Tissue cryopreservation liquid as well as preparation method and application thereof

Abstract

The invention provides a tissue cryopreservation solution as well as a preparation method and application thereof. The tissue cryopreservation solution comprises CDP-choline, roxburgh rose polysaccharide and poloxamer. With the adoption of the tissue cryopreservation liquid, ice crystal damage caused by tissue cryopreservation as well as cell membrane damage and oxidative stress damage in a resuscitation stage can be effectively prevented, and the tissue survival rate is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a tissue cryopreservation solution and a preparation method and application thereof. BACKGROUND

[0002] Tissue cryopreservation is a key supporting technology for the development of the fields of regenerative medicine, tissue engineering and clinical transplantation. The core is to use tissue cryopreservation solution (cryoprotectant) and combine programmed cooling to make biological tissues enter a metabolic arrest state in a super-low temperature environment, thereby realizing long-term and non-destructive preservation. However, the existing conventional tissue cryopreservation solution and technology still have significant defects, which seriously restrict the activity and functional integrity of the preserved tissues.

[0003] These challenges mainly come from the following aspects: Ice crystal damage: In the conventional slow freezing process, extracellular water freezes first, causing the concentration of intracellular unfrozen solutes to increase, forming a hypertonic environment, and causing intracellular water to exosmose and cause dehydration damage. More importantly, intracellular and extracellular ice crystals are inevitably formed during the cooling process, which physically pierce the cell membrane and organelle membrane, causing fatal mechanical damage to the cells.

[0004] Cell membrane damage: During the process of cryopreservation and recovery, the cell membrane will undergo a dramatic phase change and osmotic stress. Traditional osmotic cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are toxic to cells at high concentrations and can cause protein denaturation and membrane instability. During the addition and removal of DMSO, cells will experience severe volume expansion and contraction, causing "osmotic shock", damaging the lipid bilayer structure of the cell membrane, and further causing apoptosis and necrosis of the cells.

[0005] Oxidative stress damage: The process of low-temperature cryopreservation and recovery is a stress process that produces reactive oxygen species (ROS). The antioxidant system in cells is less active at low temperatures, resulting in the accumulation of ROS, causing lipid peroxidation, protein oxidation and DNA damage, ultimately leading to loss of cell function and apoptosis.

[0006] Therefore, there is an urgent need in the art to develop a tissue cryopreservation solution and a preparation method and application thereof. The cryopreservation solution should effectively inhibit ice crystal formation, reduce osmotic pressure and chemical toxicity damage, and provide strong antioxidant protection, thereby maximizing the maintenance of cell activity, three-dimensional structure integrity and tissue-specific function after cryopreservation and recovery. SUMMARY

[0007] Therefore, the present application provides a tissue cryopreservation solution and a preparation method and application thereof to reduce ice crystal damage, cell membrane damage and oxidative damage of tissues during the process of cryopreservation and recovery.

[0008] The technical scheme of the present application is implemented as follows: In a first aspect, the present application provides a tissue cryopreservation solution, comprising CDP-choline, rose hip polysaccharide and poloxamer.

[0009] The freezing-thawing process can cause serious damage to the cell membrane, leading to degradation of phospholipids, changes in membrane fluidity and increased membrane permeability, ultimately causing cell death. CDP-choline itself has the effect of stabilizing the phospholipid bilayer structure of the cell membrane, which can protect the integrity of the membrane.

[0010] Rose hip polysaccharide can increase the viscosity of the cryopreservation solution, thereby inhibiting ice crystal growth and recrystallization during the cooling process. Rose hip polysaccharide has excellent antioxidant capacity, which helps to reduce oxidative stress damage during the freezing-thawing process.

[0011] Poloxamer can reduce the surface tension of the solution, improve the wettability and penetration efficiency of the tissue.

[0012] On the basis of the above technical scheme, further comprising DMSO, antioxidant, trehalose, balanced salt solution and apoptosis inhibitor.

[0013] On the basis of the above technical scheme, further comprising PBS and DPBS.

