CN111657267A - Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus - Google Patents
Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus Download PDFInfo
- Publication number
- CN111657267A CN111657267A CN202010554905.9A CN202010554905A CN111657267A CN 111657267 A CN111657267 A CN 111657267A CN 202010554905 A CN202010554905 A CN 202010554905A CN 111657267 A CN111657267 A CN 111657267A
- Authority
- CN
- China
- Prior art keywords
- cartilage
- tendon
- meniscus
- solution
- preservation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
Abstract
The invention discloses an ice crystal-free cryopreservation liquid and a freezing method for cartilage, tendon and meniscus preservation, wherein the cryopreservation liquid comprises 1.0-6.0mol/L of ethylene glycol, 1.0-7.0mol/L of propylene glycol, 0.1-2.5mol/L, Caspase inhibitor Z-VAD-FMK 10-50 mu mol/L, 2-10mg/L of matrix metalloproteinase inhibitor GM6001 and a component X. The preservation solution disclosed by the invention can effectively improve the number of living cells and the cell survival rate in the tissues after the cartilage, tendon and meniscus tissues are frozen and preserved, and more importantly, can prevent the ice crystals from regrowing during freezing recovery and storage, and prevent devitrification. The novel ice crystal-free freezing protection solution can be used for preserving cartilage, tendon and meniscus for a longer time and can be preserved permanently at the temperature of less than-130 ℃.
Description
Technical Field
The invention belongs to the field of biomedical engineering, and particularly relates to an ice-crystal-free frozen preservation solution and a freezing method for preserving cartilage, tendon and meniscus.
Background
Cartilage, tendons, meniscus, as a structurally similar tissue, is composed of cells, matrix and fibers, and its main physiological functions are to provide a friction bearing surface, protect joint connectivity, reduce the load on the joint during activity, reduce joint contact pressure, and often also allow the same preservation method to be used during cryopreservation. Can be used for clinical repair and replacement of cartilage, tendon and meniscus injury and pathological changes. In clinical practice, the selection of cartilage, tendon, and meniscal tissue transplantation for treatment of disease usually involves ex vivo preservation of donor cartilage, tendon, and meniscal tissue. At present, in-vitro preservation methods of cartilage, tendon and meniscus tissues are divided into two types, wherein one type is cartilage, tendon and meniscus refrigeration preservation, and the other type is cartilage, tendon and meniscus refrigeration preservation. Cartilage, tendon, meniscus tissue refrigeration preservation time generally do not exceed 8 weeks, prolong cartilage, tendon, meniscus preservation time needs to use cartilage, tendon, meniscus cryopreservation technique, but the traditional conventional cartilage, tendon, meniscus tissue cryopreserving technique inevitably can produce very big destruction to cartilage, tendon, meniscus tissue, there is the literature to show that the cartilage, tendon, meniscus are cryopreserved to use conventional cryopreservation technique, and when the tendon, meniscus, cartilage, tendon, meniscus inner viable cell number is less than 50%, and spatial structure suffers serious damage. And the cartilage, tendon and meniscus which is preserved by conventional freezing usually fails to be transplanted due to the defect of the spatial structure of the cartilage, tendon and meniscus, cartilage, tendon and meniscus.
When the cells are frozen, a large amount of ice crystals are formed in the cell moisture in the cooling process, and the formation of the ice crystals can cause a series of changes in the cells, such as mechanical damage (cell membranes are punctured by the ice crystals), electrolyte concentration increase, osmotic pressure change, dehydration, pH value change, protein denaturation and the like. The freezing point of water in cytoplasm can be reduced by adding freezing protective agent (such as glycerol or DMSO), and water in cytoplasm can permeate out of cells through cell membrane in slow cooling process to reduce formation of ice crystal in cells, thereby reducing damage type and degree of cells during freezing. The ice crystal-free freezing process has increased temperature conducting speed and concentration of cryoprotectant to convert the intracellular and extracellular fluids from liquid state to non-crystallized solid state similar to glass state to form transparent glass solid, maintain the normal molecular and ion distribution of intracellular fluid, reduce ice crystal formation and raise cell survival rate. Because three types of tissue structures including cartilage, tendon and meniscus are similar in cell and interstitial components, the penetration of the cryopreservation agent, the heat conduction of tissues and the formation of ice crystals are similar in the tissues, the damage to the tissues is the same, and a method for freezing and preserving without ice crystals can be generally applied to all the tissues.
It is well known that higher concentrations of cryoprotectants cause greater toxicity to cells and tissues. Therefore, the existing vitrification cryoprotectant is used in minimum concentration, and vitrification can be formed only by freezing and cooling. However, the vitrification state is extremely unstable, and extremely small, invisible and harmless ice crystals are formed during freezing and cooling, and can grow again once meeting the change of environment, so that the ice crystals become large and large, and even devitrification can occur. This change in environment can occur at any time, either as the sample leaves the storage container, as the sample is transported, or as the storage container is opened and closed during storage, as a change in temperature. In particular, during rewarming, the cells recrystallize when passing through the freezing point of the cryoprotectant. Since the existing non-crystal frozen glass state is unstable, re-icing and de-vitrification can occur at any time, and the survival rate and the function of the stored sample are greatly influenced.
CN104938478A discloses a joint cartilage vitrification cryoprotectant and a cartilage preservation method, wherein the cryoprotectant is adopted, and each 1000ml comprises: 150-250 ml of 99.9% dimethyl sulfoxide, 110-200 g of acetamide, 70-120 ml of 1, 2-propylene glycol, 10-80 g of polyethylene glycol, 20-100 g of trehalose and the balance of deionized water. The survival rate of the chondrocytes is kept at a higher level (increased from 60% to 68% of the conventional group) at week 8 of cryopreservation.
CN108835105A discloses an articular cartilage vitrification freezing protection solution and a preparation method thereof, wherein the articular cartilage vitrification freezing protection solution comprises the following components in parts by weight: 300 parts of dimethyl sulfoxide 200-; after the articular cartilage is rewarming by the prepared cryoprotectant solution in 8 weeks, the cell survival rate of the articular cartilage still reaches 76%, and after the articular cartilage is rewarming by 10 weeks, the cell survival rate of the articular cartilage still reaches 71%.
CN109644989A discloses an articular cartilage vitrification cryoprotectant and a cartilage cryopreservation method, wherein the cryoprotectant comprises the following components in parts by weight: 18-22 parts of carboxymethyl starch, 2-3 parts of rhamnose, 1-2 parts of spermene, 2-4 parts of microcrystalline cellulose, 4-6 parts of galactomannose, 4-6 parts of muramyl dipeptide, 3-5 parts of tripalmitoyl pentapeptide, 0.5-1.5 parts of alpha-arbutin and 70 parts of deionized water. The method for freezing and preserving the cartilage by adopting the articular cartilage freezing protective solution comprises the steps of leaching, dropwise adding part of the freezing protective solution, adding the rest freezing protective solution and carrying out programmed cooling. Indicating that no cryoprotectant such as dimethyl sulfoxide harmful to cells is used; according to the articular cartilage cryopreservation method, after the articular cartilage is taken out for rewarming at the 8 th week of cryopreservation, the cell survival rate of the articular cartilage is 82-85%, and the cell survival rate of the articular cartilage at the 10 th week is 78-80%.
