CN105331582A - Stem cell recovery method of cryopreserved umbilical cord blood with high recovery rate - Google Patents
Stem cell recovery method of cryopreserved umbilical cord blood with high recovery rate Download PDFInfo
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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
The present invention relates to a kind of stem cell method for resuscitation, particularly a kind of freezing and storing umbilical hemocytoblast method for resuscitation of high anabiosis rate.
Background technology
Cord blood builds the important seed cell of tissue-engineered bone, and the deep-bed drying realizing Cord blood has great importance to bone tissue engineer.Glass frozen preservation cell and protective material solution thereof is crossed with enough fast rate of temperature fall be as cold as its second-order transition temperature; and be cured as vitreous state the method for preserving for a long time at low temperatures with this vitreous state completely; intraor extracellular completely avoid crystallization in this course, thus is avoided the various damages that cause owing to freezing.The frozen anabiosis rate of sequencing only has 5% ~ 10%, and the anabiosis rate >75% of glass frozen preservation, therefore glass frozen preservation is the important method of preserving early stage stem cell at present.
But glass frozen preservation also exists limitation.In prior art, glass frozen preservation agent adopts DMSO usually, and DMSO exists cytotoxicity, thus causes cytotoxic damage; Also likely cause protectant perviousness to damage in resuscitation process, form ice crystal simultaneously and cause physical abuse, these damages all can reduce the anabiosis rate of freeze-stored cell greatly.
Summary of the invention
Goal of the invention of the present invention is: for above-mentioned Problems existing, there is provided a kind of to cord blood stem cell low cytotoxicity, low cytotoxicity damage in glass frozen preservation resuscitation process, low-permeability damages, and improves the freezing and storing umbilical hemocytoblast method for resuscitation of the high anabiosis rate of freeze-stored cell anabiosis rate.
The technical solution used in the present invention is as follows:
The freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate of the present invention, comprises the following steps:
Step one: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Owing to have employed technique scheme, the temperature of LNG is a little more than liquid nitrogen, for-162 DEG C, heat up through pre-in LNG, slightly can improve the temperature of frozen storing liquid, for avoiding occurring little ice crystal in rapid temperature increases process below, causing physical abuse to provide guarantee to stem cell, improve the anabiosis rate of frozen stem cell.
Step 2: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 ~ 3.9 DEG C according to the temperature rise rate of average 560 DEG C/min;
Owing to have employed technique scheme, DMSO under the condition of 3.9 DEG C is minimum to cytotoxicity for temperature, but temperature is too low, may form little ice crystal, thus cause physical abuse to cell in frozen storing liquid, therefore controls temperature at 3.2 ~ 3.9 DEG C.
Under vacuum conditions, intercept air heat exchange, improve intensification efficiency, can rapid temperature increases, thus avoid in temperature-rise period, occur little ice crystal, reduce physical abuse, make the refrigerating fulid being frozen into vitreous humor comprising stem cell directly be dissolved into liquid, improve anabiosis rate.
Step 3: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the human serum albumin of the not fibrinogen of 17% is added, the gamma globulin of 3%, the monose of 0.8% ~ 1% under infrasound oscillating condition, the non-essential amino acid of 0.8% ~ 1.2%, the R-glutamine of 0.08% ~ 0.12%, the beta-mercaptoethanol of 0.03% ~ 0.04%, the pH of regulation system is 7.6 ~ 7.9, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30 ~ 45s under infrasound condition;
Owing to have employed technique scheme, cell growth factor is given as the merisis of stem cell provides catalysis hormone, thus improves anabiosis rate.
The simulation such as serum albumin and amino acid human body environment, for providing an adapt circumstance after stem cell recovery, waters down the impact of the composition in protective material on stem cell.
Monose is that stem cell growth metabolism supplies nutrients, and reduces the osmotic pressure of protective material and resuscitation fluid simultaneously, reduces in resuscitation process the osmotic injury that stem cell is caused.
Hydroxyethyl piperazine second thiosulfonic acid is pH buffer reagent, can the pH of guarantee system constant, hydroxyethyl piperazine second thiosulfonic acid no cytotoxicity, can not cause cytotoxic damage to stem cell simultaneously.
Step 4: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.21% ~ 0.23%, Eddy diffusion cell in cell precipitation.
Frozen clone's agglomerate limits clone-internal cell and is exposed to resuscitation fluid, under infrasound oscillating condition, resuscitation fluid can be spread apart rapidly, go deep into clone-internal cell, thus is also exposed in resuscitation fluid by the cell of clone agglomerate inside.
The freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate of the present invention, described infrasonic frequency is 8 ~ 13Hz.
