CN114375944A - Cryopreservation liquid and cryopreservation method for bone tissues and bone soft tissues - Google Patents
Cryopreservation liquid and cryopreservation method for bone tissues and bone soft tissues Download PDFInfo
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- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 83
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 64
- 210000004872 soft tissue Anatomy 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000007788 liquid Substances 0.000 title claims abstract description 28
- 210000001519 tissue Anatomy 0.000 claims abstract description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 17
- 239000011550 stock solution Substances 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- 238000007710 freezing Methods 0.000 claims description 13
- 230000008014 freezing Effects 0.000 claims description 13
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- 229960004063 propylene glycol Drugs 0.000 claims description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 7
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 235000013772 propylene glycol Nutrition 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- 239000012595 freezing medium Substances 0.000 claims 1
- 230000004083 survival effect Effects 0.000 abstract description 13
- 230000001988 toxicity Effects 0.000 abstract description 7
- 231100000419 toxicity Toxicity 0.000 abstract description 7
- 238000000338 in vitro Methods 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 239000003223 protective agent Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 26
- 239000002577 cryoprotective agent Substances 0.000 description 7
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 6
- 210000001612 chondrocyte Anatomy 0.000 description 6
- 230000007547 defect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000004017 vitrification Methods 0.000 description 5
- 230000000735 allogeneic effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 210000000845 cartilage Anatomy 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 210000005065 subchondral bone plate Anatomy 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 206010007710 Cartilage injury Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
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- 239000003440 toxic substance Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention belongs to the technical field of tissue cryopreservation, and particularly relates to a cryopreservation liquid and a cryopreservation method for bone tissues and bone soft tissues. The invention provides a novel cryopreservation liquid for bone tissues and bone soft tissues and also provides a method for performing cryopreservation on the bone tissues and the bone soft tissues by using the cryopreservation liquid. The cryopreservation liquid is particularly suitable for cryopreservation of bone tissues and bone soft tissues, can effectively reduce the toxicity of the low-temperature protective agent in the cryopreservation liquid to cells, improve the survival rate of the cells, and reduce the damage to the cells and the tissues, thereby being beneficial to prolonging the preservation time of the bone tissues and the bone soft tissues in vitro and having good application prospect.
Description
Technical Field
The invention belongs to the technical field of tissue cryopreservation, and particularly relates to a cryopreservation liquid and a cryopreservation method for bone tissues and bone soft tissues.
Background
In clinic, bone defects caused by various reasons such as trauma, infection, bone tumor excision and the like are one of the difficult problems of orthopedic treatment, and bone transplantation is the preferred treatment method. Although autologous bone grafting is the "gold standard" for bone defect repair, increased trauma to patients due to bone harvesting and limited bone harvesting volume limit its clinical application. The source of the allogeneic bone is wide, the generated rejection reaction is small, secondary damage does not exist, and the problem of insufficient source of autologous bone transplantation tissues is solved, so that the clinical application is gradually wide. How to preserve the allogeneic bone and bone soft tissue taken from the donor in vitro for a long time and have good cell activity becomes the focus of controversial research of the public.
The existing cryopreservation method mainly comprises a programming method and a vitrification method. The programming method is long in time consumption and easy to generate ice crystals in cells. The vitrification method can avoid the generation of ice crystals in cells, is simple to operate and becomes a hot point of cryopreservation research in recent years, but factors influencing the cryopreservation effect of the vitrification method are many, and a uniform and standard method is not formed at present. In particular to a freezing liquid which plays an important role in freezing effect. Cryoprotectants are important components of cryopreservation fluids and can protect cells from low temperature damage.
However, cryoprotectants are generally toxic substances. Therefore, how to design the formulation of the cryopreservation solution and how to set up the method for cryopreserving cells, and how to reduce the toxicity of the cryoprotectant to cells as much as possible are important research subjects in the field of cryopreserving cells. Chinese patent application CN 201910853381.0A protective solution for vitrification cryopreservation of immune cells and a method for cryopreservation of immune cells proposes a cryopreservation solution and a method aiming at immune cells, reduces the use of dimethyl sulfoxide or glycol as a cryoprotectant as much as possible, reduces the toxicity of the cryopreservation solution and improves the survival rate of cells by reasonable formula design.
