CN110558312B

CN110558312B - Cartilage tissue low-temperature refrigeration preservation solution and application thereof

Info

Publication number: CN110558312B
Application number: CN201910954720.4A
Authority: CN
Inventors: 宋云庆; 欧阳溪
Original assignee: Craibertai Biotechnology Co ltd
Current assignee: Craibertai Biotechnology Co ltd
Priority date: 2019-10-09
Filing date: 2019-10-09
Publication date: 2020-07-28
Anticipated expiration: 2039-10-09
Also published as: CN110558312A

Abstract

The invention discloses a cartilage tissue low-temperature refrigeration preservation solution and application thereof, wherein the preservation solution comprises one or more of a DMEM culture medium, HEPES 1-30 mmol/L, a component A1-20mmol/m L, a transforming growth factor β 10-100ng/m L, a component B10-100ng/m L, chondroitin sulfate 10-100 g/L and a matrix metalloproteinase inhibitor 100-1000ug/m L, wherein the component A is mircotiana acid or Q-VD-OPH, and the component B is cartilage-derived morphogenetic protein 1, BMP or PDGF.

Description

Cartilage tissue low-temperature refrigeration preservation solution and application thereof

Technical Field

The invention belongs to the field of biomedical engineering, and particularly relates to a cartilage tissue low-temperature refrigeration preservation solution and application thereof.

Background

Articular cartilage is an elastic load-bearing tissue which forms a movable joint surface, has the characteristics of reducing friction, absorbing shock, conducting and buffering load, provides the biomechanical function for a human body for decades, and has no function of replacing articular cartilage with any material at present.

In cases of more severe damage and disease of articular cartilage, replacement transplantation of articular cartilage containing active chondrocytes is an ideal treatment (meniscal and ankle cartilage transplants are more common). However, the common treatment means in the current clinical treatment process mainly comprises drug relief and artificial joint (metal composite material) replacement. The medicine is mostly adopted for alleviating the symptoms of patients who can reluctantly move, but not the root causes, and meanwhile, the irreversible damage to partial organ tissues is difficult to avoid by using the medicine for a long time. For more serious patients, the method of artificial joint replacement transplantation is often adopted for treatment.

For the replacement of the artificial joint, besides reliable mechanical strength, the defects are also obvious: 1. the movement is limited after replacement, and the movement performance of the artificial joint cannot be compared with the movement performance of a normal joint containing cartilage; 2. after the artificial joint is implanted, the patient needs to remove or destroy a large amount of normal bone tissues of the patient in the operation process to fix the artificial joint, in the process, a large amount of blood vessels and nerve tissues at the joint are damaged, and the artificial joint can hardly regenerate after the operation. Patients can feel numbness and pain at the operation position for a long time in the future life; 3. the service life is limited, the annual wear consumption of the artificial joint under the load movement is increased, and the common service life is between 10 and 20 years under the condition of not being influenced by large external force. After the service life is approached, the risks of dislocation, joint surface fracture and the like are easy to occur, and at the moment, secondary operation is needed; 4. the operation cost is high, the price of the artificial joint is usually the highest price of all bone grafting materials, and the cost of single joint replacement can reach tens of thousands of yuan.

With the intensive research on joint and articular cartilage in modern medicine, it is proved that the formation and maintenance of articular cartilage are dependent on the metabolic activity of chondrocytes, which ensures the stabilization of cartilage matrix so that articular cartilage can be always maintained in a normal state. All the nutrient sources of mature chondrocytes are obtained by diffusion of the cartilage matrix, relying solely on joint synovial fluid. Maintaining cartilage permeability is therefore a joint factor that ensures survival of chondrocytes. The normal articular cartilage does not contain blood vessels and lymphatic vessels, and does not depend on nerves to transmit information, so that the immunogenicity of the articular cartilage is low, and the possibility of transplantation among allogenic bodies is brought. Chondrocytes are very sensitive, and although usually stable, they respond to and even die when stimulated by many environmental factors, and also change the permeability of cartilage. Such as: growth factors, interleukins, matrix molecules, drugs, mechanical loads, changes in fluid pressure, loss of moisture, changes in temperature, etc.