Owing to have employed technique scheme, this frequency and human organ vibration frequency are close, thus make stem cell better adapt to frozen storing liquid environment, improve resuscitation fluid osmotic effect.
The freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate of the present invention, described cell growth factor is Urogastron, Prostatropin and vascular endothelial growth factor.
Owing to have employed technique scheme, Urogastron, Prostatropin and vascular endothelial growth factor can promote that differentiation of stem cells grows.
The freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate of the present invention, the concentration of described Urogastron is 50 ~ 75 μ g/L, the concentration of described Prostatropin is 15 ~ 20 μ g/L, and the concentration of described vascular endothelial growth factor is 4 ~ 6 μ g/L.
Owing to have employed technique scheme, concentration is suitable scope in above-mentioned scope, can not cause the too fast growth of cell because hormone-content is too high.
The freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate of the present invention, described monose is the mixing solutions of otan and L-Glycerose.
The freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate of the present invention, described monose comprises the otan of 0.3% ~ 0.5% and the L-Glycerose of 0.5% ~ 0.7%.
In sum, owing to have employed technique scheme, the invention has the beneficial effects as follows:
1, reduce the cytotoxic damage that protective material and resuscitation fluid cause freeze-stored cell in resuscitation process, the merisis for stem cell provides catalysis hormone, simultaneously for stem cell recovery creates adapt circumstance, improves frozen anabiosis rate on the whole.
2, adopt infrasound oscillating condition, resuscitation fluid is spread apart rapidly, gos deep into clone-internal cell, thus the cell of clone agglomerate inside is also exposed in resuscitation fluid, increase the contact area of resuscitation fluid and stem cell, improve the anabiosis rate of freeze-stored cell.
Accompanying drawing explanation
Fig. 1 is non-freeze-stored cell and frozen rear recovery microcytoscope observation figure.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in detail.
In order to make the object of invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
As shown in Figure 1, a kind of freezing and storing umbilical hemocytoblast method for resuscitation of high anabiosis rate, comprises the following steps:
Step one: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Step 2: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 DEG C according to the temperature rise rate of average 560 DEG C/min;
Step 3: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.2 DEG C, the human serum albumin of the not fibrinogen of 17% is added under infrasound vibration (frequency is 8Hz) condition, the gamma globulin of 3%, the human serum albumin of the not fibrinogen of 17%, the gamma globulin of 3%, the monose of 0.8%, the cell growth factor of 0.21%, the non-essential amino acid of 0.8%, the R-glutamine of 0.08%, the beta-mercaptoethanol of 0.03%, the pH of regulation system is 7.6, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30s under infrasound condition,
Step 4: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.21%, Eddy diffusion cell in cell precipitation.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard operating procedure aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculation culture 48h, at the dexamethasone containing 8mmol/L, in the nutrient solution of the β-phospho-glycerol sodium of 2.16g/L and the 2-phosphoric acid xitix of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA nutrient solution of the trypsinase-0.02% with 0.25% carries out the 1st time and goes down to posterity; With 1.3 × 10
4/ cm
2cell density inoculation, be cultured to P2 generation.
Step 2: P2 is fully immersed in the salt solution of 0.85% ~ 0.9% for Cord blood group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the perviousness protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumin of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1% ~ 2.3%, continue to vibrate 30 ~ 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed solution that obtains in step 2 under vacuum conditions, in direct plunge into Liquid Nitrogen, be cooled to-196 DEG C rapidly according to the rate of temperature fall of average 600 DEG C/min;
Step 4: preserve 24h stored in liquid nitrogen container.
Take out frozen storing liquid and carry out recovery cultivation by aforementioned method for resuscitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell cultured continuously of individual recovery 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after anabiosis rate=cultivation
5× appreciation rate), obtain the anabiosis rate of freeze-stored cell; Simultaneously by 3 × 10
5individual P2, for stem cell cultured continuously 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after appreciation rate=cultivation
5).
Experimental result: to record anabiosis rate be the upper figure of 96.5%(Fig. 1 is not frozen stem cell, and figure below is the stem cell recovered after frozen 24h)
Embodiment 2
A freezing and storing umbilical hemocytoblast method for resuscitation for high anabiosis rate, comprises the following steps:
Step one: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Step 2: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.9 DEG C according to the temperature rise rate of average 560 DEG C/min;
Step 3: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.9 DEG C, the human serum albumin of the not fibrinogen of 17% is added under infrasound (frequency is 13Hz) oscillating condition, the gamma globulin of 3%, the monose of 1%, the cell growth factor of 0.23%, the non-essential amino acid of 1.2%, the R-glutamine of 0.12%, the beta-mercaptoethanol of 0.04%, the pH of regulation system is 7.9, adds the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continues to vibrate 45s under infrasound condition;
Step 4: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.23%, Eddy diffusion cell in cell precipitation.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard operating procedure aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculation culture 48h, at the dexamethasone containing 8mmol/L, in the nutrient solution of the β-phospho-glycerol sodium of 2.16g/L and the 2-phosphoric acid xitix of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA nutrient solution of the trypsinase-0.02% with 0.25% carries out the 1st time and goes down to posterity; With 1.3 × 10
4/ cm
2cell density inoculation, be cultured to P2 generation.