Although the above patent has made some progress in the vitrification of the cryo-preserved articular cartilage, the following disadvantages still remain:
(1) the cell survival rate had large fluctuations (68%, 76% to 82-85% at week 8; 71% to 78-80% at week 10), indicating that the vitrification state was very unstable. During storage and recovery phenomena of Devitrification (Devitrification) and/or ice crystal regrowth (recrystalization) occur, leading to a change in the cell viability rate;
(2) the cell survival rate of the articular cartilage is still low after recovery, and the preservation time is short.
(3) It is difficult to adapt to a transportation environment having a long-term vibration state. Under shock conditions, the specimen is also susceptible to Devitrification (development) and/or ice crystal regrowth (Recrystallization).
In addition, there is no vitrification freezing protection liquid and preservation method for tendon and meniscus tissues disclosed at present.
Disclosure of Invention
The invention aims to provide a novel crystal-free frozen storage solution and a freezing method for storing articular cartilage, tendon and meniscus for a long time.
The invention provides a cartilage, tendon and meniscus cryo-preservation solution without crystal ice, which comprises 1.0-6.0mol/L of ethylene glycol, 1.0-7.0mol/L of propylene glycol, 0.1-2.5mol/L, Caspase of hydroxyethyl piperazine ethanethiosulfonic acid inhibitor Z-VAD-FMK 5-80 mu mol/L, 0.5-20mg/L of matrix metalloproteinase inhibitor GM6001 and a component X.
Preferably, the ice crystal-free cartilage cryopreservation solution comprises 2.0-5.0mol/L of ethylene glycol, 0.5-5.5mol/L of propylene glycol, 0.1-1.0mol/L, Caspase mol of hydroxyethyl piperazine ethanethiosulfonic acid inhibitor Z-VAD-FMK 10-50 mu mol/L, 2-10mg/L of matrix metalloproteinase inhibitor GM6001 and a component X.
GM6001 of the present invention is a broad-spectrum inhibitor of matrix metalloproteinases with CAS number 142880-36-2.
In some embodiments, component X of the present invention is preferably one or more of 0.05-1% (w/v) polyvinyl alcohol, 0.1-5% (w/v) polyethylene glycol, 0.5-10% (w/v) dextran, or 0.3-7% (w/v) hydroxyethyl starch; further preferably 0.05 to 2% (w/v) of polyvinyl alcohol. The inventor finds that the addition of the component X can obviously improve the preservation quality of cartilage, tendon and meniscus and obviously reduce the formation of ice crystals.
As a further improvement of the invention, the ethylene glycol content is 2.5 mol/L.
As a further improvement of the invention, the propylene glycol content is 2.5 mol/L.
As a further improvement of the invention, the content of the hydroxyethylpiperazine ethanethiosulfonic acid is 0.5 mol/L.
In a further improvement of the present invention, the other component of the non-ice crystal cryopreservation liquid is a solvent, and more preferably, the solvent is a Euro-Collin solution.
The invention also provides a method for preserving and recovering cartilage, tendon and meniscus, wherein during cryopreservation, the non-crystal cryopreservation liquid is introduced into cartilage, tendon and meniscus tissues in a stepping mode; during resuscitation, the non-crystal frozen storage solution is eluted from cartilage, tendon and meniscus tissues in a stepping mode.
In some embodiments, after introducing the non-crystal cryopreservation liquid of the present invention during the cryopreservation process, the present invention may further use one or more of liquids Y to cover the surface layer and the periphery of the cryopreservation system, so as to prevent the cryopreservation liquid and the cryopreserved sample from contacting with air and devitrification, wherein the component Y is a liquid that is not easily formed into a solid at a low temperature, and in some embodiments, the component Y is one or more selected from methyl propane, isobutane, bromopropane, 2-methylbutane, dibromopropane, and bromooctane. Further preferred is 2-methylbutane.
In some embodiments, the step-by-step method for introducing the non-crystal cryopreservation liquid is to add the non-crystal cryopreservation liquid into cartilage, tendon and meniscus tissues in 4-15 steps in a step-by-step manner of 10-25 min at-5 to +22 ℃.
In some preferred embodiments, the step-by-step method for introducing the non-crystal cryopreservation solution is to add the non-crystal cryopreservation solution to the cartilage, tendon and meniscus tissues in 6 steps in 15min step-by-step manner at 2-4 ℃, and introduce the non-crystal cryopreservation solution to the cartilage, tendon and meniscus tissues 15min at a time.
In some embodiments, after introducing the cryo-preservation solution without ice crystal in a stepwise manner, the cartilage, tendon and meniscus preservation method of the present invention first reduces the temperature at a rate of-20 ℃ to-70 ℃/min to-80 ℃ to-120 ℃ for the first time, then reduces the temperature at a rate of-2 ℃ to-40 ℃/min to-120 ℃ to-180 ℃ for the second time, and preserves the cartilage, tendon and meniscus at the temperature.
In some preferred embodiments, the first temperature reduction of the invention is carried out at a rate of-38 ℃ to-50 ℃/minute to-90 ℃ to 110 ℃; further preferably, the temperature is reduced to-100 ℃ at a rate of-43 +/-2 ℃/min.
In some preferred embodiments, the second temperature reduction rate of the present invention is a second temperature reduction rate of-2 ℃ to-10 ℃/minute to-120 ℃ to-150 ℃; further preferably, the temperature is reduced to-135 ℃ at a rate of-3 +/-0.2 ℃/min.
In some embodiments, the step-by-step method for eluting the non-crystal cryopreservation solution is to replace and elute the non-crystal cryopreservation solution for cartilage, tendon and meniscus in 6-25 steps by step and increment of 2-25 min under the condition of-5 to +22 ℃.
In some preferred embodiments, the step-by-step method for eluting the non-crystal cryopreservation solution is to gradually remove the non-crystal cryopreservation solution from the cartilage, the tendon and the meniscus tissues in 6 steps at 2-4 ℃ for 5 min.
In some embodiments, during resuscitation, after the cryopreserved sample is taken out of the storage environment, the temperature is raised to-70 ℃ to-120 ℃ for the first time at the speed of 10-50 ℃/min, then raised to 1-10 ℃ for the second time at the speed of 140-350 ℃/min, and then the non-crystal cryopreservation liquid is eluted from cartilage, tendon and meniscus tissues in a stepping mode.
In some embodiments of the invention, the first temperature ramp of the present invention is a ramp to-100 ℃ at a rate of 30 ± 0.2 ℃/minute.
In some embodiments of the invention, the second temperature ramp of the present invention is a rapid ramp back to 4 ℃ at a rate of 225 ± 15 ℃/minute.
The invention also provides application of the non-crystal frozen preservation solution in frozen preservation of cartilage, tendon or meniscus.
