Method for preserving large-volume biological sample at low temperature

A technology for biological samples and protective agents, applied in the field of biomedicine, can solve problems such as influence effect and survival rate, biological sample structure, mechanical and biological characteristics damage, complicated operation, etc., so as to avoid osmotic damage and biochemical toxicity, avoid reaction Vitrification and recrystallization, excellent biological functions

Pending Publication Date: 2019-06-18
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The existing cryopreservation scheme for large-scale biological samples such as blood vessels is to put the samples into cryopreservation tubes after processing. The biological samples will curl up and bend after freezing, which will affect the structure, mechanics and biological properties of the biological samples. cause damage
Moreover, most of the existing preservation techniques use the method of gradually adding protective agents, using a programmed cooling process, and adopting the method of passive rewarming while shaking in a constant temperature water bath, which takes a long time and is cumbersome to operate, which seriously affects large-volume biological samples The effect of saving
[0005] Due to the macroscopic size of the cryopreservation tube and the conduction mode of heat from the outside to the inside, the speed of heat transfer is limited, the temperature difference of each position inside the biological material sample is large during the cooling and rewarming process, and the temperature gradient inside and outside the sample is large. Will lead to large thermal stress, easy to cause fracture and microcracks
If the rewarming rate is not fast enough, it will lead to severe devitrification and recrystallization, which will seriously affect the preservation effect and survival rate; at the same time, high concentrations of cryoprotectants may cause uncontrollable osmotic damage and biochemical toxicity, which is harmful to biological materials. deadly for

Method used

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  • Method for preserving large-volume biological sample at low temperature
  • Method for preserving large-volume biological sample at low temperature
  • Method for preserving large-volume biological sample at low temperature

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, a kind of method of cryopreservation of large-volume biological sample

[0064] The stem cell hydrogel structure of the core-shell structure is added with a protective agent in two steps. The first step is to add a protective agent containing a medium solution of 1mol / L ethylene glycol and 1mol / L1,2-propanediol to balance for 25 minutes; the second Add the protective agent of the culture medium solution containing 1mol / L ethylene glycol, 1.5mol / L 1,2-propanediol, 0.002mol / L dextran and 1mol / L trehalose step by step and equilibrate for 15 minutes. Then it was divided into two parts and added to a polytetrafluoroethylene tube (1.2 mm inner diameter) and a straw (1.84 mm inner diameter), respectively, and each was frozen in liquid nitrogen for 24 hours. The frozen samples were rewarmed in a 37°C water bath. Fluorescent staining was used to test the survival rate of the stem cell hydrogel constructs preserved in the straw and tetrafluoroethylene tube containe...

Embodiment 2

[0068] Embodiment 2, a kind of method of cryopreservation of large-volume biological sample

[0069] The specific operation is as follows:

[0070] 1) adding a protective agent to the stem cell hydrogel construct with a core-shell structure in two steps, the first step is to add a protective agent in a culture medium solution containing 1mol / L ethylene glycol and 1mol / L 1,2-propanediol to balance for 25 minutes; In the second step, a protective agent was added to a culture medium solution containing 1 mol / L ethylene glycol, 1.5 mol / L 1,2-propanediol, 0.002 mol / L dextran and 1 mol / L trehalose to equilibrate for 15 minutes.

[0071] 2) Divide the stem cell hydrogel construct into two parts, one part is inhaled into a polytetrafluoroethylene tube, put into liquid nitrogen for 24 hours, and the frozen sample is rewarmed in a water bath; the other part is added with Fe 3 o 4After the nanoparticles (in g / ml, the mass volume ratio of the magnetic nanoparticles to the protective age...

Embodiment 3

[0076] Embodiment 3, a kind of method of cryogenic preservation of large-volume biological samples

[0077] The specific operation is as follows:

[0078] 1) Obtain fat stem cells from animals as biological samples;

[0079] 2) Add the protective agent in three steps. In the first step, add 1mol / L ethylene glycol + 1mol / L 1,2-propanediol to the culture medium solution to balance the protective agent for 20 minutes; in the second step, add 1.5mol / L ethylene glycol + 1.5mol / L1,2-Propanediol The protective agent of the culture medium solution is balanced for 15 minutes, and the third step is to add 2mol / L ethylene glycol+2mol / L1,2-Propanediol+0.5mol / L trehalose to protect the culture medium solution Dose equilibration for 15 minutes;

[0080] 3) Add 0.05% Fe 3 o 4 Nanoparticles are added to the equilibrated biological sample suspension;

[0081] 4) A polytetrafluoroethylene tube with a specification of 1.2mm×1.6mm (inner diameter×outer diameter) is selected and wound on the ...

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Abstract

The invention belongs to the field of biomedicine, and discloses a method for preserving a large-volume biological sample at a low temperature. According to the specific method, the biological sampleis pretreated, then a protective agent is added in the biological sample, the biological sample is injected into a polytetrafluoroethylene tube for freezing preservation, and the biological sample iswarmed in a water bath when needed. By the method for preserving the large-volume biological sample at the low temperature, freezing preservation of the large-volume biological sample is successfullyrealized under the action of a low-concentration protective agent, the method is a long-term safe, stable, reliable, simple and effective method for preserving the biological sample, and the biological sample with complete microstructure, strong cell viability and excellent biological function can be obtained.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for low-temperature preservation of large-volume biological samples. Background technique [0002] The cryopreservation of biological materials refers to the use of specific methods to cool biological materials to low temperature and store them for a long time; when necessary, the biological materials can be rewarmed to physiological temperature according to specific methods, and still maintain sufficient activity. With the development of modern biomedicine, the role of cryopreservation has become increasingly prominent, especially for the cryopreservation of large-volume biological samples such as tissues and organs. [0003] Typical cryopreservation mainly includes five steps: adding a protective agent, cooling, long-term storage, rewarming, and removing the protective agent. In the process of cryopreservation, biological materials will inevitably suffer from va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 赵刚陈中嵘程跃曹媛马超白雪飞
Owner UNIV OF SCI & TECH OF CHINA
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