Cartilage tissue, a more complex tissue, is composed of chondrocytes, matrix and fibers. In clinical practice, cartilage tissue is generally selected for direct transplantation treatment of diseases due to the limitations of growth activity of chondrocytes in cartilage tissue, doubling time, culture period, and other factors, and allogeneic articular cartilage produced by commercial companies is also used in the conventional surgical process, but the effect is not good after use, and the transplanted cartilage is worn and seriously cracked in a short time (within 1 year). This indicates that cartilage cells in the transplanted articular cartilage are not alive or the material itself has no cell activity, and the preparation and implementation of the transplantation of the articular cartilage have more problems to be solved, the most important of which include: 1. the source of the material, in addition to the screening of the donor for medical history and disease status, requires strict control of the age, occupation and joint health of the donor; 2. the preparation of the material, as an implantable medical material, needs to be sterile, pyrogen-free and basically non-immunogenic, does not damage cartilage cells and does not change the permeability of articular cartilage; 3. and (4) preserving cartilage. There are two methods for in vitro preservation of cartilage tissue, one is cartilage cryopreservation, and the other is cartilage cryopreservation.

CN 104837339 a discloses a method for storing compositions of biomaterials including cartilage and preservation of the properties of biomaterials. The composition comprises matrix metalloproteinase inhibitors, wherein the matrix metalloproteinase inhibitors include doxycycline, TIMP, compounds that up-regulate endogenous TIMP, PCK3145, BB-2516, BB-94, and the like, at a concentration of 1.0nM to 1000. mu.M. The composition comprises extracellular solution such as DMEM and intracellular solution such as nutrient mixture. The use of 0-300uM doxycycline preserves pig cartilage for one month at 4 ℃, and doxycycline improves cartilage conductivity and permeability retention in the presence of viable cells.

CN 109566601A discloses a cartilage preservation solution and its application method, the cartilage preservation solution comprises potassium dihydrogen phosphate, histidine or histidine salt, lactobionic acid, sucrose, allopurinol, low molecular dextran-40, NaCl, KCl, magnesium sulfate, penicillin, reduced glutathione, adenosine, trehalose, catechin, vitamin E and chondroitin sulfate, the solvent is deionized water, the pH value is 7.35-7.52, and the cartilage preservation solution is used for preserving cartilage at 0-4 ℃. The preservation solution group has the best preservation effect in the aspects of cartilage morphology, cartilage cell morphology, collagen fiber integrity and uniform thickness compared with the traditional-80 ℃ cryopreservation (cryopreservation group), organ preservation solution (UW solution group) and 4 ℃ conventional preservation (control group).

CN 102308787B discloses a cartilage protection solution, wherein each 1000ml cartilage preservation solution contains 20-30mmol of potassium dihydrogen phosphate, 80mmol of histidine or 15mmol of histidine salt, 75-95mmol of lactobionic acid, 50-70mmol of sucrose, 0.8-1.2mmol of allopurinol, low molecular dextran-40: 45-55g, 45-55mmol of NaCl, 25-35mmol of KCl, 3-7mmol of magnesium sulfate, 70-90U of penicillin, 2-4mmol of reduced glutathione, 3-7mmol of adenosine, 2-5g of trehalose and the balance of deionized water, wherein the pH value of the cartilage preservation solution is 7.33-7.53. The preservation time is 5 weeks, and an electron microscope shows that the preservation speed of the preservation solution for preserving the chondrocytes and the cartilage structure at 4 ℃ is obviously lower than that of a traditional freezing method preservation group.

CN 104938476B discloses osteochondral graft preservation solution and a preparation method thereof, which comprises the following steps: 80-125 mmol of glucose, 0.1-2.0 mmol of amino acid, 0.2-2.0 mmol of antioxidant, 5-60 nmol of basic fibroblast growth factor, 1-120 mmol of inorganic salt, 1-9 mmol of vitamin, 1-2 mmol of sodium pyruvate, 50-70U of penicillin and the balance of deionized water; preparing a solution, adjusting the pH value to 7.30-7.50, and filtering for sterilization; cleaning fresh allogeneic osteochondral graft with sterile normal saline for 3-5 times, and soaking in prepared osteochondral graft preservation solution; storing at 4 ℃. The survival rate of the chondrocytes is 85 percent after 21 days of storage.