For the cryopreservation of bone tissues and bone soft tissues, the existing cryopreservation liquid and cryopreservation method have the problems of over-strong toxicity and low cell survival rate, so that a new cryopreservation liquid and a new cryopreservation method for bone tissues and bone soft tissues need to be developed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a cryopreservation liquid and a cryopreservation method for bone tissues and bone soft tissues, and aims to provide the following steps: by optimizing the formula of the frozen stock solution and the freezing method, the toxicity of the frozen stock solution is reduced, and the survival rate of the frozen cells is improved.
A cryopreservation liquid for bone tissue and/or bone soft tissue, wherein each 1000ml of the cryopreservation liquid comprises the following components:
KOH 80-120mmol、
HCl 40-80mmol、
NaHCO3 10-50mmol、
NaH2PO4 0.8-1.2mmol、
MgSO4 0.8-1.2mmol、
CaCl2 0.8-1.2mmol、
2-8mmol of glucose,
TES 80-120mmol、
80-120ml of 1, 2-propylene glycol,
80-120ml of 99.9 percent dimethyl sulfoxide,
the balance being water.
Preferably, every 1000ml of the frozen stock solution consists of the following components:
KOH 100mmol、
HCl 60mmol、
NaHCO3 30mmol、
NaH2PO4 1mmol、
MgSO4 1mmol、
CaCl2 1mmol、
5mmol of glucose,
TES 100mmol、
100ml of 1, 2-propanediol,
100ml of 99.9% dimethyl sulfoxide was added,
the balance being deionized water.
Preferably, the pH of the frozen stock solution is 7.3-7.5.
The invention also provides application of the frozen stock solution in freezing and storing bone tissues and/or bone soft tissues.
The invention also provides a cryopreservation method for bone tissues and/or bone soft tissues, which comprises the following steps:
step 1, preparing the frozen stock solution;
step 2, placing the tissue to be frozen in the freezing solution for 1-3 hours at 0-4 ℃;
and 3, gradually cooling and freezing the tissues to be frozen and the frozen stock solution to-70 to-90 ℃, standing for 8 to 12 hours, and then storing in liquid nitrogen.
Preferably, in step 2, before the tissue to be cryopreserved is placed in the cryopreservation solution, the tissue to be cryopreserved is washed for 3 to 5 times by using a phosphate buffer solution.
Preferably, in step 3, the gradual cooling rate is-0.1 to-0.3 ℃/min.
In the present invention, "TES" is the compound 2- [ [ tris (hydroxymethyl) methyl ] amino ] ethanesulfonic acid.
The invention optimizes the composition of the cryopreservation solution aiming at bone tissues and bone soft tissues, only uses dimethyl sulfoxide as a cryoprotectant, and reduces the damage of the cryoprotectant to cells to the minimum. In addition, the invention also improves the freezing method, does not carry out rapid temperature rise or temperature reduction, and utilizes the characteristic that the toxicity of the cryoprotectant dimethyl sulfoxide is reduced along with the reduction of the temperature to gradually reduce the temperature when preserving bone tissues or bone soft tissues, so that tissue cells are loaded with enough dimethyl sulfoxide at low temperature finally to realize vitrification. This further reduces the damage of dimethyl sulfoxide to the cells. Therefore, the invention not only provides a novel cryopreservation liquid for cryopreservation of bone tissues and bone soft tissues, but also minimizes the damage of the cryopreservation liquid to cells, can effectively improve the cryopreservation effect of the bone tissues and the bone soft tissues, prolongs the storage time of the bone tissues and the bone soft tissues in vitro, is convenient for establishing a bone tissue and bone soft tissue bank, and expands the clinical application of allogeneic bone transplantation in repairing bone defects. Therefore, the invention has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a graph showing the results of experiments on the survival rate of cryopreserved cells in Experimental example 1;
FIG. 2 is an HE stained image of bone tissue and cartilage tissue cryopreserved according to the cryopreservation liquid and cryopreservation method of example 3 in Experimental example 1;
fig. 3 is an HE stained image of bone tissue and cartilage tissue cryopreserved according to the cryopreservation liquid and the cryopreservation method of comparative example 1 in experimental example 1.
Detailed Description
In the following examples and experimental examples, reagents and materials not specifically described are commercially available.