In some embodiments, after introducing the non-crystal cryopreservation liquid of the present invention during the cryopreservation process, the present invention may further use one or more of liquids Y to cover the surface layer and the periphery of the cryopreservation system, so as to prevent the cryopreservation liquid and the cryopreserved sample from contacting with air and devitrification, wherein the component Y is a liquid that is not easily formed into a solid at a low temperature, and in some embodiments, the component Y is one or more selected from methyl propane, isobutane, bromopropane, 2-methylbutane, dibromopropane, and bromooctane. Further preferred is 2-methylbutane.
The invention also provides a using method of the ice-free crystal cryopreservation liquid, which comprises the following steps: when cartilage, tendon and meniscus tissues are preserved, introducing the non-crystal frozen preservation solution into the cartilage, tendon and meniscus tissues in a stepping mode; the step-by-step mode is specifically that the non-crystal frozen storage solution is added to cartilage, tendon and meniscus tissues in increments of 4-15 steps in a step-by-step manner of 10-25 min at the temperature of-5 to +22 ℃.
In some preferred embodiments, the stepwise manner is to add the cryo-free cryopreservation solution to the cartilage, tendon and meniscus tissues in 6 steps in 15min stepwise increments at 2-4 ℃, and introduce the cryo-free cryopreservation solution to the cartilage, tendon and meniscus tissues 15min at a time.
In some embodiments, after introducing the non-crystal cryopreservation liquid of the invention in a stepwise manner, the non-crystal cryopreservation liquid is first cooled to-80 ℃ to-120 ℃ at a rate of-20 ℃ to-70 ℃/min, then cooled to-120 ℃ to-180 ℃ at a rate of-2 ℃ to-40 ℃/min, and then stored at the temperature.
In some preferred embodiments, the first temperature reduction of the invention is carried out at a rate of-38 ℃ to-50 ℃/minute to-90 ℃ to 110 ℃; further preferably, the temperature is reduced to-100 ℃ at a rate of-43 +/-2 ℃/min.
In some preferred embodiments, the second temperature reduction rate of the present invention is a second temperature reduction rate of-2 ℃ to-10 ℃/minute to-120 ℃ to-150 ℃; further preferably, the temperature is reduced to-135 ℃ at a rate of-3 +/-0.2 ℃/min.
The unit w/v of the invention represents mass volume fraction, such as 0.05-2% (w/v) represents mass concentration of 0.5-20 g/L.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention discloses an ice crystal-free cryoprotectant, which can effectively improve the number of living cells and the cell survival rate in tissues after the freezing preservation of cartilage, tendon and meniscus tissues, and more importantly, can prevent the ice crystals from regrowing during the freezing recovery and the storage period, and prevent the devitrification. The novel ice crystal-free freezing protection solution can be used for preserving cartilage, tendon and meniscus for a longer time and can be preserved permanently at the temperature of less than-130 ℃.
(2) The frozen and vitrified sample has the possibility of ice crystal regrowth during freezing, storing and unfreezing, which causes secondary damage to cells. For a vitrified sample, ice can recrystallize by approaching the freezing point of the cryoprotectant solution, and such crystal regrowth can cause mechanical damage to the cells, as well as damage to the cell membrane by a sharp change in the osmotic pressure inside and outside the cell due to ice crystal regrowth. The present invention, while employing stepwise cooling and rewarming, controls this process by use of ice recrystallization inhibitors have been shown to enhance cell recovery after thawing, greatly improve the survival rate of cells after tissue resuscitation, thereby improving the overall function of the tissue.
(3) The cryoprotectants of the present invention have low toxicity and function of inhibiting ice crystal regrowth similar to antifreeze proteins, and they do not bind to ice to cause dynamic ice formation of crystals, resulting in needle-like growth, puncturing cells and damaging cell membranes, thereby limiting their effectiveness. On the contrary, the method can greatly reduce the reformation of ice crystals in the freezing storage and recovery processes, particularly the recrystallization and devitrification of the sample in the recovery process, thereby reducing the secondary damage of the ice crystals to cells and effectively improving the transportation and preservation effects of the stored sample compared with the prior art.
(4) The low-temperature protective agent provided by the invention has much higher contents of ethylene glycol and propylene glycol than those of the common freezing protective agent, and can permeate into cells. Compared with DMSO, ethylene glycol and propylene glycol have low toxicity. The inventor finds that the high-concentration osmotic cryopreservation protective agent can effectively prevent the formation of ice crystals inside and outside cells in the cryopreservation process and reduce the damage and death of the cells.
(5) After the cartilage is preserved, the great factors of the undesirable transplantation effect come from the death of cartilage, tendon and meniscus cells and the damage of cartilage, tendon and meniscus matrixes, so whether the activity of the cartilage, tendon and meniscus cells and the functional integrity of the matrixes can be maintained is the key of the preservation of the cartilage, tendon and meniscus. The invention effectively retains the activity of cartilage, tendon and meniscus cells through the synergy of all the components, inhibits the degradation and damage of cartilage, tendon and meniscus matrixes, promotes the growth of cartilage, tendon and meniscus, and is beneficial to the performance preservation and the subsequent transplantation of cartilage, tendon and meniscus. The Caspase inhibitor Z-VAD-FMK in the cartilage, tendon and meniscus preservation solution provided by the invention can inhibit apoptosis of cartilage, tendon and meniscus cells, improve the survival rate of the cartilage, tendon and meniscus cells, and improve survival and functional recovery of transplanted cartilage, tendon and meniscus; GM6001 is a beneficial matrix metalloproteinase inhibitor, can effectively inhibit matrix metalloproteinase from decomposing extracellular matrix, avoids the damage of the tissue functions of cartilage, tendon and meniscus, and improves the preservation of the properties of the cartilage, tendon and meniscus; the hydroxyethyl piperazine ethanethiosulfonic acid buffer solution is used as a beneficial buffer solution, and has the functions of controlling a constant pH range for a long time and maintaining the osmotic pressure of liquid inside and outside cells.
(6) In the traditional cooling and freezing process, particularly in the low-temperature storage process, a sample is placed in the air, when a container is stored by any switch, the temperature of the sample is changed instantly by convection of the air inside and outside the container through heat conduction, so that ice crystals are formed in a vitrification state, and are grown and vitrified again, so that the sample is frozen again, and cells are damaged secondarily. The present invention can use a liquid 2-methylbutane that does not freeze at low temperatures to coat the surface of the sample. During the cooling and freezing process, the sample is placed in 2-methylbutane liquid during the storage process at the temperature of less than-130 ℃. The liquid which does not freeze at low temperature isolates air, so that the change of the temperature of the sample along with the environment can be reduced, the sample can be further protected from freezing in the long-term storage process, and the damage to cells is reduced.
Drawings
FIG. 1 samples of porcine cartilage (left), tendon (middle), and meniscus (right) were taken for the experiments in the examples and controls.
FIG. 2 tissue morphology after cryopreservation in example 1 and control 1.