Margie S in the article "Enhanng Osteochondral alloy viablility" showed that cartilage was preserved at 4 ℃ for 3 weeks, and that the addition of cytokines IGF-1 and ZVAD to the preservation solution increased the chondrocyte activity to 56.4% and 52.4%, respectively, and the survival rate of the control group was 31.2%.

Although the above prior art has made some progress in preserving articular cartilage, the following disadvantages still remain:

(1) the cartilage preservation time is short and is less than 5 weeks;

(2) chondrocyte survival remains low for long-term storage.

Disclosure of Invention

The invention aims to provide a cartilage tissue cryo-preservation solution and an application thereof, aiming at the defects in the prior art, the cartilage tissue cryo-preservation solution can prolong the preservation time of fresh cartilage, and the cartilage still meets the activity and functionality required by clinical transplantation after being preserved for 8 weeks.

The purpose of the invention is realized by the following technical scheme:

a cartilage tissue preservation solution comprises a DMEM culture medium, HEPES 1-30 mmol/L, a component A1-20mmol/m L, a transforming growth factor β (TGF- β)10-100ng/m L, a component B10-100ng/m L, chondroitin sulfate 10-100 g/L, and one or more Matrix Metalloproteinase (MMP) inhibitor 100-1000ug/m L, wherein the component A is miroacrobic acid (Bongkrekie acid) or Q-VD-OPH, and the component B is cartilage-derived morphogenetic protein 1(CDMP-1), BMP or PDGF.

DMEM medium and HEPES (meaning 4-hydroxyethylpiperazine ethanesulfonic acid) in the preservation solution of the present invention can be purchased conventionally from the market.

In one embodiment of the present invention, the cartilage tissue preservation solution of the present invention is weakly alkaline, and preferably has a pH of 7.30 to 7.50. The weakly alkaline environment is favorable for maintaining the activity and the permeability of the chondrocytes.

In a preferred embodiment of the invention, the HEPES content is 20 mmol/L.

In a preferred embodiment of the invention, component A is present in an amount of 5 to 20mmol/m L. Q-VD-OPH of the invention is a potent caspase-7 inhibitor, CAS 1135695-98-5. component A is preferably Mimejic acid (Bongkrekkic acid) for better shelf life.

In a preferred embodiment of the present invention, the content of transforming growth factor β is 10-20ng/m L.

In a preferred embodiment of the invention, component B is present in an amount of 50ng/m L. for better shelf life, component B is preferably cartilage derived morphogenetic protein 1 (CDMP-1).

In a preferred embodiment of the invention, the chondroitin sulfate is present in an amount of 50-100 g/L, more preferably 75-100 g/L.

In a preferred embodiment of the invention, the matrix metalloproteinase inhibitor is GM6001 or SB-3CT, and in a specific embodiment, the matrix metalloproteinase inhibitor is present in an amount of 200-600ug/m L.

The invention also provides the application of the cartilage tissue preservation solution in cartilage preservation, and in a specific embodiment, the cartilage tissue preservation solution is applied in cold preservation at 3-4 ℃.

The invention also provides a low-temperature cartilage tissue refrigeration method, which is characterized in that sterile fresh cartilage tissue is placed into the low-temperature refrigeration preservation solution, is aseptically sealed and is preserved in a low-temperature environment of 3-4 ℃.

Sterile fresh cartilage tissue according to the present invention may be obtained according to methods conventional in the art.

The application of the invention can be used as a method for preserving fresh cartilage tissue by refrigeration for a long time in scientific research and clinic.

The invention has the beneficial effects that:

after cartilage preservation, the great factor of undesirable transplantation results from chondrocyte death and cartilage matrix destruction, so whether the chondrocyte viability and matrix functional integrity can be maintained is the key of cartilage preservation. The invention effectively retains the activity of chondrocytes, inhibits the degradation and destruction of cartilage matrixes, promotes the growth of cartilage, and is beneficial to the preservation of the performance of the cartilage and the subsequent transplantation.

The mircotoxicillin in the cartilage preservation solution can inhibit apoptosis of chondrocytes, improve survival rate of the chondrocytes, improve survival and function recovery of transplanted cartilages, the transforming growth factor β (TGF- β) and the cartilage-derived morphogenetic protein 1(CDMP-1) are beneficial to regeneration of the chondrocytes, the chondroitin sulfate and the GM6001 are used as beneficial matrix metalloproteinase inhibitors and can effectively inhibit matrix metalloproteinase from decomposing extracellular matrix, the function of cartilage tissues is prevented from being damaged, the performance of the cartilages is improved to be preserved, and the HEPES buffer solution is used as a beneficial buffer solution and has the function of controlling constant pH range for a long time and maintaining osmotic pressure of liquid inside and outside the cells.