Example 1
The bone tissue and bone soft tissue freezing solution contains 80-120mmol of KOH, 40mmol of HCl and NaHCO per 1000ml3 10mmol、NaH2PO4 0.8mmol、MgSO4 0.8mmol、 CaCl20.8mmol, glucose 2mmol, TES 80mmol, 1,2 propylene glycol 80ml, 99.9% dimethyl sulfoxide 80ml, and the balance deionized water. The pH value of the bone tissue and soft tissue frozen stock solution is 7.3-7.5. Subpackaging, sealing, and storing at 0-4 deg.C.
Example 2
The bone tissue and bone soft tissue freezing solution contains KOH 120mmol, HCl 80mmol and NaHCO in every 1000ml3 50mmol、NaH2PO4 1.2mmol、MgSO4 1.2mmol、CaCl21.2mmol, glucose 8mmol, TES 120mmol, 1, 2-propanediol 120ml, 99.9% dimethyl sulfoxide 120ml, and the balance deionized water. The pH value of the bone tissue and soft tissue frozen stock solution is 7.3-7.5. Subpackaging, sealing, and storing at 0-4 deg.C.
Example 3
The frozen stock solution for bone tissues and bone soft tissues contains 100mmol of KOH, 60mmol of HCl and NaHCO per 1000ml330mmol、NaH2PO4 1mmol、MgSO4 1mmol、CaCl21 mmol, glucose 5mmol, 2- [ [ tris (hydroxymethyl) methyl ] methyl]Amino group]100mmol of ethanesulfonic acid, 100ml of 1, 2-propanediol, 100ml of 99.9% dimethyl sulfoxide and the balance of deionized water, and the frozen stock solution of bone tissues and soft tissuesThe pH value is 7.3-7.5. Subpackaging, sealing, and storing at 0-4 deg.C.
The method for freezing and storing bone tissues and bone soft tissues by using the freezing and storing liquid of the embodiment comprises the following steps:
step 1, preparing the frozen stock solution;
step 2, cleaning the tissue to be frozen for 3-5 times by adopting a phosphate buffer solution, and then incubating and placing the tissue in the frozen solution for 2 hours at 4 ℃;
and 3, gradually cooling and freezing the tissue to be frozen and the frozen solution to-80 ℃ at the speed of-0.3 ℃/min, standing for 12 hours, and then storing in liquid nitrogen.
The technical effects of the present invention will be further described below by experiments.
Experimental example 1
1. Experimental methods
Osteochondral tissues were cryopreserved using the cryopreservation solution (10% dimethylsulfoxide as a control) and the cryopreservation method of example 3, and chondrocyte viability of the cryopreserved osteochondral tissues was examined and HE stained images thereof were collected.
The specific method for obtaining the survival rate of the chondrocytes is as follows: directly soaking the frozen tube in a water bath at 37 ℃ for rewarming, taking out osteochondral blocks, making sections, dyeing, and observing chondrocytes under a fluorescence microscope. The survival rate of chondrocytes was the number of green-stained cells/(number of green-stained cells + number of orange-stained cells).
2. Results of the experiment
The survival rate of the cryopreserved cells is shown in fig. 1, and it can be seen from the graph that the survival rate of the cells at week 2 of the control group is 84%, the survival rate of the cells at week 4 is 79%, and the survival rate of the cells at week 6 is 72%; the cell viability of the invention group (using the cryopreservation solution and method of example 3) was 89% at week 2, 84% at week 4 and 79% at week 6. Therefore, the invention can effectively improve the survival rate of cells.
HE staining images of bone tissue and cartilage tissue cryopreserved according to the cryopreservation solution and the cryopreservation method of example 3 are shown in fig. 2, and HE staining images of the control group are shown in fig. 3. As can be seen from the figure, the articular surface of the control group is obviously damaged, the arrangement of chondrocytes is disordered, the aggregation is more, the cartilage damage is obvious, and the subchondral bone trabecular structure is reduced, slender and disordered. The subchondral bone of the joint is significantly reduced in number and results in microstructural damage. The experimental group of example 3 had a more complete articular surface, more regular chondrocyte morphology, and more orderly arranged subchondral bone trabecular structures. The cryopreservation liquid and the cryopreservation method have smaller damage to bone tissues and bone soft tissues and are more favorable for long-term storage of the bone tissues and the bone soft tissues.