FIG. 3 shows the results of the fluorescence-labeled cell viability assay. Fluorescence live cell staining (Calcein-AM) showed viable cartilage cells in control 2 (left), example 1 (middle), and control 1 (right).
FIG. 4 percent fluorescence-labeled cell viability.
Figure 5 relative fluorescence values per mg tissue viability for alamar blue staining stored for 8 months.
FIG. 6 biomechanical testing; control 2, example 1 and control 1, load-indentation depth of the meniscal tissue samples at a peak penetration load of 10 mN.
FIG. 7 is a gross anatomical observation of the porcine knee cartilage defect model taken 12 weeks after cartilage implantation. The porcine knee cartilage defect model is shown implanted in example 1 (fig. 7B) and control 2-fresh cartilage (fig. 7D) versus control 1-conventional frozen cartilage (fig. 7A and C).
FIG. 8O' Driscoll histological score chart after 12 weeks of 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. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In the following examples, the samples or tissues used were porcine cartilage (left), tendon (middle), and meniscus (right), unless otherwise specified.
Example 1
1. Mixing 2.5mol of ethylene glycol, 2.5mol of propylene glycol, 0.5mol of hydroxyethyl piperazine ethanethiosulfonic acid, 25 mu mol of caspase inhibitor Z-VAD-FMK, 6mg of matrix metalloproteinase inhibitor GM6001 and 0.8g of polyvinyl alcohol, adding Euro-Collin solution, and diluting to a constant volume of 1L to form a first storage solution.
2. Taking a proper amount of the first preservation solution obtained in the step 1, and cooling to 2-4 ℃. And adding the cooled first preservation solution into the tissue to be frozen at the temperature of 2-4 ℃ in 6 steps, and finishing the introduction within 90min (adding the first preservation solution into the tissue in 6 steps in 15 steps at the temperature of 2-4 ℃ for 15min each time).
3. Covering the tissue which is introduced into the first preservation solution in the previous step with 2-methylbutane. The container covered with the 2-methylbutane preservation solution one and the tissue was placed in a liquid bath of 2-methylbutane to be frozen.
4. And (3) leading the preservation solution I and the tissue covered with the 2-methylbutane in the previous step to be cooled to-100 ℃ at the speed of-43 ℃/min, then cooling the sample to be less than-130 ℃ at the speed of-3 +/-0.2 ℃/min, and then preserving the sample in a liquid bath of the 2-methylbutane for 8 months.
5. Taking out a sample to be resuscitated, and recovering to-70 ℃ at the rate of 30 +/-0.2 ℃/minute and then recovering to 4 ℃ at the rate of 225 +/-15 ℃/minute;
6. gradually reducing and removing the first preservation solution from the tissue by 6 steps at 2-4 deg.C for 5min, and replacing and eluting the first preservation solution within 30 min.
In this example, a portion of the preserved sample of the preservation solution cooled to less than-130 ℃ through steps 2-4 was taken out from the storage place at less than-130 ℃, placed in a Dry Shipper (a vapor phase liquid nitrogen transfer tank, which can be maintained at a temperature of less than-130 ℃ for one week), and placed on a shaker to shake slowly, simulating the transport of the sample preserved without freezing. Preservation solution-preserved samples were stored in a Dry Shipper for 48 hours under this simulated sample transport and then transferred back to the previous-130 ℃ storage location. Thereafter sample resuscitation was performed as in steps 5 and 6.
Example 2
1. Mixing 2.5mol of ethylene glycol, 2.5mol of propylene glycol, 0.5mol of hydroxyethyl piperazine ethanethiosulfonic acid, 25 mu mol of caspase inhibitor Z-VAD-FMK and 6mg of matrix metalloproteinase inhibitor GM6001, adding Euro-Collin solution, and diluting to a constant volume of 1L to form a second storage solution.
2. Taking a proper amount of the second preservation solution obtained in the step 1, and cooling to 2-4 ℃. And adding the cooled second preservation solution into the tissue to be frozen at the temperature of 2-4 ℃ by 6 steps, and finishing the introduction within 90min (adding the second preservation solution into the tissue by 6 steps by 15min at the temperature of 2-4 ℃ for 15min each time).
3. And (3) cooling the tissue introduced into the second preservation solution in the previous step to-100 ℃ at the speed of-43 ℃/min, cooling the sample to be less than-130 ℃ at the speed of-3 +/-0.2 ℃/min, and preserving the sample in a liquid bath of 2-methylbutane for 8 months.
4. Taking out a sample to be resuscitated, and recovering to-70 ℃ at the rate of 30 +/-0.2 ℃/minute and then recovering to 4 ℃ at the rate of 225 +/-15 ℃/minute;
5. gradually reducing and removing the second preservative solution in 5min at 2-4 deg.C, and replacing and eluting the second preservative solution within 30min for 5min every time.
In this example, a portion of the sample preserved in preservative solution two, which was cooled to less than-130 ℃ through steps 2 and 3, was taken out from the storage place at less than-130 ℃, placed in a Dry Shipper (a vapor phase liquid nitrogen transfer tank, the temperature of which could be maintained at less than-130 ℃ for one week), and the Dry Shipper was placed on a shaker to shake slowly, simulating the transport of the sample preserved in preservative solution two. The samples stored in preservative solution two were stored in a Dry Shipper for 48 hours under this simulated sample transport condition and then transferred back to the previous-130 ℃ storage location. Thereafter sample resuscitation was performed as in steps 4 and 5.
Example 3
1. Mixing 2.5mol of ethylene glycol, 2.5mol of propylene glycol, 0.5mol of hydroxyethyl piperazine ethanethiosulfonic acid, 6mg of matrix metalloproteinase inhibitor GM6001 and 0.8g of polyvinyl alcohol, adding Euro-Collin solution, and diluting to a constant volume of 1L to obtain a third storage solution.
The rest is the same as example 1.
Example 4
1. Mixing 2.5mol of ethylene glycol, 2.5mol of propylene glycol, 0.5mol of hydroxyethyl piperazine ethanethiosulfonic acid, 25 mu mol of caspase inhibitor Z-VAD-FMK and 0.8g of polyvinyl alcohol, adding Euro-Collin solution to a constant volume of 1L to form a fourth preservation solution.
The rest is the same as example 1.
Control 1 (conventional freezing control)
A conventional cryoprotectant solution is provided which contains 10% DMSO by volume percent. Adding the cryoprotectant into a tissue to be subjected to cryopreservation at room temperature, transferring the tissue into a freezing solution containing 10% DMSO, balancing for 1 minute, placing the tissue into a programmed cooling instrument, cooling to-90 ℃ at the speed of-1 ℃/min, and transferring to liquid nitrogen at-196 ℃ for long-term storage.
In this example, a portion of the cryopreserved sample was removed from storage at less than-130℃, placed in a Dry Shipper (a vapor phase liquid nitrogen transfer tank that could be maintained at less than-130℃ for one week), and placed on a shaker and shaken slowly to simulate the transport of the cryopreserved sample. The cryopreserved samples were stored in a Dry Shipper for 48 hours under this simulated sample transport and then transferred back to the previous liquid nitrogen storage at-196 ℃.
