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A kind of cryopreservation liquid and its preparation method and application

A cryopreservation solution and cryopreservation technology, applied in the field of cryopreservation and cryopreservation of biological materials

Active Publication Date: 2017-10-27
北京玳衲芈珂生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the field of freezing technology, programmed freezing and vitrification have been developed. The latter uses high-concentration cryoprotectant to solidify in an ultra-low temperature environment to form irregular vitrified solids without ice crystals, so that cells can be frozen in severe conditions. To overcome the ice crystal damage, the vitrified sample will still produce a large number of ice crystals during the recovery process.

Method used

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  • A kind of cryopreservation liquid and its preparation method and application
  • A kind of cryopreservation liquid and its preparation method and application
  • A kind of cryopreservation liquid and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Preparation of Graphene Oxide

[0046] 1. Pre-oxidized graphite

[0047] Weigh 2.5g of phosphorus pentoxide and 2.5g of potassium persulfate, and add them into a 250mL Erlenmeyer flask. Add 20mL of concentrated sulfuric acid under constant stirring, heat to 80°C, slowly add 3g of graphite powder to the solution, keep the temperature of the solution at 80°C for 4.5h, then cool to room temperature. Then slowly add deionized water. During this process, ensure that the temperature of the solution does not exceed 80°C. The mixture is allowed to stand overnight, filtered to remove excess concentrated sulfuric acid, and dried at 60°C.

[0048] 2. Preparation of graphite oxide

[0049] Add the dried pre-oxidized graphite into 120mL of concentrated sulfuric acid, and slowly add 15g of potassium permanganate while stirring continuously. This process is carried out in an ice bath, and the temperature of the whole process is guaranteed to be below 80°C, and the stirring is contin...

Embodiment 2

[0055] Preparation of cryopreservation solution of the present invention

[0056] 1. Weigh 0.5g glucose, 9g lactose, 0.3g raffinose, 1.1g 4-hydroxyethylpiperazineethanesulfonic acid, 1.2gNaH 2 PO 4 2H 2 O, 1.6gNa 2 HPO 4 2H 2 O, 1.5g of sodium chloride, 3g of potassium chloride, 0.02g of magnesium sulfate, 0.07g of calcium chloride, and 0.6g of casein were added to 1L of deionized water, fully mixed and dissolved, and stored in a thermostat at 37°C. spare. For the convenience described in the following examples, the cryopreservation solution prepared in this step is referred to as cryopreservation solution A for short.

[0057] 2. Preparation of cryopreservation solution containing graphene oxide

[0058] Weigh a certain amount of graphene oxide prepared in Example 1, physically crush it in an ultra-clean room, grind it into a fine powder in an agate mortar, 100W ultrasonic 30min, and disperse it in the cryopreservation solution A prepared in the first step, so that Th...

Embodiment 3

[0062] Inhibitory effect of different samples on ice crystal growth

[0063] Using the method reported in the literature (Deller R C, Vatish M, Mitchell D A, et al.Syntheticpolymers enable non-vitreous cellular cryopreservation by reducing icecrystal growth during thawing[J].Nature communications,2014,5.) to evaluate the different samples in low temperature environment Inhibition of ice crystal growth.

[0064] 1. Sample preparation: select 0.7% physiological saline, the cryopreservation solution A (excluding graphene oxide) of Example 2 of the present invention, 20% glycerin, and a commercially available graphene oxide aqueous dispersion (Sigma-Aldrich Co.LLC. 763713, the size distribution of its graphene oxide is 200nm-12μm, 2~3 layers, C / O is 1.9) and the graphene oxide aqueous dispersion prepared in embodiment 1 (the concentration of the aqueous dispersion of two kinds of graphene oxide is set Determined as the mass percent concentration of 0.01%, 0.1% and 0.5%). At the ...

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Abstract

The invention provides a cryopreservation solution containing a two-dimensional material-graphene oxide, and its preparation and application belong to the technical field of cryopreservation of biological materials. In the cryopreservation solution, by adding graphene oxide, the growth of tiny ice crystals during the recovery process of the cryopreservation solution can be inhibited, thereby avoiding the damage to the cells during the ice crystal growth process, thereby greatly improving the survival rate of biological materials after cryopreservation and recovery and cell activity. Because this method focuses on inhibiting the growth of ice crystals, this cryopreservation solution has high universality and can be used for cryopreservation of cells, tissues, etc., and meets the needs of practical use to the greatest extent.

Description

technical field [0001] The present invention relates to cryopreservation, especially the application of graphene oxide in cryopreservation of biological materials, and a cryopreservation solution containing graphene oxide, a preparation method of the preservation solution, and the application of the preservation solution to cell, tissue or organ A cryopreservation method belongs to the technical field of cryopreservation of biological materials. Background technique [0002] Low temperature or cryopreservation technology is a technology developed rapidly in the 1970s to preserve biological materials such as biological tissues, organs and cells under the condition of dry ice or liquid nitrogen. Under low temperature conditions, biological tissues are in a "dormant state", which ensures the genetic and morphological stability of biological materials, and is an effective method for preserving biological materials. In recent decades, the application of this technology has focus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07
Inventor 耿洪亚刘凯王建君吕建勇
Owner 北京玳衲芈珂生物科技有限公司