CN114467913B

CN114467913B - Long-term cryopreservation and recovery method for animal tumor tissue, clinical tumor tissue and biopsy tumor tissue sample

Info

Publication number: CN114467913B
Application number: CN202111388072.4A
Authority: CN
Inventors: 刘红亚; 王前进; 刘霞; 李继新
Original assignee: Wuhan Saier Langling Technology Co ltd
Current assignee: Wuhan Saier Langling Technology Co ltd
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-11-11
Anticipated expiration: 2041-11-22
Also published as: CN114467913A

Abstract

The invention discloses a long-term cryopreservation and recovery method of animal tumor tissues, clinical tumor tissues and biopsy tumor tissue samples, and belongs to the technical field of animal cell cryopreservation. The freezing method is to wash fresh tissues and freeze and store the tissues by using special freezing solution; thawing frozen tissue in water bath, rinsing with special thawing liquid, treating with erythrocyte lysate, centrifuging, and rinsing with thawing liquid to obtain thawed tissue cell. The method provided by the invention can achieve very good cryopreservation and recovery effects aiming at different types of animal tissue cells; after freezing storage for up to two years, the cell culture still has very good passage activity after recovery.

Description

Long-term cryopreservation and recovery method for animal tumor tissue, clinical tumor tissue and biopsy tumor tissue sample

Technical Field

The invention belongs to the technical field of animal cell cryopreservation, and particularly relates to a long-term cryopreservation and recovery method for animal tissues, clinical tissues and biopsy samples.

Background

The cryopreservation of animal tissue, clinical tissue and biopsy sample is a method of washing and trimming fresh tissue, placing the fresh tissue in cryopreservation liquid, and transferring the fresh tissue into liquid nitrogen for long-term preservation. The recovery of animal tissue, clinical tissue and biopsy sample is to obtain active tissue cell from the frozen tissue in the frozen tube through special treatment process.

At present, the activity of tissue cells obtained by resuscitation of a plurality of animal tissues through the existing cryopreservation and resuscitation technology is obviously reduced compared with that before cryopreservation, and the tissue cells cannot be stably passaged or even completely passaged. Moreover, most of the cryopreservation and resuscitation techniques are only specific to a certain animal cell, and different cryopreservation and resuscitation techniques are often required for different animal tissues and cells.

For example, the adipose stem cell cryopreservation solution and the cryopreservation method thereof provided by the chinese patent application CN108739795A, or the porcine fetal fibroblast cryopreservation solution provided by the chinese granted patent CN107173382A, are all directed to adipose stem cells or porcine fetal fibroblasts. More importantly, these cryopreservation and resuscitation techniques, with an optimal protection period of only about 30 days, beyond which the integrity of the resuscitated cells will be significantly reduced; the activity of some recovered cells can not be recovered by 100 percent, and the cells can not be effectively passaged for a long time.

Disclosure of Invention

In view of the above deficiencies of the prior art, the present invention provides a method for long-term cryopreservation and resuscitation of animal tumor tissue, clinical tumor tissue and biopsy tumor tissue samples, which is specifically realized by the following techniques.

The long-term cryopreservation and recovery method of animal tissues, clinical tissues and biopsy samples comprises a long-term cryopreservation method and a corresponding recovery method; the long-term cryopreservation method comprises the following steps:

s1, taking fresh animal tissues, clinical tissues or biopsy tissues, cleaning, trimming and cutting to be minced; adding a freezing solution with the volume at least 3 times of the tissue volume, and uniformly mixing to obtain a minced tissue suspension; said frozen stock solution comprises 10-40wt% FBS, 5-15wt% DMSO, 0.1-10wt% sucrose, 0.1-10wt% trehalose, 30-70wt% primary cell complete medium;

s2, subpackaging the minced tissue suspension, carrying out program cooling at-80 ℃, and finally transferring into liquid nitrogen to finish cryopreservation;

the recovery method of the frozen tissues comprises the following steps:

p1, taking the frozen tissues, carrying out water bath at 37 ℃ until the frozen tissues are completely melted, rinsing for 1 time by using a resuscitation solution, and centrifuging at a low speed to collect tissue precipitates; then digesting with digestive juice, filtering and collecting cell suspension, and centrifugally collecting cell sediment; adding erythrocyte lysate, mixing and lysing the mixture by gently inverting the mixture from top to bottom at room temperature, and centrifuging the mixture to collect cell sediment; adding 5ml of 1XPBS (phosphate buffer solution), adjusting the pH value to 7.2-7.4, rinsing for 1 time by using a resuscitation solution, and centrifuging again to collect cell precipitates;

and P2, suspending the cell sediment obtained in the step P1 by using the primary cell complete culture medium, and inoculating the cell sediment into a cell culture bottle for culture, namely completing the recovery of the frozen tissue.

Preferably, the primary cell basal medium comprises DMEM/F12 medium supplemented with 1 XN 2, 1mM sodium pyruvate, 1 Xnon-essential amino acids, 10ng/ml sodium selenate, 5. Mu.g/ml transferrin, 10ng/ml human epidermal growth factor, 100. Mu.g/ml heparin, 5ng/ml progesterone and 100U/ml penicillin, 100. Mu.g/ml streptomycin, 0.25. Mu.g/ml amphotericin B, and further comprising 1nM complex growth factor specific for animal tissues, clinical tissues or biopsy tissues. The compound growth factor used is different for different kinds and parts of animal tissues, for example, when the primary cells of the human high-grade glioma are frozen, the compound growth factor can refer to 10ng/ml human fibroblast growth factor and 10ng/ml basic fibroblast growth factor disclosed in the grant publication of about 08 days 09 of 2020; for another example, when cryopreserving prostatic adenocarcinoma tissue or dog penile carcinoma tissue, the complex growth factor used may be 1nM synthetic androgen. These complex growth factors can be purchased directly from zeimer feishel corporation.

Preferably, in the method for resuscitating cryopreserved tissue, the resuscitation fluid is: the resuscitation solution is: DMEM medium was supplemented with 5 Xnonessential amino acids, 20mM glutathione as a reducing agent, 0.21ug/ml fatty methyl linoleate, 20mM L-alanyl-L-glutamine, 25ug/ml transferrin, 25ng/ml progesterone, and 5nM complex growth factors corresponding exclusively to animal tissues, clinical tissues or biopsy tissues.

Preferably, in the method for resuscitating cryopreserved tissue, said digestive juice of step P1 comprises 1 XT-type collagenase, 0.25% pancreatin-0.2% EDTA.

Preferably, in the method for recovering the frozen tissues, the centrifugation conditions of the steps P1-P2 are 800-1000rm and 4-10min.

Preferably, the method for resuscitating a cryopreserved tissue, wherein the culture conditions in the cell culture flask of step P2 are 37 ℃,5% CO ₂ 。

Compared with the prior art, the invention has the advantages that: the method provided by the invention can realize very good cryopreservation and recovery effects aiming at different types of animal tissue cells; after freezing storage for nearly two years at most, the cells extracted after recovery still have very good activity and passage activity.

Drawings

FIG. 1 is a cell morphology map of primary isolated cells of the human high-grade glioma primary of example 1, magnified 100;

fig. 2 is a cell morphology map of primary cells isolated after one and a half year of cryopreservation of human high grade glioma tissue of example 1, at magnification: 100, respectively;

FIG. 3 is a graph showing the growth curves of primary human glioma cells G02-3 and G02-5 of example 1;

fig. 4 is a cell morphology map of primary isolated cells of human prostate cancer of example 2, at magnification: 100, respectively;

fig. 5 is a cell morphology map of primary cells isolated after twenty-two months of cryopreservation of human prostate cancer tissue of example 2, at magnification: 100, respectively;

FIG. 6 is a graph of the growth of primary human prostatic adenocarcinoma cells PC01 and PC01-2 of example 2;

FIG. 7 is a cell morphology map of primary dog penile cancer cells of example 3, at 100 magnification;

fig. 8 is a cell morphology map of primary cells isolated two months after cryopreservation of the dog penile cancer tissue of example 3, on a magnification: 100;

FIG. 9 is a graph of the growth of primary dog penile cancer cells CTVT01 and CTVT01-2 of example 3.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. The experimental procedures, which are carried out under the conditions not specified in the following examples, are generally carried out under the conventional experimental conditions or under the conditions recommended by the manufacturer, said room temperature being 26 ℃ and the starting materials or reagents used in the present invention being commercially available unless otherwise specified.

Example 1: primary isolation culture, freezing storage and recovery of human high-grade glioma primary cells, isolation culture and subculture of recovery primary cells

1. Primary isolation culture of human high-grade glioma primary cells

(1) Fresh clinical glioma resection specimens, of the WHOIV grade, glioblastoma, were obtained from wuhan council hospitals by the hospital ethics committee, with consent from the patient or patient guardian, and after informed consent was issued.

(2) The excised specimens were immediately placed into a collection tube containing pre-chilled sterile tissue preservation solution (containing 1000U/ml penicillin, 1000. Mu.g/ml streptomycin sulfate, 2.5. Mu.g/ml amphotericin, and 50. Mu.g/ml gentamicin) and immediately placed into a 4 ℃ specimen transport box for transport to the laboratory for cell separation within 4 hours.

(3) Primary separation culture: rapidly washing the obtained tissue in a biological safety cabinet for 1 time by using absolute ethyl alcohol, rapidly washing the tissue for 2 times by using 1xPBS (pH7.2-7.4), removing blood vessels, fat and necrotic tissue parts under a microscope by using a dissecting forceps, and shearing the tissue into a minced shape by using dissecting scissors; digesting the tumor tissue with 20ml of a digestion solution containing 1 XI type collagenase and 0.25% pancreatin-0.2% EDTA for 1.5 hours, filtering with a 100 μm filter membrane to collect the cell pellet, 1000rpm,4min, centrifuging, and collecting the cell pellet; 10ml of erythrocyte lysate (Solarbio, R1010) was added to the cell pellet, and the cell pellet was collected by centrifugation after lysis at room temperature for 5min. Adding 5ml of 1XPBS (pH value 7.2-7.4) into the cell sediment, 1000rpm for 4 minutes, and centrifuging to collect the cell sediment; resuspending the cell pellet in DF31 complete medium, placing in a cell culture flask, and culturing at 37 deg.C under 5% CO2;

the DF31 complete medium comprises the following components: DMEM/F12 medium supplemented with 1 XN 2, 1mM sodium pyruvate, 1 Xnon-essential amino acids, 10ng/ml sodium selenate, 5. Mu.g/ml transferrin, 10ng/ml human epidermal growth factor, 10ng/ml human fibroblast growth factor, 10ng/ml basic fibroblast growth factor, 100. Mu.g/ml heparin, 5ng/ml progesterone and 100U/ml penicillin, 100. Mu.g/ml streptomycin, 0.25. Mu.g/ml amphotericin B; the same applies to the DF31 medium used in example 1 below.

The primary human glioma cells successfully isolated and cultured according to the method are observed under a microscope, and the cell morphology is shown in figure 1, wherein the cells are fusiform and have irregular branches or bifurcations. The cell is classified and named as human high-grade glioma primary cell XHHG-02, and is preserved in the China center for type culture Collection (China, wuhan university) in 12 months and 12 days in 2018, with the preservation number of CCTCC NO: C2018215. the human glioma primary cell is G02-3.

2. Cryopreservation of human high-grade glioma tissues

(1) Fresh clinical glioma resection specimens, of the WHOIV grade, glioblastoma, of the same clinical organization as in example 1, were obtained from the wuhan cooperation hospital by the ethical committee of the hospital, after consent from the patient or patient guardian and written informed consent.

(2) The excised specimens were immediately placed into a collection tube containing pre-cooled sterile tissue preservation solution (containing 1000U/ml penicillin, 1000. Mu.g/ml streptomycin sulfate, 2.5. Mu.g/ml amphotericin, and 50. Mu.g/ml gentamicin), and immediately placed into a 4 ℃ sample transport box, transported to the laboratory within 4 hours for cell separation;

(3) Freezing and storing tissues: tissue harvesting in biosafety cabinet, rapidly rinsing with absolute ethanol 1 time, rapidly rinsing with 1 × PBS (pH 7.2-7.4) 2 times, removing blood vessel, fat and necrotic tissue under microscope with dissecting forceps, and cutting tissue to minced shape with dissecting scissors. Adding special glioma tissue cryopreservation liquid with the volume 3 times that of the tissue, uniformly mixing, subpackaging 1ml of each tube into a cryopreservation tube, and transferring into liquid nitrogen for long-term storage after programmed cooling; wherein the glioma tissue frozen stock solution is as follows: 50% FBS, 10% DMSO, 5% sucrose, 5% trehalose, 30% DF31 complete medium.

3. Resuscitation and isolated culture of human high-grade glioma tissue frozen for one and a half years

Taking out the cryopreservation tube containing the human high-grade glioma cryopreservation tissue (namely the cryopreservation tissue of the embodiment 2) from the liquid nitrogen, and rapidly and completely thawing in water bath at 37 ℃; after complete thawing (about 1 min), rinsing once with 10ml of resuscitating solution, centrifuging at low speed to collect the tissue pellet, adding 20ml of digestive juice containing 1 × I type collagenase and 0.25% pancreatin-0.2% EDTA for 1.5h, filtering with 100 μm filter membrane to collect the cell pellet, 1000rpm for 4min, centrifuging and collecting the cell pellet; 10ml of erythrocyte lysate (Solarbio, R1010) was added to the cell pellet, and the cell pellet was collected by centrifugation after lysis at room temperature for 5min. Adding 5ml of 1XPBS (pH value 7.2-7.4) into the cell sediment, 1000rpm for 4min, and centrifuging to collect the cell sediment; the cell pellet was resuspended in DF31 complete medium, placed in a cell culture flask, and cultured at 37 ℃ under 5% CO2.

The primary human glioma cells successfully cultured and isolated according to the method are observed under a microscope, and the cell morphology is shown in figure 2, and the cells are spindle-shaped or fibrous and are irregularly branched or forked. The cell classification was named human high-grade glioma primary cells, i.e., G02-5.

4. Subculturing of revived primary human glioma cells

(1) When the abundance of cells cultured in T25 flasks reached 80%, the cells were rinsed 2 times with 1XPBS (pH 7.2-7.4) and the monolayer cells were digested with 1ml of 0.05% trypsin-EDTA for 2-3min.

(2) Digestion was stopped by addition of 2ml DF31 complete medium.

(3) Centrifuging at 1000rpm for 4min, removing supernatant, collecting cell suspension, resuspending with 1ml DF31 complete medium, supplementing medium, and culturing in T25 culture flask at a ratio of 1 to 2.

Human high-grade glioma primary cells G02-3 and G02-5 subcultured as described above were passaged for approximately 50 days in succession. The primary cells extracted from the one-year and semi-frozen human glioma tissues can still keep the normal growth of the proliferation state, and the cell growth curve established by culture is shown in figure 3.

Example 2: primary isolation culture, freezing storage and recovery of human prostate cancer adenocarcinoma primary cells, isolation culture and subculture of recovery primary cells

1. Primary isolation culture of human prostate cancer adenocarcinoma primary cells

(1) Fresh clinical prostate cancer resection specimens were obtained from the Wuhan Tongji Hospital as prostate adenocarcinoma (Gleason score 4+4= 8) with patient or patient guardian consent and informed consent by the Hospital ethics Committee.

(2) The excised specimens were immediately placed into a collection tube containing pre-chilled sterile tissue preservation fluid (containing 1000U/ml penicillin, 1000. Mu.g/ml streptomycin sulfate, 2.5. Mu.g/ml amphotericin, and 50. Mu.g/ml gentamicin), and immediately placed into a 4 ℃ specimen transport box for transport to the laboratory for cell separation within 4 hours.

(3) Primary separation culture: tissue harvesting in biosafety cabinet, rapidly rinsing with absolute ethanol 1 time, rapidly rinsing with 1 × PBS (pH 7.2-7.4) 2 times, removing blood vessel, fat and necrotic tissue under microscope with dissecting forceps, and cutting tissue to minced shape with dissecting scissors. The tumor tissue was digested with 2.5 hours by adding 20ml of a digestion solution containing collagenase type 1X I and pancreatin-0.2% EDTA, and the cell pellet was collected by filtration through a 100 μm filter, 1000rpm,4 minutes, and collected after centrifugation. 10ml of erythrocyte lysate (Solarbio, R1010) was added to the cell pellet, lysed at room temperature for 5min, and the cell pellet was collected by centrifugation. Adding 5ml of 1XPBS (pH value 7.2-7.4) into the cell sediment, 1000rpm for 4 minutes, and centrifuging to collect the cell sediment; resuspending the cell pellet in DF41 complete medium, placing in a cell culture flask, 5% CO at 37 deg.C ₂ Culturing under the condition;

the DF41 complete culture medium comprises the following components: DMEM/F12 medium supplemented with 1 XN 2, 1mM sodium pyruvate, 1 Xnonessential amino acids, 10ng/ml sodium selenate, 5. Mu.g/ml transferrin, 10ng/ml human epidermal growth factor, 1nM artificial androgen, 100. Mu.g/ml heparin, 5ng/ml progesterone and 100U/ml penicillin, 100. Mu.g/ml streptomycin, 0.25. Mu.g/ml amphotericin B; the medium composition of DF41 in the following examples was the same.

The primary human prostatic adenocarcinoma cells successfully cultured and isolated according to the method are observed under a microscope, and the cell morphology is shown in figure 4, and the cells are fusiform, irregularly branched or forked. The cell classification is named as human prostatic adenocarcinoma primary cells TJPC-01, which is preserved in China center for type culture Collection (China, wuhan university) at 26 months 10 in 2021, with the preservation number of CCTCC NO: C2021287. the primary human prostatic adenocarcinoma cell is PC01.

2. Cryopreservation of human prostate cancer adenocarcinoma tissue

(1) Fresh prostate adenocarcinoma resection specimens of prostate adenocarcinoma (Gleason score 4+4= 8) from the wuhan congratsu hospital were obtained as the same clinical tissue as example 8, with consent from the patient or patient guardian and informed consent by the hospital ethics committee.

(3) Freezing and storing tissues: the tissue was harvested in a biosafety cabinet, rapidly rinsed 1 time with absolute ethanol, rapidly rinsed 2 times with 1 × PBS (pH 7.2-7.4), microscopically removed blood vessels, fat and necrotic tissue portions with dissecting forceps, and minced with dissecting scissors. Adding special prostatic cancer tissue freezing solution with the volume 3 times that of the tissue, uniformly mixing 1ml of the special prostatic cancer tissue freezing solution per tube, subpackaging the mixture into freezing tubes, performing programmed cooling, and transferring the mixture into liquid nitrogen for long-term storage. The prostatic cancer tissue freezing solution is as follows: 10% FBS, 5% DMSO, 5% sucrose, 10% trehalose, 70% DF41 complete medium.

3. Recovery and separation culture of human prostatic cancer adenocarcinoma tissue frozen for twenty-two months

Taking out the cryopreservation tube containing the human prostatic adenocarcinoma cryopreservation tissue from the liquid nitrogen seeds, and rapidly thawing in water bath at 37 ℃; rinsing with 10ml resuscitation solution once after completely thawing, centrifuging at low speed to collect tissue precipitate, adding 20ml 1 × I type gelatinProenzyme and 0.25% pancreatin-0.2% EDTA digestion of the tumor tissue for 2.5h; and the cell pellet was collected by filtration through a 10 μm filter at 1000rpm for 4 minutes, and collected by centrifugation. 10ml of erythrocyte lysate (Solarbio, R1010) was added to the cell pellet, and the cell pellet was collected by centrifugation after lysis at room temperature for 5min. Adding 10ml of recovery solution into the cell sediment, rinsing once at 1000rpm for 4min, and centrifuging to collect the cell sediment; resuspending the cell pellet with DF41 complete medium, placing in a cell culture flask, at 37 ℃ 5% ₂ Culturing is carried out under the conditions.

The primary human prostatic adenocarcinoma cell PC01-2 successfully isolated and cultured according to the method is shown in FIG. 5 under the microscope.

4. Subculturing recovered primary human prostate cancer adenocarcinoma cells

(1) When the abundance of cells cultured in T25 flasks reached 80%, the cells were rinsed 2 times with 1XPBS (pH 7.2-7.4) and the monolayer cells were digested with 1ml of 0.1% trypsin-EDTA for 5min.

(2) Digestion was stopped by addition of 2ml DF41 complete medium.

(3) Centrifuging at 1000rpm for 4min, removing supernatant, collecting cell suspension, resuspending with 1ml DF41 complete medium, supplementing medium, and culturing in T25 culture flask at a ratio of 1 to 2.

Carrying out continuous passage on primary human high-grade glioma cells PC01 and PC01-2 cultured by passage according to the method for about 40 days; primary cells extracted from the human prostatic adenocarcinoma tissue frozen and stored in twenty-two months can still keep normal growth in a proliferation state, and a cell growth curve established by culture is shown in figure 6.

Example 3: primary isolation culture, cryopreservation and resuscitation of dog penile cancer primary cells, isolation culture and subculture of resuscitation primary cells

1. Primary isolation culture of dog penis cancer primary cells

(1) The excised specimens were immediately placed into a collection tube containing pre-chilled sterile tissue preservation fluid (containing 1000U/ml penicillin, 1000. Mu.g/ml streptomycin sulfate, 2.5. Mu.g/ml amphotericin, and 50. Mu.g/ml gentamicin), and immediately placed into a 4 ℃ specimen transport box for transport to the laboratory for cell separation within 4 hours.

(2) Primary separation culture: rapidly washing the obtained tissue in a biological safety cabinet for 1 time by using absolute ethyl alcohol, rapidly washing the tissue for 2 times by using 1XPBS (pH value of 7.2-7.4), removing blood vessels, fat and necrotic tissue parts under a microscope by using a dissecting forceps, and shearing the tissue into a minced shape by using dissecting scissors; the tumor tissue was digested by adding 20ml of a digestion solution containing collagenase type 1X I and pancreatin 0.25% to 0.2% EDTA for 2.5 hours, and the cell pellet was collected by filtration through a 100 μm filter, 1000rpm,4min, and collected after centrifugation. Adding 10ml of erythrocyte lysate (Solarbio, R1010) into the cell sediment, carrying out lysis for 5min at room temperature, and centrifuging to collect the cell sediment; 5ml of 1XPBS (pH 7.2-7.4) was added to the cell pellet at 1000rpm for 4min, and the cell pellet was collected by centrifugation. Resuspending the cell pellet with DF41 complete medium, placing in a cell culture flask, at 37 ℃ 5% ₂ Culturing under the condition;

DF41 complete medium: DMEM/F12 medium supplemented with 1 XN 2, 1mM sodium pyruvate, 1 Xnonessential amino acids, 10ng/ml sodium selenate, 5. Mu.g/ml transferrin, 10ng/ml human epidermal growth factor, 1nM artificial androgen, 100. Mu.g/ml heparin, 5ng/ml progesterone and 100U/ml penicillin, 100. Mu.g/ml streptomycin, 0.25. Mu.g/ml amphotericin B; the medium composition of DF41 in the following examples was the same.

The primary dog penile cancer cells successfully isolated and cultured according to the above method are named as CTVT01, and the cell morphology observed under a microscope is shown in FIG. 7.

2. Cryopreservation of penis cancer tissues of dogs

(1) The excised specimen is immediately put into a collecting tube containing precooled sterile tissue preservation solution (containing 1000U/ml penicillin, 1000 mug/ml streptomycin sulfate, 2.5 mug/ml amphotericin and 50 mug/ml gentamicin), and is immediately put into a 4 ℃ sample transport box, and is transported to a laboratory for cell separation within 4 h;

(2) Freezing and storing tissues: rapidly washing the obtained tissue in a biological safety cabinet for 1 time by using absolute ethyl alcohol, rapidly washing the tissue for 2 times by using 1XPBS (pH value of 7.2-7.4), removing blood vessels, fat and necrotic tissue parts under a microscope by using a dissecting forceps, and shearing the tissue into a minced shape by using dissecting scissors; adding special prostatic cancer tissue freezing solution with the volume 3 times that of the tissue, uniformly mixing 1ml of the special prostatic cancer tissue freezing solution per tube, subpackaging the mixture into freezing tubes, performing programmed cooling, and transferring the mixture into liquid nitrogen for long-term storage. Wherein the frozen stock solution of the penis cancer tissue of the dog is as follows: 10% of FBS, 5% of DMSO, 5% of sucrose, 10% of trehalose, 70% of DF41 complete medium, exactly the same as the formulation of the frozen stock solution of human prostate cancer adenocarcinoma primary cells of example 2.

3. Resuscitation and isolated culture of dog penis cancer tissue frozen for two months

Taking out the cryopreservation tube containing the prostatic adenocarcinoma cryopreservation tissue from the liquid nitrogen seeds, and rapidly thawing in water bath at 37 ℃; rinsing once with 10ml of resuscitating solution after completely thawing, centrifuging at low speed to collect the tissue pellet, adding 20ml of digestive juice containing 1 × I type collagenase and 0.25% pancreatin-0.2% EDTA for 2.5h, filtering with 100 μm filter membrane to collect the cell pellet, 1000rpm for 4min, centrifuging, and collecting the cell pellet; adding 10ml erythrocyte lysate (Solarbio, R1010) into the cell sediment, cracking for 5min at room temperature, and centrifuging to collect the cell sediment; adding 10ml of recovery solution into the cell sediment for rinsing once at 1000rpm for 4min, and centrifuging to collect the cell sediment. Resuspending the cell pellet in DF41 complete medium, placing in a cell culture flask, 5% CO at 37 deg.C ₂ Culturing is carried out under the conditions.

The primary dog penile cancer cell CTVT01-2 cultured successfully according to the method is isolated, and the cell morphology observed under a microscope is shown in FIG. 8.

4. Subculturing of revived primary cells of canine penile cancer

(2) Digestion was stopped by addition of 2ml DF41 complete medium.

The primary dog penile tumor cells CTVT01 and CTVT01-2 subcultured according to the method can keep the normal growth of the primary cells extracted from the dog penile cancer tissues frozen for two months after continuous passage for about 40 days, and the cell growth curve established by culture is shown in figure 9.

As can be seen from the above examples and the accompanying drawings, various animal tissues after long-term cryopreservation (currently, the longest period of 22 months) still have very good cell passage activity after recovery by using the cryopreservation method of the present invention, and it can be seen that the cryopreservation and recovery method provided by the present invention has very good cryopreservation and recovery effects on various animal tissue cells.

Claims

1. The long-term cryopreservation recovery method of animal tumor tissues, clinical tumor tissues and biopsy tumor tissue samples is characterized in that the long-term cryopreservation method comprises the following steps:

s1, taking fresh animal tumor tissues, clinical tumor tissues and biopsy tumor tissue samples, cleaning, trimming and cutting to be minced; adding a freezing solution with the volume at least 3 times of the tissue volume, and uniformly mixing to obtain a minced tissue suspension; said frozen stock solution comprises 10-40wt% FBS, 5-15wt% DMSO, 0.1-10wt% sucrose, 0.1-10wt% trehalose, 30-70wt% primary cell complete medium;

the recovery method of the frozen tissues comprises the following steps:

p2, suspending the cell sediment obtained in the step P1 by using the primary cell complete culture medium, inoculating the cell sediment into a cell culture bottle for culture, and completing the recovery of the frozen tissue;

the primary cell complete culture medium contains DMEM/F12 culture medium added with 1 XN 2, 1mM sodium pyruvate, 1 Xnonessential amino acids, 10ng/ml sodium selenate, 5 mu g/ml transferrin, 10ng/ml human epidermal growth factor, 100 mu g/ml heparin, 5ng/ml progesterone, 100U/ml penicillin, 100 mu g/ml streptomycin, 0.25 mu g/ml amphotericin B and 1nM composite growth factor specially corresponding to animal tissues, clinical tissues or biopsy tissues;

the resuscitation solution is: DMEM medium was supplemented with 5 Xnonessential amino acids, 20mM glutathione as a reducing agent, 0.21ug/ml fatty methyl linoleate, 20mM L-alanyl-L-glutamine, 25ug/ml transferrin, 25ng/ml progesterone, and 5nM complex growth factors corresponding exclusively to animal tissues, clinical tissues or biopsy tissues.

2. The method for resuscitating frozen tissue in accordance with claim 1, wherein said digestive juice of step P1 is edta-0.2% containing 1 xl collagenase, 0.25% pancreatin.

3. The method for resuscitation from frozen tissue according to claim 1, wherein the centrifugation conditions of steps P1-P2 are 800-1000rm,4-10min.

4. The method for resuscitating long-term cryopreserved tissue of claim 1, wherein the culture conditions in the cell culture flask of step P2 are 37 ℃,5% co ₂ 。