The invention can be used for preserving fresh cartilage tissue without cryopreservation of cartilage tissue, thereby reducing freezing injury and perfusion injury. The invention can be used for preserving fresh cartilage tissue, and has high cartilage tissue cell activity and good tissue functionality. Compared with the traditional cartilage cryopreservation method, the method disclosed by the invention can effectively prolong the cartilage preservation time from 5 weeks reported in the prior art to 8 weeks. After 8 weeks of preservation, the biological activity of the chondrocytes and the permeability of the cartilage are not obviously changed, and the cartilage still meets the activity and the functionality required by clinical transplantation. Provides a longer time window for transplanting fresh cartilage, and simultaneously, the prolonging of the preservation time is beneficial to improving the accuracy of pathogenic microorganism detection before transplantation and improving the cartilage transplantation safety. Therefore, the long-term cartilage preservation technology has important significance for cartilage transplantation.

Drawings

FIG. 1 a human cartilage sample;

FIG. 2 is a graph of chondrocyte fluorescence RFU values comparing chondrocyte viability and proliferation for 4, 6 and 8 weeks of chondrocyte storage;

FIG. 3 calculated chondrocyte viability at 4, 6, 8 weeks;

FIG. 4 compares the change in cartilage osmolarity for chondrocyte storage at 4, 6, 8 weeks;

FIG. 5 compares the cartilage swelling pressure changes at 4, 6, 8 weeks of chondrocyte storage;

FIG. 6 compares the cartilage proteoglycan changes of chondrocytes stored for 4, 6, 8 weeks;

FIG. 7 is a schematic representation of a porcine knee cartilage defect model implanted with fresh cartilage and cryopreserved cartilage;

FIG. 8 is a pathological diagram of a porcine knee joint cartilage defect model implanted in cartilage for 12 weeks;

FIG. 9 is a safranin O staining pattern of a porcine knee cartilage defect model after 12 weeks of cartilage implantation;

figure 10O' Driscoll histological score after cartilage transplantation.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art.

Example 1

1. 500m L DMEM cell culture medium was sampled in a biosafety cabinet for sterility testing.

2. The cell culture medium DMEM with the chondroitin sulfate content of 75 g/L is prepared by adding the chondroitin sulfate.

3. HEPES was added in the previous step to prepare a homogeneous cartilage preservation solution containing 20 mmol/L of HEPES.

4. Adding fermentation starter acid, transforming growth factor β, cartilage originated morphogenetic protein 1 to obtain fermentation starter acid 10mmol/m L, transforming growth factor β 50ng/m L, cartilage originated morphogenetic protein 110 ng/m L, and pH 7.30-7.50.

5. And (3) placing the solution in the last step into a sterile container, sealing, and storing in a low-temperature environment at 4 ℃ in a dark place.

Example 2

4. DMEM cell culture medium with GM6001 content of 400ug/m L was prepared by adding 200mg of GM6001 to 500m L which was aseptically qualified.

5. HEPES was added in the previous step to prepare a homogeneous cartilage preservation solution containing 20 mmol/L of HEPES.

6. Adding fermentation starter acid, transforming growth factor β, cartilage originated morphogenetic protein 1, and making into fermentation starter acid 5mmol/m L, transforming growth factor β 10ng/m L, cartilage originated morphogenetic protein 150 ng/m L, and pH 7.30-7.50.

7. And (3) placing the solution in the last step into a sterile container, sealing, and storing in a low-temperature environment at 4 ℃ in a dark place.

Example 3 (more different embodiment):

2. Adding the chondroitin sulfate to prepare a DMEM cell culture medium with the chondroitin sulfate content of 100 g/L.

4. DMEM cell culture medium with the content of GM6001 being 800ug/m L is prepared by taking 500m L and adding 400mg GM6001 into DMEM cell culture medium which is qualified by aseptic inspection.

3. HEPES was added in the previous step to prepare a uniform cartilage preservation solution containing HEPES in an amount of 10 mmol/L.