Control 2 (fresh control): fresh tissue without freezing treatment is obtained and placed in Euro-Collin solution, and stored in environment of 2-10 deg.C for no more than 12 hr.
In addition, aiming at the currently disclosed ice-free freezing method, the method is divided into two types according to the convention of the professional field: the first is a protective agent which can penetrate into cells, has high efficacy, but has great toxicity at high concentration; the second type is a carbohydrate, which has low toxicity, but large molecule, which cannot enter the cell, and low efficacy. For each class we set a representative control (the first class is represented by control 3 and the second class is represented by control 4):
control 3 (ice-free frozen control-with intracellular cryoprotectant): the cryoprotectant used contained, per 1000 ml: 200ml of 99.9 percent dimethyl sulfoxide, 160g of acetamide, 100ml of 1, 2-propylene glycol, 50g of polyethylene glycol, 60g of trehalose and the balance of deionized water. The rest is the same as example 1.
Control 4 (ice-free frozen control-with extracellular cryoprotectant): the adopted refrigeration protective solution comprises the following components in parts by weight: 20 parts of carboxymethyl starch, 2.5 parts of rhamnose, 1.5 parts of spermene, 3 parts of microcrystalline cellulose, 5 parts of galactomannose, 5 parts of muramyl dipeptide, 4 parts of tripalmitoyl pentapeptide, 1.0 part of alpha-arbutin and 70 parts of deionized water. The rest is the same as example 1.
Effect experiment:
the decision whether ice-free freezing is effective in different tissues is primarily a matter of the thermal conductivity of the tissue, the permeability of the cells to the ice-free cryoprotectant. Although the effect test was performed in one tissue or two tissues or in three tissues of cartilage, tendon and meniscus, which were developed simultaneously. However, since cartilage, tendon, meniscus tissue structures, cells, and interstitial components are similar, and ice crystal formation is similar in these tissues, according to clinical knowledge, this invention is effective in one tissue and generally effective in the other two tissues.
Effect experiment 1: cartilage, tendon and meniscus tissue organization survival rate test experiment
The Alamar-Blue (Almaryland) detection method is adopted in the experiment: the main component is a redox indicator which exhibits violet-blue non-fluorescence in the oxidized state and in the reduced state is converted into a reduction product which fluoresces pink or red, with an absorption peak of 530-560nm and a scattering peak of 590 nm.
Collected fresh pig articular meniscus, cartilage and tendon tissue samples (figure 1) are stored at reduced temperature by using the storage solution and the method respectively prepared in examples 1-4 and control groups 3-4. After 2 days of transport and 8 months of storage, tissue viability test experiments were performed by alamar blue staining, measuring at fixed wavelengths in a spectrophotometer, comparing control 2-normal fresh tissue, calculating the cell fluorescence per mg of tissue and assessing the cell viability in the stored tissue (table 1; fig. 5).
Table 1. survival of non-iced frozen cartilage, meniscus, tendon transported for 2 days and stored for 8 months as in examples 1-4 and controls 1, 3, 4 (%) -absolute relative fluorescence of cartilage, meniscus, tendon tissue/percentage of control 2-fresh cartilage, meniscus, tendon of 100.
As can be seen by the Amar blue fluorescent labeling method, the cell activity of the tissue preserved by the method of example 1 after 2 days of transportation and 8 months of preservation after rewarming is more than 90% of that of the control group 2-fresh tissue. In contrast, the tissue cryopreserved in the control 1-conventional manner showed a severe decrease in cell activity, which was only 20% or less of that of the fresh tissue. The tissue preserved by the method of example 2 is less stable in viability from 50% to 60% after transportation and storage, and has an average viability rate of 60% after 8 months of storage; the tissue preserved by the method of the control group 3-4 was also low in tissue viability between 50% and 60% after transportation and storage. These results may be due to unstable vitrification conditions, the occurrence of ice crystal reformation (recrystalization) or Devitrification (development) to damage tissue.
Effect experiment 2: experiment of presence or absence of ice crystal formation in specimen transportation and storage
The sample adopts a cryo-substitution (cryo-stabilization) technology, 1% osmium tetroxide is used as a solvent to replace water which is frozen, and the tissue is fixed at a deep low temperature, specifically, the sample is dehydrated at-20 ℃ overnight, stored for 1 hour at 4 ℃, further dehydration is completed at room temperature, then resin is added into 100% acetone solution, toluidine blue is used for dyeing, observation is carried out under a light microscope, uranium acetate milling and lead citrate dyeing are used for observation under an electron microscope, and thus, the position and the size of the ice crystal in the tissue are displayed in a tissue section. All examples and controls tested samples for the presence or absence of ice crystal formation during transport and storage, except for fresh tissue (control 2), and the results are shown in fig. 2 and table 2.
Table 2. frozen cartilage without ice crystals, meniscus and tendon were tested for the formation of ice crystals by transporting the frozen cartilage without ice crystals for 2 days and storing the frozen cartilage without ice crystals for 8 months according to examples 1 to 4 and controls 1, 3 and 4.
As table 2 shows, no ice crystal formation was observed in examples 1, 3, 4. However, there was ice crystal formation in control 1, trace ice crystal formation in example 2 and control 3, and small ice crystal formation in control 4.
As can be seen from FIG. 2, the tissue preserved by the preservation solution and method of control 1 was clearly seen to be distributed over cartilage (upper left of FIG. 2), ice crystals of different sizes within cells of meniscus (upper right of FIG. 2) and on extracellular matrix, white irregular-sized voids were left when the ice crystals occupied, chondrocytes were occupied by the ice crystals, intracellular material was pushed aside, and the cartilage matrix was filled with fine ice crystals. The interstitial ice crystals of meniscal tissue form more cartilage, and it may be that the meniscal tissue is looser than the cartilage tissue. The freezing obviously changes the structure of the frozen meniscus, and the formation of ice crystals in the gaps between layers destroys the overall function of the meniscus, especially the fibrous tissue, because the tissue is loose and the ice crystals are larger, which is of great significance for maintaining the integrity of the fibrous tissue after the meniscus is transplanted. Cartilage (lower left in fig. 2) and meniscal tissue (lower right in fig. 2) preserved using example 1 were high in integrity and fibrous tissue morphology was intact relative to control 1.
Effect experiment 3: fluorescence labeling cell viability test experiment
Calcein (Calcein AM) is a cell staining reagent that can fluorescently label living cells, and it penetrates cell membranes and enters cells, and is cut by intracellular esterase to form Calcein, which is then retained in the cells and emits strong green fluorescence. The cartilage sample cell viability of the fresh tissue of example 1 and control 1, which had been stored for 8 months and control 2, was tested using Calcein-AM cell viability assay kit (fluorescence method) (fig. 3). The cell viability rate of the control group 2-fresh tissue (fig. 3 left), example 1-frozen without ice crystal (fig. 3) and the control group 1-traditional frozen tissue (fig. 3 right) is much higher in example 1 than in control group 1. The cell viability rate was calculated as the ratio of viable cell count to total cells in the tissue (FIG. 4), and it can be seen that the viable cell count in the tissue preserved in example 1 was much higher than that in control 1, and was close to that in control 2, a fresh tissue.