[0014] On the basis of the above technical scheme, further comprising ROCK inhibitor.

[0015] On the basis of the above technical scheme, further comprising Azaindole1.

[0016] On the basis of the above technical scheme, further comprising water-soluble vitamin E, glutathione.

[0017] On the basis of the above technical scheme, the content of each component is: CDP-choline 0.1 μM~2 μM, rose hip polysaccharide 0.2 μM~1 μM, poloxamer 1%~3% (w / v), DMSO 1%~2% (w / v), antioxidant 0.5%~1% (w / v), trehalose 0.05M~0.25M, balanced salt solution 40%~70% (w / v), apoptosis inhibitor 0.1~5 μM.

[0018] On the basis of the above technical scheme, further comprising the extraction method of rose hip polysaccharide: drying rose hip fresh fruit, grinding to obtain rose hip dry powder; Mixing the rose hip dry powder with ethyl acetate at a mass-volume ratio of 1g: (2~3) mL, soaking for 8h, repeating 2 times; After removing the ethyl acetate, the Rosa roxburghii Tratt dry powder is mixed with acetone at a mass-volume ratio of 1g:(2-3)mL, and then soaked for 8h, and the operation is repeated twice. After removing the acetone, the Rosa roxburghii Tratt dry powder is mixed with physiological saline at a mass-volume ratio of 1g:(20-30)mL, and then ultrasonicated, and the supernatant is recovered. The decolorization is performed by adding 30% hydrogen peroxide solution at a volume ratio of 1:(1.5-2) to the supernatant. The Sevage method is used to remove protein to obtain the Rosa roxburghii Tratt polysaccharide supernatant. The Rosa roxburghii Tratt polysaccharide supernatant is dialyzed, and then freeze-dried to obtain the Rosa roxburghii Tratt polysaccharide.

[0019] In a second aspect, the present application further provides a preparation method of the tissue cryopreservation solution, which comprises the following steps: uniformly mixing CDP-choline, poloxamer, Rosa roxburghii Tratt polysaccharide and balanced salt solution, then uniformly mixing trehalose, an antioxidant and an apoptosis inhibitor, and finally uniformly mixing DMSO to obtain the tissue cryopreservation solution.

[0020] In a third aspect, the present application further provides application of the tissue cryopreservation solution in tissue cryopreservation.

[0021] The method for tissue cryopreservation comprises the following steps: injecting the tissue cryopreservation solution into a cryopreservation tube, then placing the tissue into the cryopreservation tube, transferring into a programmed cooling instrument, and cooling to-80℃ according to a set cooling program, and then transferring into liquid nitrogen for cryopreservation; the cooling program is as follows: maintaining at 4℃ for 5min; then reducing to 0℃ at a rate of 1℃ / min, and maintaining for 5-10min; then reducing to-10℃ within 5-10min; reducing to-40℃ within 30-50min, and then rapidly reducing to-80℃ at a rate of 40-60℃ / min; after reducing to the target temperature, standing for 4-6h, and then transferring into liquid nitrogen.

[0022] The method for tissue recovery comprises the following steps: before starting the experiment, precooling PBS on ice, placing a gun head in contact with the matrix glue in a-20℃ freezer for 1h, taking the tissue cryopreservation tube out of liquid nitrogen, quickly placing it into a 37℃ water bath, gently melting, collecting all the solution into a 15ml centrifuge tube, adding 2mL of conventional tissue culture solution to flush the cryopreservation tube once, collecting the liquid into the centrifuge tube, centrifuging at 4℃ and 1300rpm for 5min, and then removing the supernatant.

[0023] Compared with the prior art, the present application has the following beneficial effects: (1) The present application improves extracellular viscosity by adding rosehip polysaccharide, thereby reducing ice crystal damage, and the rosehip polysaccharide has an antioxidant effect, which can reduce oxidative damage together with water-soluble vitamin E. The present application adds CDP-choline to stabilize the cell membrane and reduce cell membrane damage during the recovery stage. The components and contents of the tissue cryopreservation solution of the present application cooperate with each other to maintain osmotic pressure, reduce cell membrane damage and oxidative stress damage, and improve tissue survival rate.

[0024] (2) The present application does not add animal protein, significantly reduces the amount of DMSO, and ensures safety. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field, and the reagents can be obtained commercially.

[0027] Example 1 The present embodiment provides a tissue cryopreservation solution, a preparation method and application thereof, and the content of each component is: CDP-choline 0.5 μM, rosehip polysaccharide 0.5 μM, poloxamer 2% (w / v), DMSO 1.5% (w / v), water-soluble vitamin E 0.6% (w / v), trehalose 0.1M, PBS 60% (w / v), and ROCK inhibitor 2 μM.

[0028] The preparation method comprises the following steps: mixing CDP-choline, poloxamer, rosehip polysaccharide and PBS, then adding trehalose, water-soluble vitamin E and ROCK inhibitor, and finally adding DMSO and mixing to obtain the tissue cryopreservation solution.

[0029] The tissue cryopreservation method comprises the following steps: The above tissue cryopreservation solution is injected into a cryopreservation tube, then the tissue is placed in the cryopreservation tube, transferred into a programmed temperature controller, and cooled to -80℃ according to the set cooling program, and then transferred to liquid nitrogen for cryopreservation; the cooling program is: kept at 4℃ for 5 min; then reduced to 0℃ at a rate of 1℃ / min and kept for 5-10 min; then cooled to -10℃ within 5-10 min; cooled to -40℃ within 30-50 min, and then rapidly cooled to -80℃ at a rate of 40-60℃ / min; after being reduced to the target temperature, stand for 4-6 h, and then transferred to liquid nitrogen.

[0030] The tissue thawing process includes the following steps: remove the tissue cryopreservation tube from liquid nitrogen, quickly place it in a 37°C water bath, gently thaw it, collect all the solution into a 15mL centrifuge tube, add 2mL of routine tissue culture medium to rinse the cryopreservation tube once, collect the liquid into the centrifuge tube, centrifuge at 4°C and 1500rpm for 5min, and remove the supernatant.

[0031] Example 2 This embodiment provides a tissue cryopreservation solution and its preparation method, wherein the contents of each component are as follows: CDP-choline 2μM, prickly pear polysaccharide 0.2μM, poloxamer 3% (w / v), DMSO 1% (w / v), glutathione 0.5% (w / v), trehalose 0.05M, DPBS 40% (w / v), and ROCK inhibitor 0.1μM.

[0032] The preparation method includes the following steps: CDP-choline, poloxamer, prickly pear polysaccharide and DPBS are mixed, then trehalose, glutathione and ROCK inhibitor are added and mixed, and finally DMSO is added and mixed to obtain the tissue cryopreservation solution.

[0033] Example 3 This embodiment provides a tissue cryopreservation solution and its preparation method, wherein the contents of each component are as follows: CDP-choline 0.1 μM, prickly pear polysaccharide 1 μM, poloxamer 1% (w / v), DMSO 2% (w / v), water-soluble vitamin E 1% (w / v), trehalose 0.25M, DPBS 70% (w / v), and ROCK inhibitor 5 μM.

[0034] The preparation method includes the following steps: CDP-choline, poloxamer, prickly pear polysaccharide and DPBS are mixed, then trehalose, water-soluble vitamin E and ROCK inhibitor are added and mixed, and finally DMSO is added and mixed to obtain the tissue cryopreservation solution.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that it does not contain CDP-choline, prickly pear polysaccharide, and poloxamer.

[0036] Comparative Example 2 The difference between this comparative example and Example 1 is that it does not contain CDP-choline.

[0037] Comparative Example 3 The difference between this comparative example and Example 1 is that it does not contain prickly pear polysaccharide.

[0038] Comparative Example 4 The difference between this comparative example and Example 1 is that it does not contain poloxamer.

[0039] Comparative Example 5 The difference between this comparative example and Example 1 is that the amount of prickly pear polysaccharide is too low, at 0.1 μM.

[0040] Comparative Example 6 The difference between this comparative example and Example 1 is that the amount of prickly pear polysaccharide is too high, at 10 μM.

[0041] Comparative Example 7 This comparative example provides a conventional cryopreservation solution, the formulation of which is as follows: a conventional DMSO-fetal bovine serum cryopreservation solution is prepared for later use, the specific formulation is as follows: 100 g / L dimethyl sulfoxide, 100 g / L fetal bovine serum, and DMEM / F12 basal culture medium to a final volume of 1000 mL.

[0042] Performance testing The organoid viability of kidney organoids cryopreserved for one month and then revived in Examples 1-3 and Comparative Examples 1-7 of this invention was determined. The method was to cryopreserve the organoids when they grew to 100-200 μm, count the organoids under a microscope during cryopreservation, and freeze 300 organoids per tube. After revival, they were cultured routinely for one day and then manually counted under a microscope. The organoid viability was calculated as (number of surviving organoids after revival / 300) × 100%. The results are shown in Table 1.

[0043] Table 1. Organoid survival rates of the examples and comparative examples

[0044] As shown in Table 1, the survival rate of kidney organoids obtained from the tissue cryopreservation solution of this application was higher than that of the comparative examples after one month of cryopreservation and thawing. Comparative examples 1-4 showed a significant decrease in survival rate when lacking CDP-choline, prickly pear polysaccharide, and / or poloxamer. This is because the lack of these three substances led to an osmotic pressure imbalance in the cryopreservation solution, making the kidney organoids highly susceptible to damage after thawing. Furthermore, the lack of prickly pear polysaccharide resulted in insufficient cellular antioxidant capacity, leading to oxidative stress damage; the lack of CDP-choline caused cell membrane damage during the thawing phase; and the lack of poloxamer resulted in excessively high viscosity in the cryopreservation solution, leading to mechanical damage to the kidney organoids, resulting in cell membrane damage and osmotic pressure damage. In comparative examples 5-6, insufficient or excessive prickly pear polysaccharide caused osmotic pressure imbalance, resulting in a decrease in organoid survival rate.

[0045] In summary, the tissue cryopreservation solution of the present invention can effectively protect tissues from ice crystal damage caused by cryopreservation, as well as cell membrane damage and oxidative stress damage during the recovery phase, thereby improving tissue survival rate.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tissue cryopreservation solution, characterized in that, It includes CDP-choline, prickly pear polysaccharide, and poloxamer.

2. The tissue cryopreservation solution as described in claim 1, characterized in that, It also includes DMSO, antioxidants, trehalose, balanced salt solutions, and apoptosis inhibitors.

3. The tissue cryopreservation solution as described in claim 2, characterized in that, The balanced salt solution includes PBS and DPBS.

4. The tissue cryopreservation solution as described in claim 2, characterized in that, The apoptosis inhibitors include ROCK inhibitors.

5. A tissue cryopreservation solution as described in any one of claims 2 to 4, characterized in that, The contents of each component are as follows: CDP-choline 0.1μM~2μM, prickly pear polysaccharide 0.2μM~1μM, poloxamer 1%~3% (w / v), DMSO 1%~2% (w / v), antioxidant 0.5%~1% (w / v), trehalose 0.05M~0.25M, balanced salt solution 40%~70% (w / v), and apoptosis inhibitor 0.1~5μM.

6. The method for preparing the tissue cryopreservation solution according to any one of claims 1 to 5, characterized in that, Includes the following steps: CDP-choline, poloxamer, prickly pear polysaccharide, and balanced salt solution were mixed, then trehalose, antioxidants, and apoptosis inhibitors were added and mixed. Finally, DMSO was added and mixed to obtain the tissue cryopreservation solution.

7. The application of the tissue cryopreservation solution as described in any one of claims 1 to 5 in tissue cryopreservation.

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