Primary tumor cell stocks

Abstract

The application discloses a mild and low-toxicity optimized formula of a cell cryopreservation solution, the cryopreservation solution contains a protective amine substance, a protein stabilizer, a sugar alcohol dipropionate, an antioxidant and a buffer, does not contain DMSO and serum, and can maintain the integrity, biological activity and stability of primary cells under mild freezing conditions. The application also discloses an optimized cell freezing storage and recovery method, the cryopreservation solution of the low-toxicity formula is used, a gradual freezing method is adopted, cells gradually enter an ice crystal state in the freezing process, cell damage is reduced, a rapid thawing method is adopted, the cells are rapidly thawed out from a low-temperature environment, and a certain proportion of a recovery liquid is added to help the cells recover.

Description

Technical Field

[0001] This invention relates to the field of cell cryopreservation, and more specifically to a mild, low-toxicity cryopreservation solution for primary tumor cells. Background Technology

[0002] Primary cells are important experimental materials, but their short lifespan and limited quantity make it difficult for researchers to obtain a sufficient number of cells from the same source at the same time. Therefore, cryopreservation and thawing techniques for primary cells are crucial, allowing the use of the same batch of cells at different time points, ensuring the consistency and accuracy of experiments.

[0003] Currently, primary cell cryopreservation technology mainly uses highly toxic DMSO (dimethyl sulfoxide) as the cryopreservation solution, which easily leads to cell death and cell membrane damage. During thawing and recovery, factors such as excessively rapid or slow temperature changes and improper centrifugation can also cause excessively high cell death rates and decreased cell viability after recovery. Therefore, existing technologies are insufficient to meet the requirements for long-term preservation and efficient recovery of primary cells, necessitating the development of a low-toxicity, high-efficiency cryopreservation solution. Summary of the Invention

[0004] To address the technical problems of high toxicity of cryopreservation solution components to cells, poor stability of cell biological characteristics during cell cryopreservation, and low cell viability after cell thawing, the present invention provides a primary tumor cell cryopreservation solution containing protective amines, protein stabilizers, sugar alcohol dipropionate, antioxidants, and buffers, but free of dimethyl sulfoxide (DMSO) and serum.

[0005] In some embodiments, the protective amine is selected from trimethylamine oxide, formamide, acetamide, or combinations thereof; preferably, the protective amine is trimethylamine oxide; more preferably, the concentration of the trimethylamine oxide is 5-15% (w / v).

[0006] In some embodiments, the protein stabilizer is selected from inositol galactoside, trehalose, maltose, sucrose, glucose, lactose, fructose, dextran, mesotriose, raffinose, sucrose trisaccharide, cellobiose, chitobiose, aspergillus trisaccharide, maltotriulose, lactulose, or combinations thereof; preferably, the protein stabilizer is inositol galactoside; more preferably, the concentration of inositol galactoside is 2-10% (w / v).

[0007] In some embodiments, the sugar alcohol dipropionate component is mannitol dipropionate; the concentration of the mannitol dipropionate is 8-15 mM; more preferably, the concentration of the mannitol dipropionate is 10 mM.

[0008] In some embodiments, the antioxidant is polyvinylpyrrolidone; preferably, the concentration of the polyvinylpyrrolidone is 0.5-2.0% (w / v); preferably, the concentration of the polyvinylpyrrolidone is 1.5%.

[0009] In some embodiments, the buffer is a 75-90% (v / v) 0.9% sodium chloride solution, RPMI 1640, or high-glucose DMEM medium.

[0010] In some embodiments, the tumor cells are one or more of gastric cancer, liver cancer, and breast cancer.

[0011] The second aspect of the present invention provides a method for cryopreservation and thawing of primary tumor cells, the method comprising the following steps: pre-cryopreservation treatment of cells, cell freezing and storage, and cell thawing and thawing; wherein the pre-cryopreservation treatment of cells includes washing, centrifugation, removal of culture medium, and addition of cryopreservation solution of primary cells before cryopreservation.

[0012] In some embodiments, the composition of the cryopreservation solution is as described in the first aspect of the present invention.

[0013] In some embodiments, the freezing process employs a gradual cooling and freezing method, allowing the cells to gradually enter an ice crystal state during freezing (reducing cell damage); and the storage process employs liquid nitrogen cryopreservation.

[0014] In some embodiments, during the thawing and revival process, a rapid thawing method is used to quickly thaw the cells from the low-temperature environment, while simultaneously adding a revival liquid to aid cell revival. After revival, the cells are gradually adapted to the cellular environment by progressively adding culture medium and reducing the cell concentration, ultimately ensuring cell growth and division. Preferably, the revival liquid is a basal culture medium supplemented with cell proliferation factors and vitamins.

[0015] The advantages of this invention over the prior art are:

[0016] 1) This invention overcomes the problem that commonly used cryopreservation solutions contain highly toxic components that adversely affect cell biological characteristics. It provides an optimized formulation of a mild and low-toxicity cell cryopreservation solution containing protective amines, protein stabilizers, sugar alcohol dipropionate antioxidants, buffers, and other components, which can maintain the integrity, biological activity, and stability of primary cells under mild freezing conditions.

[0017] 2) This invention proposes an optimized method for cell cryopreservation and storage. Before cryopreservation, primary cells undergo specific treatments, including washing, centrifugation, removal of culture medium, and addition of cryopreservation solution, which more effectively protects the biological characteristics of primary cells during freezing. During freezing, a low-toxicity cryopreservation solution is used, employing a gradual cooling and freezing method to allow cells to gradually enter an ice crystal state, reducing cell damage. During storage, liquid nitrogen cryopreservation is used to ensure long-term cell preservation.

[0018] 3) This invention proposes an optimized cell thawing and resuscitation method that minimizes cell damage and enhances cell viability after resuscitation. During thawing, a rapid thawing method is employed to quickly thaw cells from the low-temperature environment, while a certain proportion of resuscitation fluid is added to aid cell resuscitation. After resuscitation, cells are gradually adapted to the cellular environment by progressively adding culture medium and reducing cell concentration, ultimately ensuring cell growth and division. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This shows the cell proliferation after droplet embedding and culture of different types of fresh tumor cells, tumor cells frozen in cell cryopreservation solution 1 and conventional cryopreservation solution for 12 months, and the results of the culture. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise specified, the reagents, instruments, equipment, and methods used in this invention are all commercially available reagents, instruments, equipment, and methods conventionally available in this technical field.

[0023] Example 1: Preparation of Cell Cryopreservation Solution

[0024] The following cell cryopreservation solution was prepared for the cryopreservation of primary cells in Example 1.

[0025] Standard cryopreservation solution: RPMI 1640, 10% DMSO, 25% serum;

[0026] Cell cryopreservation solution 1: RPMI 1640, trimethylamine oxide 10%, inositol galactoside 5%, mannitol dipropionate 10mM, polyvinylpyrrolidone 1%.

[0027] Cell cryopreservation solution 2: RPMI 1640, formamide 10%, inositol galactoside 5%, mannitol dipropionate 10mM, polyvinylpyrrolidone 1%.

[0028] Cell cryopreservation solution 3: RPMI 1640, trimethylamine oxide 10%, trehalose 5%, mannitol dipropionate 10mM, polyvinylpyrrolidone 1%.

[0029] Cell cryopreservation solution 4: RPMI 1640, trimethylamine oxide 10%, inositol galactoside 8%, polyvinylpyrrolidone 1%.

[0030] Example 2: Primary Cell Cryopreservation

[0031] 2.1 Primary cell preparation

[0032] Fresh primary tumor tissue was transferred to cell culture dishes and washed five times with sterile saline containing 1% (v / v) antimycin A to remove non-tumor tissue. The processed tumor tissue was then transferred to culture dishes and divided into small fragments approximately 1 mm in diameter using sterile scalpels, scissors, and forceps. The fragmented tumor tissue was transferred to 50 mL centrifuge tubes and centrifuged at 1,200 rpm for 3 min. After centrifugation, the supernatant was discarded, and DMEM / F12 and cell dispersing enzyme solution (200 U / mL Collagenase) were added to the cell pellet. The mixture was then incubated at 37°C with low-speed shaking for 2 h. The digested tumor cell clusters were filtered through a 300 μm nylon filter and centrifuged at 1200 rpm for 3 min, discarding the supernatant. The primary tumor cells to be cryopreserved were checked and confirmed to be free of fungal, bacterial, and mycoplasma contamination, ensuring that the cells were in the optimal logarithmic growth phase before cryopreservation. Adherent cells were digested, washed, and centrifuged at 160 g for 5–10 min to remove the culture medium.

[0033] 2.2 Cryopreservation

[0034] First, determine the appropriate cryopreservation density and volume for the cells, and then calculate the volume of the cryopreservation solution to be used. The cryopreservation density is 4–20 × 10⁻⁶. 6 / ml, cryopreservation volume is 0.25~1ml / tube. Resuspend cells in an appropriate amount of cryopreservation solution and immediately aliquot into cell cryovials. Use a gradual cooling and freezing method to allow cells to gradually enter an ice crystal state during freezing, reducing cell damage. Specifically, first place the cryovials in an environment below 0℃ (e.g., a -20℃ freezer) for approximately 30 minutes, then place them in a -80℃ freezer for approximately 12 hours, and finally transfer the cryovials to liquid nitrogen for storage.

[0035] Cell viability was determined using the MTT assay at the following times: before cryopreservation, after 3 months of cryopreservation, after 6 months of cryopreservation, and after 12 months of cryopreservation. Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan crystals, which then deposit in the cells.

[0036] Example 3 Thawing and Recovery

[0037] A rapid thawing method was used to quickly thaw cells from the low-temperature environment, while simultaneously adding a certain proportion of resuscitation fluid to aid cell revival. After revival, the cells were gradually adapted to the cellular environment by progressively adding culture medium and decreasing the cell concentration, ultimately ensuring cell growth and division. Specifically, a pre-warmed basal medium (RPMI 1640 or DMEM) was prepared at 37°C, with a volume 15–30 times that of the cryopreservation medium. The cryovials were quickly removed from liquid nitrogen and placed in a 37°C water bath with gentle shaking. When only a grain-sized ice cube remained, the cells were quickly removed from the water bath. The cell suspension was immediately diluted with resuscitation fluid (basal medium supplemented with appropriate amounts of cell proliferation factors and vitamins), and gently pipetted about three times to mix. Then, the cells were centrifuged at 180g for 5–10 minutes to obtain a cell pellet for later use. Fresh culture medium was gradually added, and the cells were slowly resuspended using a pipette. The cells were then transfected into cell culture dishes / flasks, and their condition was observed under a microscope before being placed in a cell culture incubator. After 24 hours, the cell condition was observed, and the cell culture medium was replaced with fresh medium.

[0038] Example 4: Relationship between cryopreservation time and cell viability of different cell cryopreservation solutions

[0039] Table 1 shows the cell viability assay results of primary gastric cancer cells, primary liver cancer cells, and primary breast cancer cells cryopreserved using conventional cryopreservation solution and cell cryopreservation solution 1, respectively, before cryopreservation, at 3 months, 6 months, and 12 months of cryopreservation, using the MTT assay. With prolonged cryopreservation, the rate of decline in tumor cell viability was slower in cell cryopreservation solution 1 than in conventional cryopreservation solution. After 12 months, the cell viability of gastric cancer cells cryopreserved using cell cryopreservation solution 1 and conventional cryopreservation solution were 80% and 65%, respectively; the cell viability of liver cancer cells cryopreserved using cell cryopreservation solution 1 and conventional cryopreservation solution were 75% and 62%, respectively; and the cell viability of breast cancer cells cryopreserved using cell cryopreservation solution 1 and conventional cryopreservation solution were 68% and 54%, respectively. Compared with conventional cryopreservation solution, cell cryopreservation solution 1 is more conducive to the long-term preservation of primary cancer cells.

[0040] Table 1. Cell viability of primary cancer cells frozen in cell cryopreservation solution 1 and conventional cryopreservation solution.

[0041]

[0042]

[0043] Table 2 shows the cell viability assay results of primary gastric cancer cells cryopreserved using cell cryopreservation solution 1, cell cryopreservation solution 2, cell cryopreservation solution 4, and cell cryopreservation solution 4 before cryopreservation, 3 months after cryopreservation, 6 months after cryopreservation, and 12 months after cryopreservation, respectively, using the MTT assay.

[0044] Table 2. Cell viability of different primary gastric cancer cells cryopreserved in cell cryopreservation solutions 1-4

[0045] cryopreservation solution 3 months 6 months 12 months Cell cryopreservation solution 1 92％ 86％ 80％ Cell cryopreservation solution 2 87％ 79％ 73％ Cell cryopreservation solution 3 88％ 75％ 65％ Cell cryopreservation solution 4 75％ 68％ 58％

[0046] Example 5: Viability of cryopreserved cells after thawing in different cell cryopreservation solutions

[0047] Cells frozen for 12 months were revived using the method described in Example 3. After a period of culture, the culture medium was discarded, and 2 mL of DMEM / F12 and 100 μL of cell dispersing enzyme solution (200 u / mL Collagenase, Type I) were added to the culture flask for digestion for 15–30 min. The digested tumor cell clusters were filtered through a 125 μm nylon filter and centrifuged at 1200 rpm. Collagen gel solution was added to the resuspended cell pellet and mixed thoroughly to obtain a cell-collagen mixture. 30 μL of the cell-collagen mixture was dropped onto a 6-well culture plate to form a collagen gel drop and incubated at 37°C for 1 h. 3 mL of PCM-2 was added, and the plates were incubated at 37°C in a 5% CO2 incubator. The 0-time group was stained and fixed on day 1 of culture, and the experimental group was stained and fixed on day 7 of culture. Cells were stained with neutral red for 2 hours, washed twice with 4 mL PBS for 15 min each time, fixed with neutral formalin for 40 min, soaked in distilled water for 20 min, and then air-dried. The collagen gel droplets were scanned and analyzed using the Primage image analysis system to calculate the cell proliferation rate. Cell proliferation rate (GR) = average OD value of the experimental group / average OD value of the 0-time group. A GR ≥ 0.8 was considered as tumor cell survival. Cell culture success rate = total number of surviving samples / total number of samples × 100%.

[0048] Figure 1 This paper shows the cell proliferation after droplet embedding and culture of fresh tumor cells, cell cryopreservation solution 1, and tumor cells cryopreserved for 12 months in conventional cryopreservation solution. The results indicate that gastric cancer cells, liver cancer cells, and breast cancer cells cryopreserved in cell cryopreservation solution 1 of Example 1 provided by this invention can still proliferate after 12 months of cryopreservation, and the proliferation effect is significantly better than that of tumor cells cryopreserved in conventional cryopreservation solution.

[0049] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the scope of the invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0050] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This description is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A cryopreservation solution for primary tumor cells, characterized in that, The cell cryopreservation solution comprises a protective amine, a protein stabilizer, a sugar alcohol dipropionate, an antioxidant, and a buffer, and is free of dimethyl sulfoxide and serum. The protective amine is trimethylamine oxide, the protein stabilizer is inositol galactoside, the sugar alcohol dipropionate is mannitol dipropionate, the antioxidant is polyvinylpyrrolidone, the mass-volume ratio of trimethylamine oxide is 5-15%, the mass-volume ratio of inositol galactoside is 2-10%, the concentration of mannitol dipropionate is 8-15 mM, the mass-volume ratio of polyvinylpyrrolidone is 0.5-2.0%, and the buffer is RPMI 1640.

2. The application of the cell cryopreservation solution according to claim 1 in the cryopreservation of primary tumor cells, characterized in that, The tumor cells are one or more of the following: gastric cancer, liver cancer, and breast cancer.

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