Effect experiment 4: biomechanical testing experiment
The biomechanical properties of cartilage and meniscus, like those of other human tissues, are based on the integrity of cells and extracellular matrix components. The tissues in the joint should be able to bear pressure and have good elasticity. Various biomechanical tests can be used to assess the mechanical properties of cartilage, meniscal tissue. Conventional cartilage, meniscal mechanical property tests include compression, tension and shear tests. Most of which require sufficiently large tissue samples. Because of the need to test small cartilage, meniscal specimens, such as porcine cartilage, meniscus, a depth and load sensing microcarper was used. The load and displacement of the indenter, which is a stainless steel tapered pin with a radius of 0.25m m, a force resolution of about 0.5mN, and a displacement resolution of about 0.1 μm, was monitored and recorded by a micro-indenter. The method can evaluate indentation resistance of wet tissue samples (cartilage, meniscus) similar to indentation tests on dry solid materials (e.g., metals) to measure hardness. An indentation load-depth curve can be obtained through a micro-indentation test. The difference in indentation depth is an index for measuring the change in mechanical properties.
Figure 6 shows the load-indentation depth after 8 months of storage of cartilage and meniscus tissue samples from control 2-fresh tissue, example 1-no-cryo-and control 1-conventional cryo-cartilage storage. As can be seen in fig. 6, the resistance to indentation of example 1 is similar to the control 2 sample. Control 1 had the highest indentation depth (relatively lower resistance), indicating that example 1 had better integrity of the cells and extracellular matrix components.
Effect experiment 5: pig articular cartilage transplantation experiment
Fresh pig articular cartilage is selected for the experiment and prepared into a cylinder with the diameter of 6mm and the thickness of 4-6mm under a clean environment (the result is prevented from being inaccurate due to the pollution of microorganisms on the sample). A group of the samples were subjected to cryopreservation by using the first preservative solution and the method of example 1, and a group of the samples were subjected to cryopreservation by using the control group 1-conventional cryopreservation method for 8 months.
9 pig knee joint cartilage defect models were prepared, and three groups of 3 were each prepared by transplanting control group 2-fresh cartilage, example 1-preservation solution-frozen cartilage and control group 1-conventional frozen cartilage, respectively, and fig. 7 shows the implantation of the pig knee joint cartilage defect model with the frozen cartilage of example 1 (fig. 7B), the control group 2-fresh cartilage (fig. 7D) and the control group 1-conventional frozen cartilage (fig. 7A and C). After the cartilage defect model of the knee joint of the pig is implanted into the cartilage for 12 weeks, the cartilage is taken out for observation, and the joint healing of the transplanted ice crystal-free frozen cartilage and the transplanted fresh cartilage is good; whereas joints transplanted with conventional frozen cartilage heal poorly.
Fig. 8 is O' Driscoll histological score 12 weeks after cartilage transplantation, and transplantation example 1-non-cryo cartilage was not significantly different from transplantation control 2-fresh cartilage. The transplanted control group 1-conventional frozen cartilage was significantly different from the example 1 control group 2 cartilage.
Claims (10)
1. A cryo-preservation solution for cartilage, tendon and meniscus without crystal ice is characterized by comprising 1.0-6.0mol/L of ethylene glycol, 1.0-7.0mol/L of propylene glycol, 0.1-2.5mol/L, Caspase of hydroxyethyl piperazine ethanethiosulfonic acid inhibitor Z-VAD-FMK 10-50 mu mol/L, 2-10mg/L of matrix metalloproteinase inhibitor GM6001 and a component X.
2. The cartilage, tendon and meniscus cryo-preservation solution without ice crystal according to claim 1 comprising 2.0-5.0mol/L ethylene glycol, 2.5-5.5mol/L propylene glycol, 0.1-1.0mol/L, Caspase hydroxyethylpiperazine ethanethiosulfonic acid inhibitor Z-VAD-FMK 10-50 μmol/L, 2-10mg/L matrix metalloproteinase inhibitor GM6001 and component X; the component X is selected from one or more of 0.05-2% (w/v) polyvinyl alcohol, 0.1-5% (w/v) polyethylene glycol, 0.5-10% (w/v) dextran or 0.3-7% (w/v) hydroxyethyl starch; preferably 0.05-1% (w/v) polyvinyl alcohol.
3. The cartilage, tendon and meniscus cryo-preservation solution without ice crystals according to claim 1, wherein the ethylene glycol content is 2.5 mol/L; the content of the propylene glycol is 2.5 mol/L; the content of the hydroxyethyl piperazine ethanesulfonic acid is 0.5 mol/L.
4. The cartilage, tendon and meniscus cryo-preservation solution according to claim 1 wherein the other component is a solvent, preferably the solvent is Euro-Collin solution.
5. Use of the cryo-preservation solution according to any of claims 1 to 4 for the cryo-preservation of cartilage, tendons or menisci.
6. The use according to claim 5, wherein the non-crystal cryopreservation solution according to any one of claims 1 to 4 is introduced into cartilage, tendon and meniscus tissue in a stepwise manner when the cartilage, tendon and meniscus tissue is preserved; the step-by-step mode is specifically that the non-crystal frozen storage solution of any one of claims 1 to 4 is added to cartilage, tendon and meniscus tissues in increments of 4 to 15 steps in 10 to 25min under the condition of-5 to +22 ℃, then the temperature is firstly reduced to-80 to-120 ℃ at the speed of-20 to-70 ℃ per minute, and then is reduced to-120 to-180 ℃ at the speed of-2 to-40 ℃ per minute, and the frozen storage solution is stored at the temperature.
7. The use according to claim 6, wherein after introducing the non-crystal cryopreservation liquid according to any one of claims 1 to 4, one or more liquids Y are used to cover the surface layer and the periphery of the cryopreservation system, and the component Y is one or more selected from methyl propane, isobutane, bromopropane, 2-methylbutane, dibromopropane and bromooctane; 2-methylbutane is preferred.
8. The use according to claim 6, wherein the stepwise manner is to add the cryo-free preservation solution to the cartilage, tendon and meniscus tissue in 6 steps in 15min stepwise increments at 2-4 ℃, and introduce the cryo-free preservation solution to the cartilage, tendon and meniscus tissue 15min at a time.
9. The use of claim 6, wherein the first temperature reduction is carried out at a rate of-38 ℃ to-50 ℃/min to-90 ℃ -110 ℃; preferably, the temperature is reduced to-100 ℃ at a rate of-43 +/-2 ℃/min.
