Novel preservation scheme with propylene glycol as low-temperature protective agent

Abstract

The invention relates to a novel preservation scheme taking propylene glycol as a low-temperature protective agent, and provides a low-temperature preservative and a preservation method for biological large tissues. The preservative takes propylene glycol as a main low-temperature protective agent, and comprises fetal calf serum and a culture medium, the pH value is adjusted to be 7.30 to 7.45. The preservation method comprises the following steps: carrying out gradient perfusion on tissues by using the preservation agent, sequentially carrying out balanced cooling in an environment of-15 DEG C and profound hypothermia transition in an environment of-80 DEG C, and transferring the tissues into liquid nitrogen for long-term preservation. According to the scheme, the characteristic that the freezing point of a propylene glycol aqueous solution mixture is reduced is utilized, a specific formula and a step-by-step cooling procedure are combined, pre-cooling is conducted at the temperature of 0-15 DEG C in a non-freezing state, ice crystal damage is effectively reduced, the activity and structural integrity of large biological tissues are better maintained, and the method is suitable for low-temperature preservation of various in-vitro biological tissues.

Description

Technical Field

[0001] This invention relates to the field of cryopreservation technology for biological samples, specifically to a preservative with propylene glycol as the main cryoprotectant, its preparation method, and a method for vitrifying large biological tissues using the preservative. Background Technology

[0002] Long-term viability preservation of ex vivo biological tissues (such as skin, cornea, blood vessels, and ovarian tissue) is a core challenge in the fields of regenerative medicine, organ transplantation, and biobanks. Currently, traditional cryoprotectants, represented by dimethyl sulfoxide (DMSO), are widely used in achieving vitrification preservation, but they have drawbacks such as high cytotoxicity, long osmotic equilibrium time, and potential impact on tissue cell function after washing and thawing.

[0003] Slow programmed cooling is another commonly used technique, but it is complex, the equipment is expensive, and ice crystals are difficult to completely avoid, posing a high risk of damage to complex biological tissues (especially complex tissues containing multiple cell types).

[0004] Therefore, there is an urgent need in the field for a cryopreservation method that is less toxic, relatively simple in procedure, and can effectively reduce ice crystal formation, in order to improve the resuscitation activity and structural integrity of large biological tissues. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a biological tissue preservation agent and method using propylene glycol as the main cryoprotectant. This solution, through optimized formulation and a unique cooling procedure, aims to reduce the toxicity of the cryoprotectant, decrease the ice crystal formation range, and promote vitrification, thereby preserving large biological tissues more safely and efficiently.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a cryopreservation agent for large biological tissues. Its components, by weight percentage, include: Propylene glycol: 30-35%; Fetal bovine serum: 10%-20%; The remainder is basal culture medium (RPMI-1640).

[0007] After preparation, the pH of the preservative needs to be precisely adjusted to the physiological range of 7.30-7.45, and then sterilized by filtration through a 0.22 μm filter membrane before use.

[0008] Secondly, the present invention provides a method for vitrifying and preserving large biological tissues using the above-mentioned preservative, comprising the following steps: Gradient perfusion: Two-step gradient perfusion is performed on the excised large biological tissue. First, perfusion with a pre-equilibrated solution containing 15% propylene glycol is carried out, and it is left standing for about 30 minutes to allow the tissue to achieve preliminary osmotic balance; subsequently, perfusion with the above-mentioned complete preservative containing 30% propylene glycol is carried out, and it is left standing for about 30 minutes again to ensure that the protective agent fully penetrates.

[0009] Initial cooling: The perfused tissue is completely immersed in the preservative and transferred together to an environment at -15°C (such as a programmable freezer or a low-temperature refrigerator) and maintained for 2 - 3 hours, so that the temperature of the tissue and the preservative evenly and slowly drops to -15°C and remains in a supercooled state without freezing.

[0010] Deep cryogenic vitrification transition: The tissue is directly transferred from the -15°C environment to an -80°C deep cryogenic refrigerator overnight (usually 8 - 12 hours). During this process, the water in the preservative and the tissue rapidly crosses the critical stage of ice crystal formation and directly achieves vitreous solid transformation.

[0011] Long-term preservation: The tissue that has completed vitrification transition is taken out of the -80°C refrigerator and quickly transferred to the gas phase or liquid phase of liquid nitrogen (-196°C) for permanent or long-term preservation.

[0012] Preferably, when reviving the tissue, the protective agent is removed by the reverse gradient washing method: First, perfusion and washing are carried out with a 15% propylene glycol solution, and it is left standing for about 30 minutes; subsequently, perfusion is carried out with a medium containing 5% - 10% fetal bovine serum; finally, repeated washing is carried out with the basic medium at least 3 times to thoroughly remove the residual protective agent.

[0013] In a third aspect, the present invention also provides a method for reviving biological tissue配套 with the above preservation method, including: Gradient dilution revival: When it is necessary to take out and use the preserved tissue, the operation is carried out using the principle of reverse gradient dilution. First, the tissue is transferred from the preservation temperature to a lower-level dilution solution (the concentration is reduced by 10% - 15%) and balanced for a period of time, and then gradually transferred to a medium serum mixture without propylene glycol, and finally, the basic medium or physiological buffer solution is used to wash at least three times to thoroughly remove the residual preservative components inside and outside the tissue and complete the revival process. Beneficial effects

[0014] Low toxicity and high efficiency: Compared with traditional DMSO, propylene glycol has lower cytotoxicity and better biocompatibility. By reducing the freezing point of the preservation solution and reducing the ice crystal formation time and range, the present invention can greatly improve the preservation effect.