Step 2: P2 is fully immersed in the salt solution of 0.85% ~ 0.9% for Cord blood group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the perviousness protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumin of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1% ~ 2.3%, continue to vibrate 30 ~ 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed solution that obtains in step 2 under vacuum conditions, in direct plunge into Liquid Nitrogen, be cooled to-196 DEG C rapidly according to the rate of temperature fall of average 600 DEG C/min;
Step 4: preserve 24h stored in liquid nitrogen container.
Take out frozen storing liquid and carry out recovery cultivation by aforementioned method for resuscitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell cultured continuously of individual recovery 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after anabiosis rate=cultivation
5× appreciation rate), obtain the anabiosis rate of freeze-stored cell; Simultaneously by 3 × 10
5individual P2, for stem cell cultured continuously 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after appreciation rate=cultivation
5).
Experimental result: recording anabiosis rate is 96.9%
Embodiment 3
A freezing and storing umbilical hemocytoblast method for resuscitation for high anabiosis rate, comprises the following steps:
Step one: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Step 2: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.6 DEG C according to the temperature rise rate of average 560 DEG C/min;
Step 3: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.6 DEG C, the human serum albumin of the not fibrinogen of 17% is added under infrasound (frequency is 10Hz) oscillating condition, the gamma globulin of 3%, the monose of 0.9%, the cell growth factor of 0.22%, the non-essential amino acid of 1.1%, the R-glutamine of 0.1%, the beta-mercaptoethanol of 0.032%, the pH of regulation system is 7.7, adds the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continues to vibrate 40s under infrasound condition;
Step 4: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.22%, Eddy diffusion cell in cell precipitation.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard operating procedure aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculation culture 48h, at the dexamethasone containing 8mmol/L, in the nutrient solution of the β-phospho-glycerol sodium of 2.16g/L and the 2-phosphoric acid xitix of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA nutrient solution of the trypsinase-0.02% with 0.25% carries out the 1st time and goes down to posterity; With 1.3 × 10
4/ cm
2cell density inoculation, be cultured to P2 generation.
Step 2: P2 is fully immersed in the salt solution of 0.85% ~ 0.9% for Cord blood group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the perviousness protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumin of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1% ~ 2.3%, continue to vibrate 30 ~ 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed solution that obtains in step 2 under vacuum conditions, in direct plunge into Liquid Nitrogen, be cooled to-196 DEG C rapidly according to the rate of temperature fall of average 600 DEG C/min;
Step 4: preserve 24h stored in liquid nitrogen container.
Take out frozen storing liquid and carry out recovery cultivation by aforementioned method for resuscitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell cultured continuously of individual recovery 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after anabiosis rate=cultivation
5× appreciation rate), obtain the anabiosis rate of freeze-stored cell; Simultaneously by 3 × 10
5individual P2, for stem cell cultured continuously 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after appreciation rate=cultivation
5).
Experimental result: recording anabiosis rate is 97.6%
Embodiment 4
A freezing and storing umbilical hemocytoblast method for resuscitation for high anabiosis rate, comprises the following steps:
Step one: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Step 2: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.4 DEG C according to the temperature rise rate of average 560 DEG C/min;
Step 3: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.4 DEG C, the human serum albumin of the not fibrinogen of 17% is added under infrasound oscillating condition, the gamma globulin of 3%, the otan of 0.3%, the L-Glycerose of 0.5%, the cell growth factor of 0.21%, the non-essential amino acid of 1.2%, the R-glutamine of 0.08%, the beta-mercaptoethanol of 0.04%, the NaCl of 0.68% and the deionized water of 75.49%, wherein cell growth factor is Urogastron, the mixing solutions of Prostatropin and vascular endothelial growth factor 1:1:1:1, the concentration of Urogastron is 50 μ g/L, the concentration of acid fibroblast growth factor is 15 μ g/L, the concentration of Prostatropin is 15 μ g/L, the concentration of vascular endothelial growth factor is 4 μ g/L, the pH of regulation system is 7.8, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 40s under infrasound condition,
Step 4: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.21%, Eddy diffusion cell in cell precipitation.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard operating procedure aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculation culture 48h, at the dexamethasone containing 8mmol/L, in the nutrient solution of the β-phospho-glycerol sodium of 2.16g/L and the 2-phosphoric acid xitix of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA nutrient solution of the trypsinase-0.02% with 0.25% carries out the 1st time and goes down to posterity; With 1.3 × 10
4/ cm
2cell density inoculation, be cultured to P2 generation.