10. The use of claim 6, wherein the second temperature reduction is carried out at a rate of-2 ℃ to-10 ℃/minute to a second temperature of-120 ℃ to-150 ℃; preferably, the temperature is reduced to-135 ℃ at a rate of-3 +/-0.2 ℃/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010554905.9A CN111657267B (en) | 2020-06-17 | 2020-06-17 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010554905.9A CN111657267B (en) | 2020-06-17 | 2020-06-17 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111657267A true CN111657267A (en) | 2020-09-15 |
| CN111657267B CN111657267B (en) | 2021-02-02 |
Family
ID=72388491
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010554905.9A Active CN111657267B (en) | 2020-06-17 | 2020-06-17 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111657267B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113331176A (en) * | 2021-06-01 | 2021-09-03 | 北京戴域生物技术有限公司 | Mesenchymal stem cell cryopreservation liquid |
| CN114375944A (en) * | 2021-12-31 | 2022-04-22 | 四川大学华西医院 | Cryopreservation liquid and cryopreservation method for bone tissues and bone soft tissues |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024013571A1 (en) * | 2023-02-03 | 2024-01-18 | Tavakoli Seyedamirhossein | Processing of human hyaline cartilage |
Citations (31)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992008347A1 (en) * | 1988-04-18 | 1992-05-29 | Cryolife, Inc. | Cryoprotective agent |
| WO1997039104A1 (en) * | 1996-04-17 | 1997-10-23 | Osiris Therapeutics, Inc. | Cryopreservation and extensive subculturing of human mesenchymal stem cells |
| WO2004052098A1 (en) * | 2002-12-11 | 2004-06-24 | Cryolife, Inc. | Radical retardant cryopreservation solutions |
| CA2857051A1 (en) * | 2004-03-17 | 2005-09-29 | David Ayares | Tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase |
| CN101720753A (en) * | 2009-12-09 | 2010-06-09 | 中国人民解放军第四军医大学 | Cryopreservation solution of tissue engineering products and application method thereof |
| WO2011047469A1 (en) * | 2009-10-19 | 2011-04-28 | The Governors Of The University Of Alberta | Cryopreservation of articular cartilage |
| CN102308787A (en) * | 2011-07-06 | 2012-01-11 | 中国人民解放军第四军医大学 | Cartilage preservation solution |
| CN102550541A (en) * | 2010-12-23 | 2012-07-11 | 潘峰 | Organ vitrification freezing and preserving fluid |
| CN104837339A (en) * | 2012-10-18 | 2015-08-12 | 生命线科学有限公司 | Preservation of biomaterial properties and methods of storing |
| CN104938478A (en) * | 2015-05-20 | 2015-09-30 | 泰山医学院 | Cartilago articularis vitrification cryoprotectant and cartilage preservation method |
| CN105211053A (en) * | 2015-10-29 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution for tendon stem cells derived from human achilles tendon and preparation method thereof |
| CN105230610A (en) * | 2015-11-13 | 2016-01-13 | 中国科学院化学研究所 | Cryopreservation solution as well as preparation method and application thereof |
| WO2016109828A1 (en) * | 2014-12-31 | 2016-07-07 | Applied Biologics, Llc | Injectable amniotic membrane tissue graft |
| CN106614526A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Vitrified cryopreservation method for cartilage |
| CN108041023A (en) * | 2017-12-14 | 2018-05-18 | 泰山医学院 | A kind of mechanical stimulation method for improving articular cartilage storage in vitro effect |
| US20180326122A1 (en) * | 2017-05-09 | 2018-11-15 | Vivex Biomedical, Inc. | Biological composition in a protectant shroud and methods |
| CN108835105A (en) * | 2018-09-10 | 2018-11-20 | 刘亚莉 | A kind of articular cartilage glass freezing protection liquid and preparation method |
| WO2018232445A1 (en) * | 2017-06-19 | 2018-12-27 | Deakin University | Scaffolds for cell culture and tissue regeneration |
| WO2019006328A1 (en) * | 2017-06-30 | 2019-01-03 | Allosource | Cellular bone grafts, and methods of manufacture and use |
| CN109566601A (en) * | 2018-12-29 | 2019-04-05 | 中国人民解放军第四军医大学 | A kind of cartilage preservation solution and its application method |
| CN109644989A (en) * | 2019-01-28 | 2019-04-19 | 徐艳红 | A kind of articular cartilage frozen solution and cartilage freezing and storing method |
| CN109788752A (en) * | 2016-07-22 | 2019-05-21 | 组织测试技术有限公司 | It is saved with glycolipid enhancing cell freezing |
| CN110250162A (en) * | 2019-07-05 | 2019-09-20 | 北京太东生物科技有限公司 | Frozen Biological protective agent and its application |
| CN110402918A (en) * | 2018-04-27 | 2019-11-05 | 庄明华 | A method of saving bone tissue material |
| CN110545665A (en) * | 2016-12-20 | 2019-12-06 | 组织测试技术有限公司 | Ice-free preservation of large volume tissue samples for live functional tissue banks |
| CN110558312A (en) * | 2019-10-09 | 2019-12-13 | 科瑞百奥泰州生物技术有限公司 | Cartilage tissue low-temperature refrigeration preservation solution and application thereof |
| US20200054789A1 (en) * | 2018-08-17 | 2020-02-20 | Vivex Biomedical Inc. | Infused fibers |
| WO2020037416A1 (en) * | 2018-08-22 | 2020-02-27 | The Governors Of The University Of Alberta | Cryopreservation of cell monolayers |
| US20200107935A1 (en) * | 2017-05-30 | 2020-04-09 | Trauma Care Consult | Cartilage graft scaffolds |
| CN111088224A (en) * | 2019-12-31 | 2020-05-01 | 广东唯泰生物科技有限公司 | Method for promoting directional differentiation of umbilical cord mesenchymal stem cells to chondroblasts |
| CN111264516A (en) * | 2020-02-20 | 2020-06-12 | 科瑞百奥泰州生物技术有限公司 | Cryo-free blood vessel preservation solution and blood vessel preservation method |
-
2020
- 2020-06-17 CN CN202010554905.9A patent/CN111657267B/en active Active
Patent Citations (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992008347A1 (en) * | 1988-04-18 | 1992-05-29 | Cryolife, Inc. | Cryoprotective agent |
| WO1997039104A1 (en) * | 1996-04-17 | 1997-10-23 | Osiris Therapeutics, Inc. | Cryopreservation and extensive subculturing of human mesenchymal stem cells |
| WO2004052098A1 (en) * | 2002-12-11 | 2004-06-24 | Cryolife, Inc. | Radical retardant cryopreservation solutions |
| CA2857051A1 (en) * | 2004-03-17 | 2005-09-29 | David Ayares | Tissue products derived from animals lacking any expression of functional alpha 1,3 galactosyltransferase |
| WO2011047469A1 (en) * | 2009-10-19 | 2011-04-28 | The Governors Of The University Of Alberta | Cryopreservation of articular cartilage |
| CN101720753A (en) * | 2009-12-09 | 2010-06-09 | 中国人民解放军第四军医大学 | Cryopreservation solution of tissue engineering products and application method thereof |
| CN102550541A (en) * | 2010-12-23 | 2012-07-11 | 潘峰 | Organ vitrification freezing and preserving fluid |
