Formula and preparation method of dimethyl-sulfoxide-free protein-free -80 DEG C cell freezing medium

A technology of dimethyl sulfoxide and cryopreservation solution, which is applied to the preservation, application, and animal husbandry of human or animal bodies. The effect of high recovery rate

Inactive Publication Date: 2016-06-29
南京三生生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The dimethyl sulfoxide in the cell cryopreservation solution used in domestic clinics often uses imported pharmaceutical grade reagents, which are very e

Method used

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  • Formula and preparation method of dimethyl-sulfoxide-free protein-free -80 DEG C cell freezing medium
  • Formula and preparation method of dimethyl-sulfoxide-free protein-free -80 DEG C cell freezing medium
  • Formula and preparation method of dimethyl-sulfoxide-free protein-free -80 DEG C cell freezing medium

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Experimental program
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Embodiment 1

[0020] 1. Synthesis of modified polylysine

[0021] A, Synthesis of acetyl-modified polylysine: Control the 20% polylysine aqueous solution below 10 degrees, adjust the pH value to 9 with 5N NaOH, continuously add acetic anhydride, and control with sodium hydroxide The pH value of the reaction solution is different according to the amount of acetic anhydride added, and the acetylated polylysine modified in different degrees is obtained. The obtained product was gel-filtered to remove salts and by-products, and freeze-dried to obtain a white solid.

[0022] B, Synthesis of maleylated polylysine: control the 20% polylysine aqueous solution below 10 degrees, adjust the pH value to 9 with 5N NaOH, continuously add maleic anhydride, and use sodium hydroxide to The pH value of the reaction solution is controlled, and maleylated polylysine modified to varying degrees is obtained according to the amount of maleic anhydride added. The obtained product was gel-filtered to remove salts...

Embodiment 2

[0024] 1. The preparation of dimethyl sulfoxide-free protein-free cell freezing solution at -80°C includes the following steps:

[0025] (1) Weigh 6.0 g of dextran and dissolve it in 70 ml of RPMI-1640 cell basal medium;

[0026] (2) Add 7.0 g of 60% maleylated polylysine to the above solution;

[0027] (3) Finally, add RPMI-1640 cell basal medium to make the volume to 100ml;

[0028] 2. Under non-sterile conditions, the above mixture is first clarified and filtered with a 0.45 μm filter membrane, and then sterilized with a 0.22 μm filter membrane under sterile conditions to obtain dimethyl sulfoxide-free and protein-free -80°C cell freezing solution.

Embodiment 3

[0030] 1. Preparation of dimethyl sulfoxide-free protein-80°C cell freezing medium, including the following steps:

[0031] (1) Weigh 6.0 g of dextran and dissolve it in 70 ml of RPMI-1640 cell basal medium;

[0032] (2) Add different amounts of acetylated polylysine with a degree of modification of 60% to the above solution, the amount of acetylated polylysine added makes the final concentration respectively 1%, 3%, 7%, 9% %;

[0033] (3) Finally, add RPMI-1640 cell basal medium to make the volume to 100ml;

[0034] 2. Under non-sterile conditions, first clarify and filter the above mixed solution with a 0.45 μm filter membrane, and then filter and sterilize it with a 0.22 μm filter membrane under sterile conditions to obtain a protein-free cell cryopreservation solution.

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Abstract

The invention belongs to the field of biotechnology, and specifically relates to a dimethyl sulfoxide-free protein-80°C cell freezing solution and a preparation method thereof. Every 100ml of dimethyl sulfoxide-free protein-80°C cell freezing solution contains 6g of dextran, 1.0-9.0g of maleylated polylysine with a modification degree of 60%, and the balance being RPMI-1640 cell basal medium. The dimethyl sulfoxide-free protein-80°C cell cryopreservation solution of the present invention is prepared from chemical components, does not contain dimethyl sulfoxide and protein, has high cell recovery rate and good stability.

Description

technical field [0001] The invention relates to a dimethyl sulfoxide-free protein-80°C cell freezing liquid formula and a preparation method thereof, belonging to the technical field of tissue cell culture. Background technique [0002] Cell cryopreservation technology is a very important method in cell biology technology. In the conventional cell cryopreservation solution, it is often necessary to add 10% concentration of dimethyl sulfoxide and serum or protein of human or animal origin. With the development of stem cell therapy technology and immune cell therapy technology in recent years, there is an increasing demand for the clinical use of cell cryopreservation fluid. Theoretically, the cryopreserved cells need to be washed after recovery to remove the secondary substances in the cell cryopreservation fluid. Methyl sulfoxide and other animal-derived components, but this process is easy to operate in the laboratory, but it is very troublesome for clinical use, and dimeth...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 徐念沁郁庆明徐炜华
Owner 南京三生生物技术股份有限公司
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