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Ice-crystal-free cryopreservation solution for cartilage, tendon and meniscus preservation and freezing method thereof

A technology for cryopreservation of liquid and meniscus, which is applied in the field of biomedical engineering, can solve the problems of difficult to adapt to a long-term vibration state transportation environment, low articular cartilage cell viability, short storage time, etc., so as to improve the cell viability and reduce the Ice crystal reformation, low toxicity effect

Active Publication Date: 2020-09-15
江苏科瑞百奥生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] (2) After resuscitation, the cell viability of articular cartilage is still low, and the storage time is short
[0011] (3) It is difficult to apply to the transportation environment with long-term vibration state
[0012] In addition, there is currently no published vitrification cryoprotective solution and preservation method for tendon and meniscus tissue

Method used

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  • Ice-crystal-free cryopreservation solution for cartilage, tendon and meniscus preservation and freezing method thereof
  • Ice-crystal-free cryopreservation solution for cartilage, tendon and meniscus preservation and freezing method thereof
  • Ice-crystal-free cryopreservation solution for cartilage, tendon and meniscus preservation and freezing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 1. Get 2.5mol ethylene glycol, 2.5mol propylene glycol, 0.5mol hydroxyethylpiperazine ethanesulfonic acid, 25 μmol Caspase inhibitor Z-VAD-FMK, 6mg matrix metalloproteinase inhibitor GM6001 and 0.8g polyvinyl alcohol, mix After uniformity, add Euro-Collin solution to make up to 1L to form preservation solution 1.

[0061] 2. Take an appropriate amount of preservation solution 1 from step 1 and cool it to 2-4°C. Add the cooled preservation solution 1 to the tissue to be frozen gradually in 6 steps at 2-4°C, and complete the introduction within 90 minutes (at 2-4°C, step by step in 15 minutes, step by step in 6 steps) Incrementally add preservation solution 1 to the tissue, 15 min each time).

[0062] 3. Cover the tissue that was introduced into Preservation Solution 1 in the previous step with 2-methylbutane. Place the 2-methylbutane-covered Preservative Solution 1 and the tissue container in the 2-methylbutane liquid bath and wait to freeze.

[0063]4. Introduce the ...

Embodiment 2

[0068] 1. Take 2.5mol ethylene glycol, 2.5mol propylene glycol, 0.5mol hydroxyethylpiperazine ethanesulfonic acid, 25μmol Caspase inhibitor Z-VAD-FMK, 6mg matrix metalloproteinase inhibitor GM6001, mix well and add Euro-Collin solution Dilute to 1L to form preservation solution 2.

[0069] 2. Take an appropriate amount of preservation solution 2 in step 1 and cool it to 2-4°C. Add the cooled preservation solution 2 gradually to the tissue to be frozen according to the 6-step method at 2-4°C, and complete the introduction within 90 minutes (at 2-4°C, step by step in 15 minutes, follow the 6-step method Incrementally add preservation solution 2 to the tissue, 15 min each time).

[0070] 3. Cool the tissue that was introduced into preservation solution 2 in the previous step to -100°C at a rate of -43°C / min, and then cool the sample to less than -130°C at a rate of -3±0.2°C / min, and then store the sample at 2 - in a liquid bath of methylbutane and kept at this temperature for 8...

Embodiment 3

[0075] 1. Take 2.5mol ethylene glycol, 2.5mol propylene glycol, 0.5mol hydroxyethylpiperazine ethanesulfonic acid, 6mg matrix metalloproteinase inhibitor GM6001 and 0.8g polyvinyl alcohol, mix well and add Euro-Collin solution to volume to 1L to form preservation solution 3.

[0076] Others are the same as embodiment 1.

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Abstract

The invention discloses an ice-crystal-free cryopreservation solution for cartilage, tendon and meniscus preservation and a freezing method thereof. The cryopreservation solution is prepared from 1.0to 6.0 mol / L of ethylene glycol; 1.0 to 7.0 mol / L of propylene glycol, 0.1 to 2.5 mol / L of hydroxyethyl piperazine ethanesulfonic acid, 10 to 50 [mu] mol / L of a Caspase inhibitor Z-VAD-FMK, 2 to 10 mg / L of a matrix metalloproteinase inhibitor GM6001 and a component X. The cryopreservation solution disclosed by the invention can effectively increase the number of living cells and the cell viabilityin cartilage, tendon and meniscus tissues after cryopreservation, and more importantly, the solution can prevent ice crystals from growing again during cryopreservation recovery and during storage, so as to prevent vitrification. According to the novel ice-crystal-free cryopreservation solution, cartilages, tendons and meniscus can be preserved for a longer time and permanently preserved in an environment with the temperature lower than -130 DEG C.

Description

technical field [0001] The invention belongs to the field of biomedical engineering, and in particular relates to an ice crystal-free cryopreservation solution and a freezing method for cartilage, tendon and meniscus preservation. Background technique [0002] Cartilage, tendon, and meniscus are structurally similar tissues composed of cells, matrix, and fibers. Its main physiological function is to provide a friction bearing surface, protect joint connectivity, reduce the load of joints during activities, and reduce joint contact Pressure, and the same preservation method can often be used in cryopreservation. It can be used for clinical repair and replacement of cartilage, tendon, meniscus injury and disease. In clinical practice, the selection of cartilage, tendon, and meniscus tissue transplantation for disease treatment usually involves the ex vivo preservation of donor cartilage, tendon, and meniscus tissue. At present, there are two methods for ex vivo preservation ...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 宋云庆
Owner 江苏科瑞百奥生物技术有限公司
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