| CN102308787A (en) * | 2011-07-06 | 2012-01-11 | 中国人民解放军第四军医大学 | Cartilage preservation solution |
| CN102308787B (en) * | 2011-07-06 | 2013-10-02 | 中国人民解放军第四军医大学 | Cartilage preservation solution |
| CN104837339A (en) * | 2012-10-18 | 2015-08-12 | 生命线科学有限公司 | Preservation of biomaterial properties and methods of storing |
| WO2016109828A1 (en) * | 2014-12-31 | 2016-07-07 | Applied Biologics, Llc | Injectable amniotic membrane tissue graft |
| CN104938478A (en) * | 2015-05-20 | 2015-09-30 | 泰山医学院 | Cartilago articularis vitrification cryoprotectant and cartilage preservation method |
| CN104938478B (en) * | 2015-05-20 | 2018-02-02 | 泰山医学院 | A kind of articular cartilage glass freezing protection liquid and cartilage store method |
| CN105211053A (en) * | 2015-10-29 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation solution for tendon stem cells derived from human achilles tendon and preparation method thereof |
| CN105230610A (en) * | 2015-11-13 | 2016-01-13 | 中国科学院化学研究所 | Cryopreservation solution as well as preparation method and application thereof |
| CN109788752A (en) * | 2016-07-22 | 2019-05-21 | 组织测试技术有限公司 | It is saved with glycolipid enhancing cell freezing |
| CN110545665A (en) * | 2016-12-20 | 2019-12-06 | 组织测试技术有限公司 | Ice-free preservation of large volume tissue samples for live functional tissue banks |
| CN106614526A (en) * | 2016-12-30 | 2017-05-10 | 潍坊医学院 | Vitrified cryopreservation method for cartilage |
| US20180326122A1 (en) * | 2017-05-09 | 2018-11-15 | Vivex Biomedical, Inc. | Biological composition in a protectant shroud and methods |
| US20200107935A1 (en) * | 2017-05-30 | 2020-04-09 | Trauma Care Consult | Cartilage graft scaffolds |
| WO2018232445A1 (en) * | 2017-06-19 | 2018-12-27 | Deakin University | Scaffolds for cell culture and tissue regeneration |
| WO2019006328A1 (en) * | 2017-06-30 | 2019-01-03 | Allosource | Cellular bone grafts, and methods of manufacture and use |
| CN108041023A (en) * | 2017-12-14 | 2018-05-18 | 泰山医学院 | A kind of mechanical stimulation method for improving articular cartilage storage in vitro effect |
| CN110402918A (en) * | 2018-04-27 | 2019-11-05 | 庄明华 | A method of saving bone tissue material |
| US20200054789A1 (en) * | 2018-08-17 | 2020-02-20 | Vivex Biomedical Inc. | Infused fibers |
| WO2020037416A1 (en) * | 2018-08-22 | 2020-02-27 | The Governors Of The University Of Alberta | Cryopreservation of cell monolayers |
| CN108835105A (en) * | 2018-09-10 | 2018-11-20 | 刘亚莉 | A kind of articular cartilage glass freezing protection liquid and preparation method |
| CN109566601A (en) * | 2018-12-29 | 2019-04-05 | 中国人民解放军第四军医大学 | A kind of cartilage preservation solution and its application method |
| CN109644989A (en) * | 2019-01-28 | 2019-04-19 | 徐艳红 | A kind of articular cartilage frozen solution and cartilage freezing and storing method |
| CN110250162A (en) * | 2019-07-05 | 2019-09-20 | 北京太东生物科技有限公司 | Frozen Biological protective agent and its application |
| CN110558312A (en) * | 2019-10-09 | 2019-12-13 | 科瑞百奥泰州生物技术有限公司 | Cartilage tissue low-temperature refrigeration preservation solution and application thereof |
| CN111088224A (en) * | 2019-12-31 | 2020-05-01 | 广东唯泰生物科技有限公司 | Method for promoting directional differentiation of umbilical cord mesenchymal stem cells to chondroblasts |
| CN111264516A (en) * | 2020-02-20 | 2020-06-12 | 科瑞百奥泰州生物技术有限公司 | Cryo-free blood vessel preservation solution and blood vessel preservation method |
Non-Patent Citations (11)
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113331176A (en) * | 2021-06-01 | 2021-09-03 | 北京戴域生物技术有限公司 | Mesenchymal stem cell cryopreservation liquid |
| CN113331176B (en) * | 2021-06-01 | 2022-04-29 | 零下十八度(北京)生物科技有限公司 | Umbilical cord mesenchymal stem cell cryopreservation liquid |
| CN114375944A (en) * | 2021-12-31 | 2022-04-22 | 四川大学华西医院 | Cryopreservation liquid and cryopreservation method for bone tissues and bone soft tissues |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111657267B (en) | 2021-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111657267B (en) | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus | |
| KR100361357B1 (en) | Cryopreservation of cultured tissue equivalents | |
| AU2002211792B2 (en) | Method of cryopreservation of tissues or organs other than a blood vessel by vitrification | |
| DE69333926T2 (en) | Method for processing and preserving collagen-based tissue parts for transplantation | |
| US5131850A (en) | Method for cryopreserving musculoskeletal tissues | |
| Capella et al. | Preservation of viable corneal tissue | |
| SK284801B6 (en) | Method for cryopreservation of mammalian tissue | |
| SCHACHAR et al. | Investigations of low-temperature storage of articular cartilage for transplantation. | |
| Finkle et al. | Cryoprotective compounds in the viable freezing of plant tissues | |
| AU2002211792A1 (en) | Method of cryopreservation of tissues or organs other than a blood vessel by vitrification | |
| Taylor | Clinical cryobiology of tissues: preservation of corneas | |
| Pegg | The preservation of tissues for transplantation | |
| Sherman et al. | Ultrastructure before freezing, while frozen, and after thawing in assessing cryoinjury of mouse epididymal spermatozoa | |
| EP3516959B1 (en) | Protocol for the cryopreservation of shrimp spermatophores/sperm mass | |
| Han et al. | Protection of osteoblastic cells from freeze/thaw cycle-induced oxidative stress by green tea polyphenol | |
| Villalba et al. | Cryopreservation of human skin with propane-1, 2-diol | |
| Pegg | Cryopreservation | |
| Chang et al. | Changes in mechanical, structural integrity and microbiological properties following cryopreservation of human cadaveric iliac arteries | |
| US11589574B2 (en) | Compositions and methods for transport and preservation of biological material | |
| Bourne | Clinical and experimental aspects of corneal cryopreservation | |
| Kruit et al. | Superior preservation of capillaries, myofibrils and mitochondria after long-term extracorporeal perfusion of free muscle flaps–A descriptive electron microscopy study | |
| Armitagetage | Feaibility of cornel vitrification | |
| Platts et al. | USE OF DIMETHYL SULPHOXIDE FOR PRESERVING CORNEAL TISSUE | |
| Yang et al. | Cryopreservation of human nasal ciliated epithelium | |
| Song et al. | Principles of cell and tissue cryopreservation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |