A method for cryopreservation of biological cells using a cryogenic tube

Abstract

The present application relates to the technical field of biology, in particular to a method for cryopreserving biological cells by using a freezing tube, which is characterized by the following specific steps: (1) collecting the cultured biological cells, (2) filling and sealing the freezing tube, (3) a cell cryopreservation procedure, and (4) cell recovery culture. The present application has the advantages that the technical solution of the present application can use the freezing tube to preserve a small amount of biological cells and ensure that the recovery activity of the cryopreserved cells reaches a degree similar to that of manual cryopreservation, thus having great promotional value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for cryopreserving biological cells by using a frozen semen tube. BACKGROUND

[0002] China has rich resources of livestock and poultry breeds, not only the world famous high-yield breeds introduced, but also a large number of excellent local breeds with strong adaptability and high production performance. Due to the lag in development, China's local breed resources have not been given enough attention, and there are also some problems in their own use, making the local breed advantage show a gradual declining trend, which needs to be protected and preserved. Biological sample preservation is an important method for germplasm resource preservation, and cryopreservation of cultured biological cells can provide materials for germplasm research and clonal recovery population, which is a high-efficiency and low-cost preservation method. However, because genetic material will become cancerous after a certain number of generations, biological cells that can be used as backup for germplasm resources cannot be cultured for a long time. At present, the methods for cryopreserving biological cells mainly include machine freezing, programmed freezing box freezing and manual freezing. Machine freezing requires the use of special equipment, mainly for semen and human embryos, and is commonly used in places with more abundant material conditions such as hospitals; programmed freezing box is simple to operate and has low requirements, and is widely used; manual freezing has the lowest requirements, and is widely used. In the common freezing method, the freezing tube has a capacity of 2.0 mL, and the number of cells contained is far more than the experimental demand of somatic cell cloning, and if the extra cells are continued to be cultured and frozen, they will not be able to adapt to the experimental demand after several generations, causing waste of sample resources. The frozen semen tube has a capacity of 300-400 μL, which is more suitable for general experimental use. Therefore, it is necessary to develop a method for preserving biological cells by using a frozen semen tube to avoid the waste of rare samples. SUMMARY

[0003] The purpose of the present application is to provide a method for cryopreserving biological cells by using a frozen semen tube, which can use a frozen semen tube to preserve a small amount of biological cells and ensure that the recovery activity of the cryopreserved cells reaches a level similar to that of manual freezing, and has great promotional value.

[0004] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is as follows:

[0005] A method for cryopreserving biological cells by using a frozen semen tube, specifically comprising the following specific steps:

[0006] (1) Collecting cultured biological cells:

[0007] When the cells grow to 70% to 80% confluence, prepare for digestion and freezing storage, use a pipette to remove the culture solution along the edge of the culture dish, add DMEM containing double antibodies for cleaning once, remove the DMEM, add 100 to 500 μl of 1% trypsin, and after observing the cell morphology under a microscope or observing the cell sheet detaching from the culture dish wall with the naked eye, add complete culture medium to terminate the digestion, uniformly blow and centrifuge at 1000 rpm for 3 min, discard the supernatant, and add cell freezing solution for resuspension;

[0008] (2) Filling and sealing of the freezing tube:

[0009] Use a 1 mL pipette to take 350 μl of the mixed cell solution, inject the freezing tube from the side without a cotton plug, slightly tilt the freezing tube to make the liquid flow to the middle of the freezing tube, and leave 1 to 1.5 cm at both ends; use a hemostat to clamp the freezing tube of about 0.5 cm in length on the side without a cotton plug, burn the hemostat on an alcohol lamp for about 2 to 3 s, clamp the freezing tube from the middle to the opening of the freezing tube that has been clamped for about 1 to 3 s, and observe whether the freezing tube is damaged by burning; repeat the burning and clamping action for 1 to 2 times, tilt the freezing tube, observe whether the liquid flows, and adjust the sealing until the liquid does not flow.

[0010] (3) Cell freezing procedure:

[0011] Freeze the filled and sealed cells according to the following steps: 4°C for 10 to 15 min, -20°C for 30 min, -80°C for 360 to 380 min, and transfer to liquid nitrogen for long-term storage.

[0012] (4) Cell recovery and culture:

[0013] Take the freezing tube out of the liquid nitrogen and place it in a 38°C water bath for 30 s; disinfect one end of the thawed freezing tube with an alcohol cotton ball and observe whether there is a liquid leakage phenomenon; in a sterile environment, cut the disinfected end of the freezing tube, vertically stand the freezing tube above the opening of the centrifugal tube, cut the other end, make the liquid flow into the centrifugal tube, centrifuge at 1500 to 3000 rpm for 5 min, discard the supernatant, add culture medium for resuspension, repeat the centrifugation once, discard the supernatant, add culture medium for resuspension, and transfer to a culture dish or a culture bottle for general cell culture.

[0014] Further, in step (1), the cells are 293T cells, buffalo skeletal muscle fibroblasts, or other animal tissue cells.

[0015] Further, in step (1), the complete culture medium is 90 mL of DMEM high-sugar culture medium + 10 mL of FBS fetal bovine serum + 10,000 U of penicillin-streptomycin.

[0016] Further illustrate that in step (1), the cell freezing solution is the complete culture medium 90 mL + DMSO 10 mL or the complete culture medium 80 mL + DMSO 20 mL.

[0017] Further illustrate that in step (2), the frozen semen fine tube is a Fuji Ping frozen semen fine tube or a Casu cow frozen semen fine tube.

[0018] Preferably, in step (3), the cell freezing procedure is: after the cell is filled and sealed, the cell is frozen, and the freezing is performed according to the following steps: 4℃, 10min, -20℃, 30min, -80℃, 360min, and then transferred to liquid nitrogen for long-term storage.

[0019] Due to the adoption of the above technical solutions, the present application has the following beneficial effects:

[0020] 1. The present application uses a frozen semen tube to store biological cells, which has the advantages of small capacity, less pollution, easy operation, and the like compared to a traditional freezing tube. At the same time, the frozen semen tube can also adapt to the needs of less experimental cells, and for experiments that do not require large quantities of cultured cells, the use of the frozen semen tube can avoid waste. For primary biological cells that are extremely difficult to sample, the frozen semen tube can limit the amount of cells taken, avoid genetic variation caused by multiple passages, and make the rare sample more fully utilized.

[0021] 2. The present application adjusts the manual freezing cell procedure for the frozen semen tube, adjusts the freezing procedure suitable for a 2.0ml freezing tube (4℃ 15min, -20℃ 60min, -80℃ 12h), and the new freezing procedure (4℃ 10min, -20℃ 30min, -80℃ 360min) is more suitable for the frozen semen tube, which can ensure the viability of the frozen cells and save the cooling time.

[0022] In summary, the 293T cells frozen by the scheme researched by the present applicant can proliferate to 60%-70% density within 48h, and the damage to the cells is minimal. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a growth condition diagram of the 293T cells in group A after being cultured for 24h in example 1;

[0024] Figure 2 is a growth condition diagram of the 293T cells in group B after being cultured for 24h in example 1;

[0025] Figure 3 is a growth condition diagram of the 293T cells in group A after being cultured for 48h in example 1;

[0026] Figure 4is a growth condition chart of the 293T cells in Group B in Example 1 after being cultured for 48h;

[0027] Figure 5 is a growth condition chart of the 293T cells in Example 2 after being cultured for 12h;

[0028] Figure 6 is a growth condition chart of the 293T cells in Example 2 after being cultured for 24h;

[0029] Figure 7 is a growth condition chart of the 293T cells in Example 2 after being cultured for 48h;

[0030] Figure 8 is a growth condition chart of the 293T cells in Group 10C in Example 3 after being cultured for 12h;

[0031] Figure 9 is a growth condition chart of the 293T cells in Group 101 in Example 3 after being cultured for 12h;

[0032] Figure 10 is a growth condition chart of the 293T cells in Group 20C in Example 3 after being cultured for 12h;

[0033] Figure 11 is a growth condition chart of the 293T cells in Group 201 in Example 3 after being cultured for 12h;

[0034] Figure 12 is a growth condition chart of the 293T cells in Group 10C in Example 3 after being cultured for 24h;

[0035] Figure 13 is a growth condition chart of the 293T cells in Group 101 in Example 3 after being cultured for 24h;

[0036] Figure 14 is a growth condition chart of the 293T cells in Group 20C in Example 3 after being cultured for 24h;

[0037] Figure 15 is a growth condition chart of the 293T cells in Group 201 in Example 3 after being cultured for 24h.DETAILED DESCRIPTION

[0038] In order to make the above objectives, features and advantages of the present application more apparent, specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. It will be apparent, however, to one skilled in the art that the present application can be practiced without some or all of these details. In other instances, well known process steps have not been described in detail in order to avoid unnecessarily obscuring the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. The techniques employed herein are generally known in the art.

[0039] Example 1:

[0040] 1. Liquid preparation

[0041] The liquid preparation method used in this example is as follows:

[0042] Complete medium: DMEM high-sugar medium is mixed with inactivated FBS (fetal bovine serum) at a volume ratio of 9:1, 10,000 U of double-antibiotic (0.1 mg / mL penicillin and 0.05 mg / mL streptomycin in phosphate buffered saline) is added to each 100 ml of the mixture, and it is stored at 4°C for future use.

[0043] Cell freezing medium: the above complete medium is mixed with DMSO (dimethyl sulfoxide) at a volume ratio of 9:1, and the resulting mixture is stored at 4°C for future use.

[0044] 1% trypsin: the trypsin solution is diluted 100 times with DMEM high-sugar medium, 500 μl is aliquoted, and it is stored at -20°C for future use.

[0045] 2. The operation method for freezing biological cells in the freezing tube used in this example is as follows:

[0046] (1) Collecting cultured biological cells: 293T cells are grown to 70% confluence and then prepared for digestion and freezing. The culture solution is removed along the edge of the culture dish using a pipette, and DMEM containing double-antibiotic is added for washing once. The DMEM is removed, 100 μl of 1% trypsin is added, and the cell morphology is observed under a microscope or the cells are observed to be detached from the culture dish wall in patches. The digestion is terminated by adding serum-containing medium, and the cells are uniformly blown after centrifugation at 1000 rpm for 3 min. The supernatant is discarded, and the cell freezing medium is added for resuspension.

[0047] (2) Filling and sealing of the freezing tube: 350 μl of the mixed cell solution is taken with a 1 mL pipette, and it is injected into the cotton plug-free side of the Fuji flat freezing tube. The freezing tube is slightly tilted so that the liquid flows to the middle of the freezing tube, and 1.5 cm is left at both ends. The cotton plug-free side of the freezing tube is clamped for about 0.5 cm in length using a hemostat, the hemostat is placed on an alcohol lamp for about 3 s, the freezing tube is clamped from the middle to the opening for about 3 s, and whether the freezing tube is damaged by the heat is observed. The burning-clamping action is repeated twice, the freezing tube is tilted, and whether the liquid is flowing is observed. The sealing is adjusted until the liquid stops flowing.

[0048] (3) Cell freezing procedure: To optimize the freezing cooling procedure, the packaged freezing tubes in this example are randomly and evenly divided into two groups, each group containing three tubes. The freezing tubes are wrapped with foam paper before cooling. The cooling procedures for the two groups are as follows: (group A) 4°C for 10 min, -20°C for 60 min, and -80°C for 360 min; (group B) 4°C for 30 min, -20°C for 120 min, and -80°C for 600 min. After the freezing procedure is completed, the freezing tubes in both groups are transferred to liquid nitrogen for long-term storage.

[0049] (4) Cell recovery culture: Take the frozen semen tube out of liquid nitrogen and place it in a 38°C water bath for 30 seconds. Disinfect one end of the thawed semen tube with an alcohol cotton ball and observe for any leakage. In a sterile environment, cut the disinfected end of the semen tube, vertically place the semen tube above the centrifuge tube opening, cut the other end, and allow the liquid to flow into the centrifuge tube. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add culture medium and resuspend by blowing, repeat centrifugation once, discard the supernatant, add complete culture medium and resuspend by blowing, transfer to a 30mm culture dish, add 1 tube of cells per dish, and 1.5ml of complete culture medium. Observe and take pictures of the growing cells at 24h and 48h.

[0050] Example 2:

[0051] 1. Liquid preparation

[0052] The liquid preparation method used in this example is as follows:

[0053] Complete culture medium: Mix DMEM high-sugar medium and inactivated FBS (fetal bovine serum) at a volume ratio of 9:1, add 10,000 U of double-antibiotic (0.1 mg / mL penicillin and 0.05 mg / mL streptomycin in phosphate buffered saline) to each 100 ml of the mixture, and store at 4°C for future use.

[0054] Cell freezing solution: Mix the above complete culture medium with DMSO (dimethyl sulfoxide) at a volume ratio of 9:1, and store the resulting mixture at 4°C for future use.

[0055] 1% trypsin: Dilute the trypsin solution 100-fold with DMEM high-sugar medium, aliquot 500μl, and store at -20°C for future use.

[0056] 2. The operation method for freezing biological cells using the semen tube in this example is as follows:

[0057] (1) Collecting cultured biological cells: 293T cells were grown to 70% confluence and prepared for digestion and freezing. Use a pipette to remove the culture medium along the edge of the culture dish, wash once with DMEM containing double-antibiotic, remove the DMEM, add 100μl of 1% trypsin, and observe the cell morphology under a microscope or the cell sheet detaching from the culture dish wall with the naked eye. Add serum-containing medium to terminate digestion, resuspend evenly by blowing, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, and resuspend in cell freezing solution.

[0058] (2) Filling and sealing of the freezing tube: 350 μl of the mixed cell solution was taken with a 1 mL pipette and injected into the freezing tube from the side without the cotton plug. The freezing tube was slightly tilted so that the liquid flowed to the middle of the freezing tube, leaving 1.5 cm at both ends. The freezing tube was clamped for about 0.5 cm in length from the side without the cotton plug using a hemostat. The hemostat was placed on an alcohol lamp and burned for about 3 s. The freezing tube was held from the middle to the opening for about 3 s, and whether the freezing tube was damaged by the heat was observed. The burning and holding operation was repeated twice. The freezing tube was tilted, and whether the liquid flowed was observed. The sealing was adjusted until the liquid stopped flowing.

[0059] (3) Cell freezing procedure: The cooling procedure of this example was as follows: 4°C for 5 min, -20°C for 10 min, and -80°C for 120 min. After the freezing procedure was completed, the freezing tube was transferred to liquid nitrogen for long-term storage.

[0060] This procedure further shortened the time used in each procedure of Example 1, and was an attempt to find the shortest time required for freezing storage. From the results, it can be concluded that shortening the freezing procedure time to the extent of Example 2 does not affect the growth of the recovered cells.

[0061] (4) Cell recovery and culture: The freezing tube was taken out of the liquid nitrogen and placed in a 38°C water bath for 30 s. One end of the thawed freezing tube was disinfected with an alcohol cotton ball, and whether there was a liquid leakage was observed. In a sterile environment, the disinfected end of the freezing tube was cut, the freezing tube was vertically placed above the opening of a centrifuge tube, and the other end was cut so that the liquid flowed into the centrifuge tube. The centrifuge tube was centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the complete culture medium was added for resuspension by blowing. The centrifugation was repeated once, the supernatant was discarded, and the complete culture medium was added for resuspension by blowing. The cells were transferred to a 30 mm culture dish, 1 tube of cells and 1.5 ml of complete culture medium were added to each dish. The growth of the cells was observed and photographed at 12 h, 24 h, and 48 h, respectively.

[0062] Example 3:

[0063] The liquid preparation method used in this example was as follows:

[0064] Complete culture medium: DMEM high-sugar medium was mixed with inactivated FBS (fetal bovine serum) at a volume ratio of 4:1. 10,000 U of double-antibiotic (0.1 mg / mL penicillin and 0.05 mg / mL streptomycin in a phosphate buffered saline solution) was added to each 100 ml of the mixed solution, and the mixture was stored at 4°C for later use.

[0065] 10% cell freezing solution: The complete culture medium and DMSO (dimethyl sulfoxide) were mixed at a volume ratio of 9:1, and the resulting mixture was stored at 4°C for later use.

[0066] 20% cell freezing solution: mix the complete medium and DMSO (dimethyl sulfoxide) at a ratio of 4:1 by volume, and store the mixture at 4°C for later use.

[0067] 1% trypsin: dilute the trypsin solution 100 times with DMEM high-sugar medium, and store 500 μl aliquots at -20°C for later use.

[0068] 2. The operation method for freezing biological cells in the freezing tube is as follows:

[0069] (1) Collecting cultured biological cells: 293T cells were grown to 70%-80% confluence before being prepared for freezing. The culture medium was removed along the edge of the culture dish using a pipette, and DMEM containing double antibodies was added for washing once. After removing the DMEM, 100 μl of 1% trypsin was added. After observing the cell morphology under a microscope or observing the cell sheet detaching from the culture dish wall with the naked eye, the digestion was terminated by adding serum-containing medium. After uniform blowing, centrifugation was performed at 1000 rpm for 3 min, and the supernatant was discarded. Different concentrations of cell freezing solution were added for resuspension.

[0070] (2) Filling and sealing of the freezing tube: 350 μl of the mixed cell solution was taken with a 1 mL pipette gun, and injected into the Fuji flat freezing tube from the side without a cotton plug. The freezing tube was slightly tilted to make the liquid flow to the middle of the freezing tube, leaving 1.5 cm at both ends. The hemostatic forceps were used to clamp the freezing tube about 0.5 cm long on the side without a cotton plug, and the hemostatic forceps were placed on the alcohol lamp for about 3 s. The freezing tube was clamped from the middle to the opening for about 3 s, and whether the freezing tube was damaged was observed. The burning-clamping action was repeated twice, and the freezing tube was tilted to observe whether the liquid was flowing. The sealing was adjusted until the liquid stopped flowing.

[0071] (3) Cell freezing procedure: To optimize the composition of the cell freezing solution, this example divided the packaged freezing tubes into 2 groups according to the concentration of the cell freezing solution, with 3 tubes in each group, and no wrapping for cooling.

[0072] The freezing solution composition and cooling procedure of the 2 groups are as follows:

[0073] (101 group) freezing with a freezing tube, 10% DMSO freezing solution, 4°C for 10 min, -20°C for 30 min, and -80°C for 360 min;

[0074] (201 group) freezing with a freezing tube, 20% DMSO freezing solution, 4°C for 10 min, -20°C for 30 min, and -80°C for 360 min. After the freezing procedure was completed, the freezing tubes in both groups were transferred to liquid nitrogen for long-term storage.

[0075] (4) Cell recovery culture: Take the frozen sperm tube out of liquid nitrogen and place it in a 38°C water bath for 30 seconds. Disinfect one end of the thawed sperm tube with an alcohol cotton ball and observe for any leakage. In a sterile environment, cut the sterilized end of the frozen sperm tube, vertically place the frozen sperm tube above the centrifuge tube opening, and cut the other end to allow the liquid to flow into the centrifuge tube. Centrifuge at 3000 rpm for 5 minutes, discard the supernatant, add 300 μL of culture medium and resuspend by blowing, repeat the centrifugation once, discard the supernatant, add complete culture medium and resuspend by blowing, transfer to a 30 mm culture dish, add 1 frozen sperm tube of resuspension (about 300 μL) and 1.5 mL of complete culture medium per dish. Observe and take pictures of the growth of the cultured cells at 12 h and 24 h, respectively.

[0076] Comparative Example:

[0077] 1. The liquid preparation method used in this example is as follows:

[0078] Complete culture medium: Mix DMEM high-sugar medium and inactivated FBS (fetal bovine serum) at a volume ratio of 4:1, add 10,000 U of double-antibiotic (0.1 mg / mL penicillin and 0.05 mg / mL streptomycin in phosphate buffered saline) to each 100 mL of the mixture, and store at 4°C for future use.

[0079] 10% cell cryopreservation solution: Mix the complete culture medium and DMSO (dimethyl sulfoxide) at a volume ratio of 9:1, and store the resulting mixture at 4°C for future use.

[0080] 20% cell cryopreservation solution: Mix the complete culture medium and DMSO (dimethyl sulfoxide) at a volume ratio of 4:1, and store the resulting mixture at 4°C for future use.

[0081] 1% trypsin: Dilute the trypsin solution 100-fold with DMEM high-sugar medium, aliquot 500 μl, and store at -20°C for future use.

[0082] 2. The operation method for freezing biological cells using frozen sperm tubes in this example is as follows:

[0083] (1) Collecting cultured biological cells: 293T cells were grown to 70%-80% confluence before being prepared for digestion and freezing. Use a pipette to remove the culture medium along the edge of the culture dish, wash once with DMEM containing double-antibiotic, remove the DMEM, add 100-500 μl of 1% trypsin, and observe the cell morphology under a microscope or the cell detachment from the culture dish wall with the naked eye. After adding serum-containing medium to terminate digestion, resuspend by blowing, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, and resuspend in different concentrations of cell cryopreservation solution.

[0084] (2) Freezing tube filling and sealing: Freezing tube is 2 mL screw tube. When using, 1 mL cell and cell freezing solution mixture is injected into the tube, and the cap is screwed tightly.

[0085] (3) Cell freezing procedure: The packaged freezing tubes are randomly and evenly divided into 2 groups, 3 tubes in each group, and cooling is performed without wrapping.

[0086] The freezing solution components and cooling procedures of the two groups are as follows:

[0087] (10C group) Freezing with ordinary 2 ml freezing tube, 10% cell freezing solution, 4°C for 20 min, -20°C for 60 min, -80°C for 12 h;

[0088] (20C group) Freezing with ordinary 2 ml freezing tube, 20% cell freezing solution, 4°C for 20 min, -20°C for 60 min, -80°C for 12 h;

[0089] After the freezing procedure is completed, the freezing tubes of the two groups are transferred to liquid nitrogen for long-term storage.

[0090] (4) Cell recovery culture: The freezing tube is taken out from the liquid nitrogen and placed in a 38°C water bath for 2 min to completely thaw. The surface of the thawed freezing tube is wiped with an alcohol cotton ball, and the cap is opened in a sterile environment. The liquid is transferred to a 1.5 mL centrifuge tube and centrifuged at 3000 rpm for 5 min. The supernatant is discarded, 1 mL of complete culture medium is added and blown to resuspend, and the centrifugation is repeated once. The supernatant is discarded, the complete culture medium is blown to resuspend, and transferred to a 30 mm culture dish. Each dish is added with 300 μL of resuspended liquid (about equal to the volume of 1 freezing tube), and 1.5 ml of complete culture medium. The cultured cells are observed and photographed at 12 h and 24 h, respectively, to record their growth conditions.

[0091] Example 4:

[0092] 1. Liquid preparation, same as example 1.

[0093] 2. The operation method of this example for freezing biological cells in freezing tubes is as follows:

[0094] (1) Collecting cultured biological cells: 293T cells are grown to 80% confluence for digestion and freezing. The culture solution is removed along the edge of the culture dish with a pipette, and 1% trypsin is added. After observing the cell morphology under a microscope or observing the cell sheet detaching from the culture dish wall with the naked eye, the digestion is terminated by adding serum-containing culture medium. After blowing and uniformity, centrifuge at 1000 rpm for 3 min, discard the supernatant, and add cell freezing solution for resuspension.

[0095] (2) Filling and sealing of the straws: 350 μl of the mixed cell solution was taken with a 1 mL pipette and injected into the straws from the side without the cotton plug. The straws were slightly tilted to allow the liquid to flow to the middle of the straws, leaving 1 cm at both ends. The straws were clamped with hemostats for about 0.5 cm from the side without the cotton plug. The hemostats were placed on an alcohol lamp for about 3 s, and the straws were clamped from the middle to the opening for about 3 s. The straws were observed to see if they were damaged by the heat. The clamping and heating were repeated twice. The straws were tilted to see if the liquid was flowing, and the sealing was adjusted until the liquid stopped flowing.

[0096] (3) Cell cryopreservation procedure: To optimize the cryopreservation cooling procedure, the packaged straws were wrapped with foam paper before cooling. The cooling procedures were as follows: 4°C for 10 min, -20°C for 60 min, and -80°C for 360 min. After the cryopreservation procedure, the straws were transferred to liquid nitrogen for long-term storage.

[0097] (4) Cell recovery and culture: The straws were taken out of the liquid nitrogen and placed in a 38°C water bath for 30 s. One end of the thawed straw was disinfected with an alcohol cotton ball, and the straw was observed to see if there was any leakage. In a sterile environment, the disinfected end of the straw was cut, and the straw was vertically placed above the opening of a centrifuge tube. The other end was cut to allow the liquid to flow into the centrifuge tube. The tube was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended by adding culture medium and repeating the centrifugation once. The supernatant was discarded, and the cells were resuspended by adding complete culture medium. The cells were transferred to a 30 mm culture dish, and 1.5 ml of complete culture medium was added to each dish.

[0098] Example 5:

[0099] 1. Liquid preparation: The same as in Example 1.

[0100] 2. The operation method for cryopreserving biological cells using straws in this example is as follows:

[0101] (1) Collection of cultured biological cells: 293T cells were grown to 80% confluence and prepared for digestion and cryopreservation. The culture medium was removed from the edge of the culture dish using a pipette, and the culture dish was washed once with DMEM containing double antibodies. The DMEM was removed, and 300 μl of 1% trypsin was added. The cells were observed under a microscope to see if they were rounded or detached from the wall of the culture dish. When the cells were rounded or detached from the wall of the culture dish, the digestion was stopped by adding serum-containing culture medium. The cells were resuspended by blowing and centrifuging at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in cell cryopreservation solution.

[0102] (2) Filling and sealing of the freezing tube: 350 μΐ of mixed cell solution was taken by 1 mL pipette and injected into the freezing tube from the side without cotton plug. The freezing tube was slightly tilted to make the liquid flow to the middle of the freezing tube, leaving 1 cm at both ends. The freezing tube was clamped by hemostatic forceps for about 0.5 cm from the side without cotton plug. The hemostatic forceps was placed on the alcohol lamp for about 3 s. The freezing tube was clamped from the middle to the opening for about 2 s. Whether the freezing tube was burnt was observed. The clamping and burning operation was repeated for 2 times. The freezing tube was tilted to observe whether the liquid flowed. The sealing was adjusted until the liquid did not flow.

[0103] (3) Cell freezing procedure: In order to optimize the freezing and cooling procedure, the packaged freezing tube was wrapped by foam paper before cooling in this embodiment. The cooling procedure was as follows: 4°C for 10 min, -20°C for 60 min, -80°C for 360 min. After the freezing procedure, the freezing tube was transferred to liquid nitrogen for long-term storage.

[0104] (4) Cell recovery and culture: The freezing tube was taken out from the liquid nitrogen and placed in a 38°C water bath for 30 s. The freezing tube was disinfected by alcohol cotton ball at one end. Whether there was liquid leakage was observed. In a sterile environment, the disinfected end of the freezing tube was cut. The freezing tube was vertically placed above the centrifuge tube. The other end was cut to make the liquid flow into the centrifuge tube. The centrifuge tube was centrifuged at 2000 rpm for 5 min. The supernatant was discarded. The complete culture medium was added to resuspend the cells. The cells were centrifuged again. The supernatant was discarded. The complete culture medium was added to resuspend the cells. The cells were transferred to a 30 mm culture dish. One tube of cells and 1.5 ml of complete culture medium were added to each dish.

[0105] Test results:

[0106] In Example 1, it was observed that at 24 h, the cells in group A adhered more and began to proliferate. The number of adhered and proliferated cells in group B was less than that in group A. At 48 h, the cells in group A grew to 50%-60% density. The growth of 293T cells in group B was significantly less than that in group A, only growing to about 40% density. It was preliminarily determined that the freezing procedure in group B was not suitable for freezing tube freezing.

[0107] In Example 2, the time of each procedure in Example 1 was further shortened, which was an attempt to explore the shortest time required for freezing. It was observed that at 12 h, the cells basically adhered. At 24 h, the cells grew to 40%-50% density. At 48 h, the cells grew to 80-90% density. From the results, it can be concluded that shortening the freezing procedure time to the extent of Example 2 will not affect the growth of recovered cells.

[0108] In Example 3, the recovery effect of cryopreservation tubes and cryopreservation tubes for freezing cells was compared in the case of using the same concentration of cell freezing solution. There is a big difference between the appearance of the cryopreservation tube and the cryopreservation tube. If the same freezing procedure is used, it will greatly affect the cell viability in the cryopreservation tube. The present patent is a study to optimize the freezing procedure of the cryopreservation tube. At 12h of recovery culture, it can be observed that the growth of 10C, 20C and 201 groups is better than that of 101 group. At 24h, it can be observed that the growth of 201 group and 101 group is significantly better than that of other groups, followed by 20C group and 10C group. It is expected that the cells can grow to the subculture density after 48h.

[0109] In summary, the 293T cells frozen using the method of the present application can proliferate to 60%-70% density within 48h, and the damage to the cells is minimal.

[0110] The present application is not limited to the foregoing specific embodiments. The present application extends to any novel one, or any new combination, of the characteristics disclosed in this specification, as well as to any novel method or process disclosed in any step, or any new combination, of the steps of the disclosed methods or processes.

Claims

1. A method for cryopreserving biological cells using sperm tubules, characterized in that: Specifically comprising the following specific steps: (1) Collecting cultured biological cells: When the cells grow to 70%-80% confluence, prepare for digestion and freezing, use a pipette to remove the culture solution along the edge of the culture dish, add DMEM containing double antibodies for cleaning once, remove the DMEM, add 100-500 μl of 1% trypsin, and then observe the cell morphology under a microscope or observe the cell sheet detaching from the culture dish wall with the naked eye. After that, add complete culture medium to terminate the digestion, blow evenly, centrifuge at 1000 rpm for 3 min, discard the supernatant, and add cell freezing solution for resuspension; the cells are 293T cells; the cell freezing solution is 90 mL of complete culture medium + 10 mL of DMSO dimethyl sulfoxide or 80 mL of complete culture medium + 20 mL of DMSO dimethyl sulfoxide; the complete culture medium is 90 mL of DMEM high-sugar culture medium + 10 mL of FBS fetal bovine serum + 10,000 U of penicillin-streptomycin; (2) Filling and sealing of the freezing tube: Use a 1 mL pipette to take 350 μl of mixed cell solution, inject it into the freezing tube from the side without a cotton plug, slightly tilt the freezing tube to make the liquid flow to the middle of the freezing tube, and leave 1-1.5 cm at both ends; use a hemostat to clamp the 0.5 cm length of the freezing tube on the side without a cotton plug, burn the hemostat on an alcohol lamp for 2-3 s, clamp the freezing tube 1-3 s from the middle to the opening of the freezing tube that has been clamped, and observe whether the freezing tube is damaged; repeat the burning and clamping action 1-2 times, tilt the freezing tube, and observe whether the liquid is flowing, adjust the sealing until the liquid stops flowing; the freezing tube is a Fuji flat freezing tube or a Casu cattle freezing tube; (3) Cell freezing procedure: The filled and sealed cells are frozen according to the following steps: 4°C for 10 min, -20°C for 30 min, -80°C for 360 min, and then transferred to liquid nitrogen for long-term storage; (4) Cell recovery and culture: Take the freezing tube out of the liquid nitrogen and place it in a 38°C water bath for 30 s; disinfect one end of the thawed freezing tube with an alcohol cotton ball and observe whether there is any leakage; in a sterile environment, cut the disinfected end of the freezing tube, vertically stand the freezing tube above the centrifuge tube opening, cut the other end, and make the liquid flow into the centrifuge tube, centrifuge at 1500-3000 rpm for 5 min, discard the supernatant, add culture medium and resuspend by blowing, repeat the centrifugation once, discard the supernatant, add culture medium and resuspend by blowing, and then transfer it to a culture dish or a culture bottle for general cell culture.

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