Method for cryopreserving erythrocytes
A technology for cryopreservation and red blood cells, which is applied in the preservation, application, and animal husbandry of human or animal bodies, and can solve the problems of unsuitability for cryopreservation and low red blood cell fragmentation rate.
Inactive Publication Date: 2012-11-28
INST OF FIELD OPERATION SURGERY NO 3 MILITARY MEDICL UNIV PLA
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Problems solved by technology
In the field of cryopreservation of red blood cells, the existing methods also pursue to maintain the integrity and function of red blood cells. After thawing, the red blood cell fragmentation rate is low, and about 8-15% of hemoglobin will be oxidized to methemoglobin, which is not suitable for cryopreservation for preparation Red blood cells required for stroma-free hemoglobin
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[0015] Example: Take 180ml of washed red blood cells and add any one of the following or a mixture of two or more reducing agents:
[0016] Ascorbic acid (or ascorbate), cysteine hydrochloride (or N-acetylcysteine), sodium borohydride, sodium sulfite (or sodium bisulfite or sodium metabisulfite), reduced glutathione;
[0017] The final concentration of the added reducing agents is 0.5-300 mM.
[0018] At the same time, glycerol with a final content of 5% to 10% by volume and ethylenediaminetetraacetic acid (or ethylenediaminetetraacetic acid salt) with a final concentration of 0.1 to 10mM were added, fully mixed and the acidity value adjusted to 6.5 ~8.0, seal the container, let it stand for 1 hour, and store it in a freezer below -40°C.
[0019] Among them, the more optimal final concentration of ascorbic acid or ascorbate is 5.5-115mM, the more optimal final concentration of cysteine hydrochloride or N-acetylcysteine is 0.5-60mM, and the more optimal final concentrati...
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The invention provides a method for cryopreserving erythrocytes, which comprises the following steps of: adding a reducing agent, glycerol and ethylene diamine tetraacetic acid or ethylene diamine tetraacetate serving as a metal ion chelating agent into the erythrocytes, wherein the reducing agent is any one of or a mixture of two or more of ascorbic acid or ascorbate, cysteine hydrochloride or N-acetylcysteine, sodium borohydride, sodium sulfite or sodium bisulfite or sodium pyrosulfite, and reduced glutathione, and the final concentration of the reducing agent is 0.5 to 300nM; the glycerol accounts for 5 to 10 percent of the final volume; and the final concentration of the ethylene diamine tetraacetic acid or ethylene diamine tetraacetate is 0.1 to 10mM; and fully and uniformly mixing, regulating acidity value to 6.5 to 8.0, sealing a container, standing for one hour and cryopreserving the erythrocytes at the temperature of below -40 DEG C. When the erythrocytes are cryopreserved bythe method and unfrozen one year later, the breakage rate of the erythrocytes is over 95 percent; and hemoglobin is not denatured and not oxidized into hemiglobin.
Description
technical field [0001] The invention belongs to the field of cell preservation, and in particular relates to a method for freezing red blood cells. Background technique [0002] Oxygen-carrying fluid based on stroma-free hemoglobin is a red blood cell substitute that has attracted widespread attention and is likely to enter clinical application. In the series of processes for preparing this type of oxygen-carrying liquid, the most upstream step is to collect, separate and preserve red blood cells. The collection and separation technology of red blood cells is relatively mature, and the part that needs to be perfected is the preservation of red blood cells. An ideal method for preserving erythrocytes for the preparation of stroma-free hemoglobin should meet the requirements of the following three aspects at the same time: (1) erythrocytes will not be corrupted after 2 to 5 years or even longer storage; (2) the hemoglobin in erythrocytes should Freezing-thawing process does ...
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IPC IPC(8): A01N1/02
Inventor 刘良明臧家涛徐竞刘建仓
Owner INST OF FIELD OPERATION SURGERY NO 3 MILITARY MEDICL UNIV PLA