the aforementioned embodiments, the serum-free freezing solution comprises serum-free culture medium, 7.5% (v / v) DMSO, 0.1 M trehalose, 5 g / L recombinant human albumin, and 2 g / L methylcellulose.

[0091] The present application will be described in more detail below by way of 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 various noncritical parameters can be changed or modified to produce substantially the same results.

[0092] Example

[0093] 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.

[0094] Example 1: Preparation of cryopreservation solution

[0095] (1) Preparation of trehalose mother solution (400g / L)

[0096] 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.

[0097] (2) Preparation of human albumin stock solution (100 g / L)

[0098] 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.

[0099] (3) Preparation of methyl cellulose mother liquor (100 g / L)

[0100] 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.

[0101] (4) Preparation of serum-free culture medium solution

[0102] i. Add 80% of the prepared volume of purified water (determine the prepared volume according to the supplier's standard preparation process), add 19.3 g of CD medium powder (Thermo Fisher Scientific, Catalog No. 12681011) to the purified water and dissolve completely;

[0103] ii. adjusting the pH of the medium obtained in step (i) to 7.00 ± 0.05 with 5M NaOH or 5M HCl;

[0104] iii. Add purified water to make up the volume (i.e., adjust the culture medium solution to the prepared volume). The default density of CD culture medium is 1 kg / L.

[0105] iv. After adjusting to volume, mix for 15 minutes and test the pH and osmotic pressure of the culture medium. The test results should be as follows: pH 7 ± 0.2 and osmotic pressure 275-320 mOsm / kg. If the test results are satisfactory, proceed.

[0106] v. Sterile filter the culture medium using a 0.2 μm filter and store it in a refrigerator at 4°C until use.

[0107] (5) In a biosafety cabinet, the serum-free medium obtained in step (4) and DMSO were mixed according to the specific volume ratio of each freezing solution formula (see Table 2 below), and pipetted evenly. The resulting mixture was placed on an ice box at 4°C or in a refrigerator for later use.

[0108] (6) In a biosafety cabinet, add the trehalose, human albumin, methylcellulose and other additives obtained in steps (1) to (3) above according to the concentration of each freezing solution formula, based on the final volume required for freezing. After pipetting and mixing evenly, place on a 4°C ice box or in a refrigerator for later use.

[0109] The volume ratios of serum-free culture medium and DMSO and the final concentrations of trehalose, human albumin and / or methylcellulose in each freezing solution formulation are shown in Table 2 below.

[0110] Table 2. Components and concentrations of cell freezing solutions

[0111] 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.

[0112] Example 2: Cryopreservation of cells and cell activity detection

[0113] 1. Cryopreservation of cells

[0114] (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.

[0115] (2) When the density of the cell suspension is about 5×10 6 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.

[0116] (3) The cell pellet obtained after centrifugation in step (2) was resuspended using the freezing solution of formula A1 in Example 1, and the cell density was adjusted to 5×10 7 cells / mL.

[0117] (4) Place the resuspended cells on a 4°C ice box.

[0118] (5) Use a multichannel screw capper 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.

[0119] (6) Place the packaged cryovials in a transport ice box (placed in a 4°C cold storage 12 hours in advance).

[0120] (7) Set the cooling program of the programmed cooling instrument (purchased from Thermofisher Scientific, model 7455) according to the "control program" in Table 3 (the "control program" is a set of cooling programs commonly used when using a programmed cooling instrument). Then, place the cryopreservation tubes in the transport ice box in step (6) into the programmed cooling instrument and cool them according to the "control program".

[0121] (8) The cryopreserved tubes cooled in step (7) are transferred to a liquid nitrogen tank for storage.

[0122] The control procedure in step (7) was replaced by the procedures 1-4 of the present application listed in Table 3 below, and the above steps (1)-(8) were repeated to freeze the cells.

[0123] Table 3. Cooling program

[0124] 2. Cell viability assay

[0125] (1) On the second day, the frozen cells were taken out of the liquid nitrogen tank and cells from three cryopreserved tubes were randomly taken out from each cooling program experiment for recovery.

[0126] (2) Cell recovery:

[0127] i. 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 approximately 2 minutes) and place it in a biosafety cabinet.

[0128] ii. Use a pipette to transfer the cell suspension in the cryovial into 40 mL of pre-warmed serum-free culture medium.

[0129] iii. Pipette about 1 mL of the cell suspension from step (ii), mix the cell suspension with trypan blue in a 1:1 ratio, and count the cells.

[0130] (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%).

[0131] (4) Results and analysis

[0132] The cell activities of cells frozen at each cooling program after thawing are shown in Table 4 below.

[0133] Table 4. Cell viability after cell recovery

[0134] Compared with the control program, the cell activity of cells frozen using programs 1-4 after thawing was higher than that of cells frozen using the control program after thawing. It can be seen that using programs 1-4 for cell freezing achieved better freezing effects.

[0135] Example 3

[0136] Different cooling programs were used for cell freezing experiments.

[0137] The steps for freezing cells were the same as those in Example 2, wherein the formula A1 in step (3) of freezing cells in Example 2 was replaced with formula A3, and the cooling program in step (7) of freezing cells in Example 2 was replaced with the control program and programs 1-5 listed in Table 3, respectively.

[0138] The cells were revived and observed using the same cell activity detection steps as in Example 2. The cell activity after resuscitation is shown in Table 5 below.

[0139] Table 5. Cell viability after cell recovery

[0140] As shown in the above post-thaw cell viability results, the cell samples containing cryopreservation solution A3 using cooling schedules 1-5 of the present invention also achieved relatively high post-thaw cell viability. This indicates that the cooling schedule of the present invention significantly improves the efficiency of cell cryopreservation.

[0141] Example 4

[0142] The cells were cryopreserved using the same steps as in Example 2, except that Formula A1 in Step (3) of Example 2 was replaced with Formulas A2-A6, respectively, and the cooling program in Step (7) of Example 2 was replaced with Program 4 listed in Table 3. The cells were revived and observed using the same cell activity assay steps as in Example 2. The cell activity after resuscitation is shown in Table 6 below.

[0143] Table 6. Cell viability after cell recovery

[0144] From the above cell activity results after thawing, it can be seen that the cooling program of the present invention also shows a relatively high-quality freezing effect for cell samples using a variety of different freezing solution formulas.

[0145] In addition, the cells were cryopreserved using the same steps as in Example 2, wherein the cooling program in step (7) of freezing the cells in Example 2 was replaced with Program 2 listed in Table 3, and cryopreservation was performed using cryopreservation solution formula A6.

[0146] The cells were cryopreserved using the same steps as in Example 2, except that the cooling program in step (7) of cryopreserving the cells in Example 2 was replaced with Program 3 listed in Table 3, and cryopreservation was performed using cryopreservation solution formula A2.

[0147] The cells were cryopreserved using the same steps as in Example 2, except that the cooling program in step (7) of cryopreserving the cells in Example 2 was replaced with Program 6 listed in Table 3, and cryopreservation was performed using cryopreservation solution Formula A6.

[0148] The corresponding activities of the frozen cells after thawing are shown in Table 7 below.

[0149] Table 7

[0150] Example 5

[0151] The same cell freezing, cell recovery and activity detection operations as in Example 2 were adopted, wherein the cooling program in step (7) of freezing cells in Example 2 was replaced with program 4 listed in Table 3, and a recombinant CHO-2 cell line (the CHO cell expresses CD147 antibody, the CD147 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 11, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 12) was used for the study. The freezing solution was prepared using the steps of Example 1. The volume ratio of serum-free medium and DMSO in the freezing solution, the final concentrations of trehalose, human albumin and methylcellulose, and the cell activity after cell recovery are shown in Table 8 below.

[0152] Table 8. Components and concentrations of cell freezing solutions

[0153] Note: "S3CD" in Table 8 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.

[0154] From the above results of cell activity after recovery, it can be seen that the cooling program of the present invention can also achieve the same good technical effect in the cryopreservation of different cell lines.

[0155] Example 6

[0156] The program 4 in Table 3 was used for program cooling, wherein the freezing solutions of Formula A3 and Formula A6 in Example 1 were used, and the recombinant CHO-1 cells and recombinant CHO-2 cells were frozen according to the steps and methods in Example 2. 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 cooling program of the present invention 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, three cryopreserved tubes were taken out and the frozen cells were revived, and indicators such as cell viability, 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. The following Tables 9 and 10 respectively list the stability investigation results of recombinant CHO-1 cells frozen using the cooling program of Program 4 and the freezing solution of Formula A3 and recombinant CHO-2 cells frozen using the cooling program of Program 4 and the freezing solution of Formula A6.

[0157] Table 9. Long-term stability study results of recombinant CHO-1 cells (using cryopreservation solution of formula A3)

[0158] Note: The expression level of anti-CD147 antibody in recombinant CHO-1 cells before freezing is 2.5±0.2g / L.

[0159] Table 10. Long-term stability study results of recombinant CHO-2 cells (using cryopreservation solution of formula A6)

[0160] Note: The expression level of anti-CD147 antibody in recombinant CHO-2 cells before freezing is 3.5±0.2g / L.

[0161] Tables 9 and 10 show that cells cryopreserved using both protocols recovered 24 hours, one month, three months, six months, one year, and three years after cryopreservation. Cell morphology was good, doubling times were stable, the target gene sequence was consistent with the theoretical sequence, and antibody expression and binding activity were minimally affected by cryopreservation. This demonstrates that both protocols effectively support the long-term cryopreservation of CHO cells.

[0162] At the same time, the cell viability of cells frozen in two experimental protocols after thawing is shown in Figure 1. As can be seen from Figure 1, the cell viability was greater than 90% after thawing for 24 hours, 1 month, 3 months, 6 months, 1 year, and 3 years after freezing, which is much higher than the cell thawing activity requirement of >80% in the Chinese Pharmacopoeia (2020 Edition, Part III).

Claims

1. A cell freezing method, comprising setting a temperature changing step to program a cell sample to cool down, wherein the cell sample comprises the cell and a freezing solution, wherein the temperature changing step comprises: (a) placing the cell sample in an environment at a first temperature, wherein the first temperature is 2°C to 8°C; (b) lowering the temperature of the cell sample to a second temperature at a temperature change rate of 2°C-10°C / min, wherein the second temperature is 0°C to -18°C; (c) lowering the ambient temperature of the cell sample to a third temperature at a temperature change rate of 30°C-80°C / min, wherein the third temperature is -60°C to -90°C; (d) The ambient temperature of the cell sample is raised to a fourth temperature at a temperature change rate of 15°C-40°C / min, wherein the fourth temperature is -10°C to -30°C; (e) The ambient temperature of the cell sample is reduced to a fifth temperature at a temperature change rate of 0.2°C-10°C / min, wherein the fifth temperature is -35°C to -50°C; (f) lowering the ambient temperature of the cell sample to a sixth temperature at a temperature change rate greater than 5°C / min, wherein the sixth temperature is below -80°C.

2. The freezing method according to claim 1, wherein the cell sample in step (a) is maintained in the first temperature environment for 1 to 60 minutes.

3. The freezing method according to claim 1 or 2, wherein the cell sample in step (a) is maintained in the first temperature environment for 30 to 60 minutes.

4. The cryopreservation method according to any one of claims 1 to 3, wherein the first temperature is 4°C.

5. The cryopreservation method according to any one of claims 1 to 4, wherein the temperature change rate in step (b) is 2°C-3°C / min.

6. The cryopreservation method according to any one of claims 1 to 5, wherein the temperature change rate in step (b) is 2°C / min.

7. The cryopreservation method according to any one of claims 1 to 5, wherein the temperature change rate in step (b) is 3°C / min.

8. The cryopreservation method according to any one of claims 1 to 7, wherein the second temperature is -4°C to -10°C.

9. The cryopreservation method according to any one of claims 1 to 8, wherein the second temperature is -7°C to -10°C.

10. The cryopreservation method according to any one of claims 1 to 9, wherein the second temperature is -7°C.

11. The cryopreservation method according to any one of claims 1 to 9, wherein the second temperature is -10°C.

12. The cryopreservation method according to any one of claims 1 to 11, wherein the temperature change rate in step (c) is 35°C-62°C / min.

13. The cryopreservation method according to any one of claims 1 to 12, wherein the temperature change rate in step (c) is 45°C-62°C / min.

14. The cryopreservation method according to any one of claims 1 to 13, wherein the temperature change rate in step (c) is 50°C-62°C / min.

15. The cryopreservation method according to any one of claims 1 to 13, wherein the temperature change rate in step (c) is 45°C / min.

16. The cryopreservation method according to any one of claims 1 to 14, wherein the temperature change rate in step (c) is 50°C / min.

17. The cryopreservation method according to any one of claims 1 to 14, wherein the temperature change rate in step (c) is 52°C / min.

18. The cryopreservation method according to any one of claims 1 to 14, wherein the temperature change rate in step (c) is 62°C / min.

19. The cryopreservation method according to any one of claims 1 to 18, wherein the third temperature is -60°C to -80°C.

20. The cryopreservation method according to any one of claims 1 to 19, wherein the third temperature is -76°C to -80°C.

21. The cryopreservation method according to any one of claims 1 to 20, wherein the third temperature is -80°C.

22. The cryopreservation method according to any one of claims 1 to 21, wherein the temperature change rate in step (d) is 17°C-33°C / min.

23. The cryopreservation method according to any one of claims 1 to 22, wherein the temperature change rate in step (d) is 23°C-33°C / min.

24. The cryopreservation method according to any one of claims 1 to 23, wherein the temperature change rate in step (d) is 23°C-30°C / min.

25. The cryopreservation method according to any one of claims 1 to 24, wherein the temperature change rate in step (d) is 23°C-26°C / min.

26. The cryopreservation method according to any one of claims 1 to 25, wherein the temperature change rate in step (d) is 23°C / min.

27. The cryopreservation method according to any one of claims 1 to 25, wherein the temperature change rate in step (d) is 24°C / min.

28. The cryopreservation method according to any one of claims 1 to 25, wherein the temperature change rate in step (d) is 25°C / min.

29. The cryopreservation method according to any one of claims 1 to 25, wherein the temperature change rate in step (d) is 26°C / min.

30. The cryopreservation method according to any one of claims 1 to 24, wherein the temperature change rate in step (d) is 30°C / min.

31. The cryopreservation method according to any one of claims 1 to 30, wherein the fourth temperature is -12°C to -26°C.

32. The cryopreservation method according to any one of claims 1 to 31, wherein the fourth temperature is -20°C to -26°C.

33. The cryopreservation method according to any one of claims 1 to 32, wherein the fourth temperature is -20°C.

34. The cryopreservation method according to any one of claims 1 to 33, wherein the temperature change rate in step (e) is 1°C / min.

35. The cryopreservation method according to any one of claims 1 to 34, wherein the fifth temperature is -40°C.

36. The cryopreservation method according to any one of claims 1 to 35, wherein the temperature change rate in step (f) is 10°C / min.

37. The cryopreservation method according to any one of claims 1 to 36, wherein the sixth temperature is -80°C.

38. The cryopreservation method according to any one of claims 1 to 36, wherein the sixth temperature is -90°C.

39. The cryopreservation method according to any one of claims 1 to 38, wherein the cell sample is cooled to below -80°C within 90 minutes.

40. The cryopreservation method according to any one of claims 1 to 39, wherein the cell sample is cooled to below -80°C within 60 to 75 minutes.

41. The cryopreservation method according to any one of claims 1 to 40, wherein the cell sample is contained in a cryopreservation tube.

42. The cryopreservation method according to any one of claims 1-41, further comprising controlling the temperature of the cell sample at the first temperature before performing the programmed cooling.

43. The cryopreservation method according to any one of claims 1-42, further comprising, after the programmed cooling, transferring the cooled cell sample to liquid nitrogen for storage.

44. The cryopreservation method according to any one of claims 1-43, wherein the cells are Chinese hamster ovary cells.

45. The cryopreservation method according to any one of claims 1 to 44, wherein the initial cell concentration in the cell sample is 1×10 7 -5×10 8 cells / mL.

46. ​​The cryopreservation method according to any one of claims 1 to 45, wherein the initial cell concentration in the cell sample is 5×10 7 cells / mL.

47. The cryopreservation method according to any one of claims 1 to 46, wherein the cryopreservation solution is a serum-free cryopreservation solution comprising serum-free culture medium, DMSO, trehalose, human albumin and methylcellulose.

48. The cryopreservation method according to claim 47, wherein the cryopreservation solution comprises 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.

49. The cryopreservation method according to any one of claims 1 to 48, wherein the cryopreservation solution comprises 7.5% (v / v) DMSO.

50. The cryopreservation method according to any one of claims 1-48, wherein the cryopreservation solution comprises 10% (v / v) DMSO.

51. The cryopreservation method according to any one of claims 1-50, wherein the cryopreservation solution comprises 0.05 M trehalose.

52. The cryopreservation method according to any one of claims 1-50, wherein the cryopreservation solution comprises 0.1 M trehalose.

53. The cryopreservation method according to any one of claims 1-52, wherein the cryopreservation solution comprises 5 g / L human albumin.

54. The cryopreservation method according to any one of claims 1-52, wherein the cryopreservation solution comprises 10 g / L human albumin.

55. The cryopreservation method according to any one of claims 1-54, wherein the cryopreservation solution comprises 2 g / L methylcellulose.

56. The cryopreservation method according to any one of claims 1-54, wherein the cryopreservation solution comprises 5 g / L methylcellulose.

57. The cryopreservation method according to any one of claims 1-56, wherein the cryopreservation solution comprises serum-free culture medium, 10% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin and 2 g / L methylcellulose.

58. The cryopreservation method according to any one of claims 1-56, wherein the cryopreservation solution comprises serum-free culture medium, 7.5% (v / v) DMSO, 0.05 M trehalose, 10 g / L human albumin and 2 g / L methylcellulose.

59. The cryopreservation method according to any one of claims 1-56, wherein the cryopreservation solution comprises serum-free culture medium, 7.5% (v / v) DMSO, 0.1 M trehalose, 5 g / L human albumin and 2 g / L methylcellulose.

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