[0015] Promoting vitrification: A 30% concentration of propylene glycol combined with a specific two-step cooling procedure (-15°C equilibration and -80°C rapid deep cooling) effectively increases solution viscosity, significantly inhibits the formation and growth of ice crystals, and makes it easier for tissues to reach an amorphous glassy state, thereby minimizing the physical damage of ice crystals to cells and extracellular matrix.

[0016] Simple to operate: This method does not require extremely complex ultra-fast cooling equipment, and the main steps can be completed in a conventional laboratory refrigerator, which reduces the technical threshold and cost and makes it easier to standardize and promote.

[0017] Excellent preservation effect: Experimental verification shows that mouse skin tissue and other tissues preserved using the method of this invention have significantly higher cell survival rate, tissue structural integrity and functional activity after revival than the control group using the traditional DMSO slow freezing method. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to embodiments, but the implementation and protection scope of the present invention are not limited thereto.

[0019] Weigh the following components by weight percentage: 30% propylene glycol, 0.3% trehalose, 0.3% proline, 25% fetal bovine serum, with the remainder being basal culture medium (e.g., DMEM). After thorough mixing, precisely adjust the pH to 7.32 using a dilute acid or alkali solution (e.g., HCl or NaOH). Then, sterilize the mixture by filtration through a 0.22 μm pore size filter, aliquot, and store at 4°C. This is the stock solution. This stock solution can be stored as a stable, non-freezing liquid at -15°C.

[0020] Table 1. Proportions for the Novel Preservative Solution Group Protectant 1640 culture medium serum freezing point Example 1 30ml propylene glycol 50ml 20ml Below -15℃ Comparative Example 1 10ml DMSO 70ml 20ml Above -5℃ The results in Table 1 show the suitable storage temperatures for different ratios of the preservation solution.

[0021] Preparation of final concentration and gradient solutions: The final concentration of propylene glycol was selected as 30%. The stock solution of the preservation solution prepared in Example 1 was diluted using basal culture medium. Let the final concentration (30%) be C, and gradient preservation solutions with concentrations of C1 (15% propylene glycol) and C2 (30% propylene glycol) were prepared sequentially.

[0022] Gradient osmosis: Fresh rat skin was taken and sequentially immersed in C1 and C2 preservation solutions for 40 minutes at each stage. The immersion process can be carried out at 4°C.

[0023] Low-temperature preservation: Transfer the tissue blocks treated with C2 solution to a culture dish, add an appropriate amount of C solution, and pre-cool in a -15℃ refrigerator.

[0024] After 40 minutes, it was moved to a -80°C freezer overnight, and then transferred to liquid nitrogen for permanent cryopreservation.

[0025] Gradual recovery: a. Transfer the tissue into C1 solution at 37°C and equilibrate for 30 minutes.

[0026] b. Transfer to a solution containing only basal culture medium and equilibrate for 30 minutes.

[0027] d. Finally, rinse three times with fresh basal culture medium, five minutes each time. The revived tissue can be used for subsequent sectioning or cell viability testing. Skin reimplantation can then be performed.

[0028] Table 2 Comparison of skin preservation activity assays (flow cytometry) As shown in Table 2, the cell viability of the cryopreservation scheme using 30% propylene glycol as a cryoprotectant was 91.5%, while the cell viability of the traditional DMSO scheme was 84.4%, indicating a significant improvement in preservation efficiency.

[0029] Compared with existing technologies, this project uses a method that lowers the freezing point of the preservative and preserves the tissue in a liquid state between 0°C and -15°C to prevent ice crystal formation. This completely avoids damage to the tissue caused by the formation of ice crystals and changes in osmotic pressure, significantly reduces the temperature range in which ice crystals form during tissue cooling, and increases the effectiveness of large tissue preservation.

[0030] The above description is merely 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 principle of lowering the freezing point and preserving the product in a liquid state as described in the present invention should be included within the protection scope of the present invention.

Claims

1. A novel preservation method using propylene glycol as a cryoprotectant.

2. A preservative for large biological tissues, characterized in that, By weight percentage, it includes the following components: Propylene glycol: 28-35%; fetal bovine serum: 10%-20%; the remainder is culture medium. After preparation, adjust the pH to 7.30-7.

45. Sterilize by high-temperature filtration before use.

3. A method for preserving large biological tissues, characterized in that, Using the preservative according to any one of claims 1 includes the following steps: a) Perform gradient perfusion of preservation solution on large biological tissues. First, perfuse with 15% propylene glycol preservation solution and let stand for 20-60 minutes to allow the osmotic pressure in the tissue to reach equilibrium. Then, perfuse with 30% propylene glycol preservation solution and let stand for 20-60 minutes to allow the osmotic pressure in the tissue to reach equilibrium. b) Immerse the perfused tissue in 30% propylene glycol preservation solution and place it in a -15℃ environment for 2-3 hours until the overall temperature drops uniformly to -15℃. At this time, both the preservation solution and the preserved tissue are in a non-freezing state. c) The tissue is directly transferred into a -80°C freezer overnight, which reduces the pre-cooling step of freezing between 0 and -80°C compared to the traditional method; d) The preserved tissues were transferred from a -80°C freezer to liquid nitrogen for permanent preservation.

4. The method according to claim 2, characterized in that, When the preserved biological tissue is removed, it is washed again with a gradient dilution solution. The method is characterized by first perfusing 15% propylene glycol preservation solution and letting it stand for 20-60 minutes to allow the osmotic pressure in the tissue to reach equilibrium; then perfusing serum culture medium mixture and letting it stand for 20-60 minutes; and finally washing with the base solution at least 3 times to remove residual preservatives.