4. Adding fermentation starter acid, transforming growth factor β, cartilage originated morphogenetic protein 1, and making into fermentation starter acid 20mmol/m L, transforming growth factor β 100ng/m L, cartilage originated morphogenetic protein 1100 ng/m L, and pH 7.30-7.50.

Example 4

Fresh human cartilage tissue was immersed in the preservation solution prepared in example 2, sealed, and stored at 4 ℃. The biological function parameters such as the character, activity and the like of the cartilage were observed at 4 time points of 0 week, 4 weeks, 6 weeks and 8 weeks, and the detection method was as follows.

Experimental methods for chondrocyte survival testing and cartilage matrix determination:

(1) cartilage cell survival rate test experiment

The cell survival rate in the cartilage tissue using the preservation solution can be measured by Alma blue staining and a spectrophotometer at a fixed wavelength, and after the sample is preserved for a preset time, the normal and fresh cartilage tissue cells are compared, and finally the cell fluorescence intensity in each milliliter is calculated.

(2) Functional assay of cartilage extracellular matrix

The functions of cartilage matrix permeability and the like are main factors determining whether chondrocyte viability and preservation are successful, and two methods are designed to measure cartilage matrix functions, namely: a. DMA test method, a method of measuring changes in an elastic material with time and external environment, which can be used to detect osmotic and oncotic pressure of cartilage matrix; b. and (4) detecting the content of proteoglycan by safranin O staining, and comparing the activity difference of the cartilage before and after storage.

The experimental process comprises the following steps: healthy human cartilage was selected and prepared as cylindrical samples (fig. 1) approximately 5-6 mm in diameter and in several quantities. The samples were stored in 4 ℃ storage solution for 8 weeks and chondrocyte activity in stored cartilage was assessed using alamar blue to test the chondrocyte fluorescence per milligram (RFU/mg) of the samples at 0, 4, 6 and 8 week time points. And simultaneously, measuring the osmotic pressure and the swelling pressure of the matrix by adopting a DMA test method, and detecting the content of proteoglycan by safranin O staining.

The results are shown in FIGS. 2-6:

chondrocyte viability was calculated by measuring the chondrocyte fluorescence RFU values of each sample by alamar blue staining (fig. 2) (fig. 3), and statistics were made that there was no significant difference in RFU values between 8 and 4 weeks of cartilage tissue preservation (P > 0.05). Chondrocyte viability was 83.46% ± 1.07% at 4 weeks, 71.26% ± 2.64% at 6 weeks, 64.21% ± 2.48% at 8 weeks. The data are expressed as mean ± standard error, n ═ 5, using one-way analysis of variance (ANOVA).

FIG. 4 shows that the osmotic pressure of the cartilage tissue was not significantly different between 8 weeks and 0 weeks of preservation (P > 0.05). The data are expressed as mean ± standard error, n ═ 5, using one-way analysis of variance (ANOVA).

The results of the test in FIG. 5 show that there was no significant difference in swelling pressure between 8 weeks and 0 weeks of cartilage tissue preservation (P > 0.05). The data are expressed as mean ± standard error, n ═ 5, using one-way analysis of variance (ANOVA).

FIG. 6 shows that cartilage was preserved for 8 weeks, and safranin O staining showed no significant difference in proteoglycan content between cartilage at 8 and 4 weeks (P > 0.05). The data are expressed as mean ± standard error, n ═ 5, using one-way analysis of variance (ANOVA).

Example 5

Fresh pig cartilage tissue was immersed in the preservation solution of example 2, sealed, and stored in an environment at 4 ℃ for 8 weeks. 6 pig knee joint cartilage defect models were prepared, each group was divided into two groups of 3, and fresh cartilage and cartilage preserved under refrigeration for 8 weeks were transplanted, respectively, and fig. 7 shows the pig knee joint cartilage defect model with fresh cartilage (left) and cartilage preserved under refrigeration for 8 weeks (right). After 12 weeks of transplantation, the cartilage at the transplantation site was subjected to pathological section observation and safranin O staining.

FIG. 8 is a pathological diagram of a pig knee joint cartilage defect model implanted with fresh cartilage for 12 weeks, with good healing; the right graph is a pathological diagram of the cartilage defect model part of the knee joint of the pig which is implanted with the cartilage preserved for 8 weeks in a refrigerating way for 12 weeks, and the healing effect is slightly lower than that of the fresh cartilage, but the cartilage still heals better, and the cartilage defect model part can be used for clinical transplantation.

