Stem cell recovery method of cryopreserved umbilical cord blood with high recovery rate

A cord blood stem cell, high resuscitation rate technology, applied to blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of reducing the resuscitation rate of cryopreserved cells, penetration damage of protective agents, cytotoxic damage, etc. Achieve the effect of reducing cytotoxic damage, reducing mechanical damage, and increasing contact area

Inactive Publication Date: 2016-02-17
黄林海
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] But vitrification has limitations
Vitrification cryopreservation agents in the prior art usually use DMSO, DMSO has cytotoxicity, thereby causing cytotoxic damage; during the resuscitation process, it may also cause osmotic damage to the protective agent, and at the same time form ice crystals to cause mechanical damage, which will greatly reduce freezing. Recovery rate of stored cells

Method used

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  • Stem cell recovery method of cryopreserved umbilical cord blood with high recovery rate

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Embodiment 1

[0034] Such as figure 1 As shown, a method for resuscitating cryopreserved cord blood stem cells with a high recovery rate comprises the following steps:

[0035] Step 1: Take out the cryopreservation solution from liquid nitrogen, put it into LNG and heat it up for 1 minute;

[0036] Step 2: Take out the cryopreserved liquid from LNG, put it into an ice bath under vacuum, and rapidly cool down to 3.2°C at an average heating rate of 560°C / min;

[0037] Step 3: Pour the cryopreservation solution into 0.9% saline, maintain the temperature at 3.2°C, add 17% human serum albumin without fibrinogen under the condition of infrasonic oscillation (frequency 8Hz), 3 % gamma globulin, 17% fibrinogen-free human serum albumin, 3% gamma globulin, 0.8% monosaccharides, 0.21% cell growth factors, 0.8% non-essential amino acids, 0.08 % R-glutamine, 0.03% β-mercaptoethanol, adjust the pH of the system to 7.6, add 1.5% hydroxyethylpiperazine ethanesulfonic acid, and continue to oscillate under...

Embodiment 2

[0049] A method for resuscitating cryopreserved umbilical cord blood stem cells with a high recovery rate, comprising the following steps:

[0050] Step 1: Take out the cryopreservation solution from liquid nitrogen, put it into LNG and heat it up for 1 minute;

[0051] Step 2: Take out the cryopreserved liquid from LNG, put it into an ice bath under vacuum, and rapidly cool down to 3.9°C at an average heating rate of 560°C / min;

[0052] Step 3: Pour the cryopreservation solution into 0.9% saline, maintain the temperature at 3.9°C, add 17% human serum albumin without fibrinogen under the condition of infrasonic (frequency 13Hz) oscillation, 3 % γ-globulin, 1% monosaccharide, 0.23% cell growth factor, 1.2% non-essential amino acid, 0.12% R-glutamine, 0.04% β-mercaptoethanol, adjust the pH of the system to 7.9 , add 1.5% hydroxyethylpiperazine ethanesulfonic acid, continue to shake under infrasonic conditions for 45s;

[0053] Step 4: Centrifuge the diluted cryopreservation so...

Embodiment 3

[0064] A method for resuscitating cryopreserved umbilical cord blood stem cells with a high recovery rate, comprising the following steps:

[0065] Step 1: Take out the cryopreservation solution from liquid nitrogen, put it into LNG and heat it up for 1 minute;

[0066] Step 2: Take out the cryopreserved liquid from LNG, put it into an ice bath under vacuum, and rapidly cool down to 3.6°C at an average heating rate of 560°C / min;

[0067] Step 3: Pour the cryopreservation solution into 0.9% saline, maintain the temperature at 3.6°C, add 17% human serum albumin without fibrinogen under the condition of infrasonic (frequency 10Hz) oscillation, 3 % γ-globulin, 0.9% monosaccharide, 0.22% cell growth factor, 1.1% non-essential amino acid, 0.1% R-glutamine, 0.032% β-mercaptoethanol, adjust the pH of the system to 7.7 , add 1.5% hydroxyethylpiperazine ethylsulfuric acid, continue to shake under infrasonic conditions for 40s;

[0068] Step 4: Centrifuge the diluted cryopreservation s...

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Abstract

The invention discloses a stem cell recovery method of the cryopreserved umbilical cord blood with the high recovery rate, and belongs to the field of stem cell recovery. The method comprises the steps that a freezing medium is taken out of liquid nitrogen and put into LNG for heating for 1 min; the freezing medium is taken out of the LNG and put into an ice bath in a vacuum state, and the temperature is decreased to 3.2 DEG C-3.9 DEG C according to the average heating rate of 560 DEG C / min. The stem cell recovery method of the cryopreserved umbilical cord blood with the high recovery rate has the advantages that the cytotoxicity to umbilical cord blood stem cells is low, in the vitrified cryopreservation recovery process, the cytotoxic damage is low, the permeability damage is low, and the cryopreserved cell recovery rate is increased.

Description

technical field [0001] The invention relates to a method for resuscitating stem cells, in particular to a method for resuscitating frozen cord blood stem cells with a high recovery rate. Background technique [0002] Umbilical cord blood is an important seed cell for constructing tissue-engineered bone, and cryopreservation of umbilical cord blood is of great significance for bone tissue engineering. Vitrification is a method in which cells and their protective agent solutions are supercooled to their glass transition temperature at a sufficiently fast cooling rate, solidified into a complete glass state and stored at low temperature for a long time in this glass state. In this process, crystallization is completely avoided inside and outside the cells, thereby avoiding various damages caused by freezing. The recovery rate of programmed cryopreservation is only 5%-10%, while the recovery rate of vitrification is >75%. Therefore, vitrification is currently an important me...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
Inventor 黄林海
Owner 黄林海
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