The invention is particularly suitable for the cryopreservation of bone tissues and bone soft tissues, can effectively reduce the toxicity of the low-temperature protective agent in the cryopreservation liquid to cells, improves the survival rate of the cells, and reduces the damage to the cells and the tissues. Thereby being beneficial to prolonging the preservation time of in vitro bone tissues and bone soft tissues, being convenient for establishing a bone tissue and bone soft tissue bank and expanding the clinical application of allogeneic bone transplantation in repairing bone defects. Therefore, the invention has good application prospect.
Claims (7)
1. A cryopreservation liquid for bone tissue and/or soft bone tissue, wherein each 1000ml of the cryopreservation liquid comprises the following components:
KOH 80-120mmol、
HCl 40-80mmol、
NaHCO3 10-50mmol、
NaH2PO4 0.8-1.2mmol、
MgSO4 0.8-1.2mmol、
CaCl2 0.8-1.2mmol、
2-8mmol of glucose,
TES 80-120mmol、
80-120ml of 1, 2-propylene glycol,
80-120ml of 99.9 percent dimethyl sulfoxide,
the balance being water.
2. The cryopreservation liquid of claim 1, wherein each 1000ml of the cryopreservation liquid is composed of:
KOH 100mmol、
HCl 60mmol、
NaHCO3 30mmol、
NaH2PO4 1mmol、
MgSO4 1mmol、
CaCl2 1mmol、
5mmol of glucose,
TES 100mmol、
100ml of 1, 2-propanediol,
100ml of 99.9% dimethyl sulfoxide was added,
the balance being deionized water.
3. The frozen stock solution according to claim 1 or 2, wherein: the pH value of the frozen stock solution is 7.3-7.5.
4. Use of the cryopreservation solution of any one of claims 1 to 3 for the cryopreservation of bone tissue and/or bone soft tissue.
5. A method for cryopreserving bone tissue and/or soft bone tissue, comprising the steps of:
step 1, preparing a cryopreservation solution according to any one of claims 1 to 3;
step 2, incubating the tissue to be cryopreserved in a cryopreservation solution at 0-4 ℃ for 1-3 hours;
and 3, gradually cooling and freezing the tissues to be frozen and the frozen stock solution to-70 to-90 ℃, standing for 8 to 12 hours, and then storing in liquid nitrogen.
6. The cryopreservation method of claim 5, wherein: in step 2, before the tissue to be frozen is put into the freezing medium, phosphate buffer solution is adopted to wash the tissue for 3 to 5 times.
7. The cryopreservation method of claim 5, wherein: in the step 3, the gradual cooling speed is-0.1 to-0.3 ℃/min.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR9003310A (en) * | 1990-07-10 | 1992-01-28 | Brasil Pesquisa Agropec | PROCESS AND COMPOSITION FOR THE CRYOPRESERVATION OF MAMMALIAN EMBRYOS |
| CN104938478A (en) * | 2015-05-20 | 2015-09-30 | 泰山医学院 | Cartilago articularis vitrification cryoprotectant and cartilage preservation method |
| CN106614526A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Vitrified cryopreservation method for cartilage |
| CN109843052A (en) * | 2016-10-04 | 2019-06-04 | 全崴生技股份有限公司 | The composition and method saved for cell freezing |
| CN111657267A (en) * | 2020-06-17 | 2020-09-15 | 科瑞百奥泰州生物技术有限公司 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
-
2021
- 2021-12-31 CN CN202111675559.0A patent/CN114375944A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR9003310A (en) * | 1990-07-10 | 1992-01-28 | Brasil Pesquisa Agropec | PROCESS AND COMPOSITION FOR THE CRYOPRESERVATION OF MAMMALIAN EMBRYOS |
| CN104938478A (en) * | 2015-05-20 | 2015-09-30 | 泰山医学院 | Cartilago articularis vitrification cryoprotectant and cartilage preservation method |
| CN109843052A (en) * | 2016-10-04 | 2019-06-04 | 全崴生技股份有限公司 | The composition and method saved for cell freezing |
| CN106614526A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Vitrified cryopreservation method for cartilage |
| CN111657267A (en) * | 2020-06-17 | 2020-09-15 | 科瑞百奥泰州生物技术有限公司 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
Non-Patent Citations (2)
| Title |
|---|
| KEZHOU WU ET AL.: ""Comparison of three multi-cryoprotectant loading protocols for vitrification of porcine articular cartilage"", 《CRYOBIOLOGY》 * |
| 关静: ""关节软骨的玻璃化冷冻与保存"", 《中国组织工程研究》 * |
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