FIG. 9 is a left image of safranin O staining of a cartilage defect model of a pig knee joint implanted with fresh cartilage for 12 weeks, wherein cartilage matrix staining is deeper, and chondrocytes are uniformly dispersed; the right image is a safranin O staining image after 12 weeks of cartilage is implanted into a cartilage defect model part of a pig knee joint and is refrigerated and stored for 8 weeks, cartilage matrix staining is deeper, and safranin O staining does not decline, which shows that matrix proteoglycan components are not lost, the content is rich, chondrocyte metabolism is active, and chondrocytes are uniformly dispersed.

FIG. 10O' Driscoll histology score 12 weeks after cartilage transplantation, no significant difference was observed between cartilage preserved by transplantation cold storage for 8 weeks and transplanted fresh cartilage (P > 0.05). The data are expressed as mean ± sem, n is 3, using T-test.

In conclusion, the preservation solution is used for preservation for 8 weeks, the survival rate of the chondrocytes and the function of extracellular matrix are not obviously changed, the cartilage transplanted by using the preservation solution for cold preservation for 8 weeks is well healed with joints after 12 weeks, and safranin O staining shows that the components of matrix proteoglycan are not lost, the number of the chondrocytes is large, and the metabolism is active. The cryopreservation method of cartilage was demonstrated to maintain viable cartilage for at least 8 weeks.

In addition, the inventor also finds that when the component proportion in the preservation solution is not in the range of 1-20mmol/m L of given mircoyomycelic acid (Bongkrekeicid), 10-100ng/m L of transforming growth factor β (TGF- β) and 10-100ng/m L of cartilage-derived morphogenetic protein 1(CDMP-1), the component proportion is lower than or higher than the range, under the same other conditions, the cartilage cell survival rate after 4 weeks is 60%, the cartilage cell survival rate after 6 weeks is 40%, the cartilage cell survival rate after 8 weeks is 30%, the content of proteoglycan is reduced, and the healing after cartilage transplantation is poor.

Claims

1. A cartilage tissue low-temperature refrigeration preservation solution is characterized by comprising a DMEM culture medium, HEPES 1-30 mmol/L, a component A1-20mmol/m L, a transforming growth factor β 10-100ng/m L, a component B10-100ng/m L, chondroitin sulfate 10-100 g/L and a matrix metalloproteinase inhibitor 100-1000ug/m L, wherein the component A is mircotiana acid, and the component B is cartilage-derived morphogenetic protein 1.

2. The cryopreservation solution for cartilage tissue according to claim 1, wherein the pH of the cryopreservation solution for cartilage tissue is 7.30-7.50.

3. The cryo-cryopreservation solution for cartilage tissue according to claim 1 wherein the HEPES content is 20 mmol/L.

4. The cryo-cryopreservation solution for cartilage tissue according to claim 1, wherein the content of component A is 5-20mmol/m L.

5. The cryo-cryopreservation solution for cartilage tissue according to claim 1, wherein the content of transforming growth factor β is 10-20ng/m L.

6. The cryopreservation solution for cartilage tissue according to claim 1, wherein the content of component B is 50ng/m L.

7. The cryopreservation solution for cartilage tissue according to claim 1, wherein the content of chondroitin sulfate is 50-100 g/L.

8. The cryopreservation solution for cartilage tissue according to claim 1 wherein the matrix metalloproteinase inhibitor is GM6001 or SB-3 CT.

9. The cryopreservation solution for cartilage tissue as claimed in claim 1, wherein the matrix metalloproteinase inhibitor is 200-600ug/m L.

10. Use of the cryo-cryopreservation solution for cartilage tissue according to any one of claims 1 to 9 for cartilage preservation.

11. Use of the cryo-cryopreservation solution for cartilage tissue according to any one of claims 1 to 9 for cryopreservation of cartilage at 3 to 4 ℃.

12. A method for cold-storing and preserving cartilage tissue at a low temperature is characterized in that sterile fresh cartilage tissue is put into the cartilage tissue cold-storing and preserving solution of any one of claims 1-9, is sealed aseptically and is preserved in a low-temperature environment at 3-4 ℃.