Step 2: P2 is fully immersed in the salt solution of 0.85% ~ 0.9% for Cord blood group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the perviousness protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumin of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1% ~ 2.3%, continue to vibrate 30 ~ 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed solution that obtains in step 2 under vacuum conditions, in direct plunge into Liquid Nitrogen, be cooled to-196 DEG C rapidly according to the rate of temperature fall of average 600 DEG C/min;
Step 4: preserve 24h stored in liquid nitrogen container.
Take out frozen storing liquid and carry out recovery cultivation by aforementioned method for resuscitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell cultured continuously of individual recovery 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after anabiosis rate=cultivation
5× appreciation rate), obtain the anabiosis rate of freeze-stored cell; Simultaneously by 3 × 10
5individual P2, for stem cell cultured continuously 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after appreciation rate=cultivation
5).
Experimental result: recording anabiosis rate is 97.3%
Embodiment 5
A freezing and storing umbilical hemocytoblast method for resuscitation for high anabiosis rate, comprises the following steps:
Step one: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Step 2: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.8 DEG C according to the temperature rise rate of average 560 DEG C/min;
Step 3: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.8 DEG C, the human serum albumin of the not fibrinogen of 17% is added under infrasound oscillating condition, the gamma globulin of 3%, the otan of 0.5%, the L-Glycerose of 0.7%, the cell growth factor of 0.23%, the non-essential amino acid of 0.8%, the R-glutamine of 0.12%, the beta-mercaptoethanol of 0.03%, the NaCl of 0.69% and the deionized water of 75.63%, cell growth factor is Urogastron, the mixing solutions of Prostatropin and vascular endothelial growth factor 1:1:1:1, the concentration of Urogastron is 75 μ g/L, the concentration of acid fibroblast growth factor is 20 μ g/L, the concentration of Prostatropin is 20 μ g/L, the concentration of vascular endothelial growth factor is 6 μ g/L, the pH of regulation system is 7.6, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 42s under infrasound condition,
Step 4: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.23%, Eddy diffusion cell in cell precipitation.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard operating procedure aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculation culture 48h, at the dexamethasone containing 8mmol/L, in the nutrient solution of the β-phospho-glycerol sodium of 2.16g/L and the 2-phosphoric acid xitix of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA nutrient solution of the trypsinase-0.02% with 0.25% carries out the 1st time and goes down to posterity; With 1.3 × 10
4/ cm
2cell density inoculation, be cultured to P2 generation.
Step 2: P2 is fully immersed in the salt solution of 0.85% ~ 0.9% for Cord blood group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the perviousness protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumin of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1% ~ 2.3%, continue to vibrate 30 ~ 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed solution that obtains in step 2 under vacuum conditions, in direct plunge into Liquid Nitrogen, be cooled to-196 DEG C rapidly according to the rate of temperature fall of average 600 DEG C/min;
Step 4: preserve 24h stored in liquid nitrogen container.
Take out frozen storing liquid and carry out recovery cultivation by aforementioned method for resuscitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell cultured continuously of individual recovery 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after anabiosis rate=cultivation
5× appreciation rate), obtain the anabiosis rate of freeze-stored cell; Simultaneously by 3 × 10
5individual P2, for stem cell cultured continuously 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after appreciation rate=cultivation
5).
Experimental result: recording anabiosis rate is 97.6%
Embodiment 6
A freezing and storing umbilical hemocytoblast method for resuscitation for high anabiosis rate, comprises the following steps:
Step 5: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Step 6: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.5 DEG C according to the temperature rise rate of average 560 DEG C/min;
Step 7: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.5 DEG C, the human serum albumin of the not fibrinogen of 17% is added under infrasound oscillating condition, the gamma globulin of 3%, the otan of 0.35%, the L-Glycerose of 0.55%, the cell growth factor of 0.22%, the non-essential amino acid of 1.1%, the R-glutamine of 0.09%, the beta-mercaptoethanol of 0.036%, the NaCl of 0.68% and the deionized water of 74.474%, cell growth factor is Urogastron, the mixing solutions of Prostatropin and vascular endothelial growth factor 1:1:1:1, the concentration of Urogastron is 63 μ g/L, the concentration of acid fibroblast growth factor is 17 μ g/L, the concentration of Prostatropin is 19 μ g/L, the concentration of vascular endothelial growth factor is 5 μ g/L, the pH of regulation system is 7.8, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 45s under infrasound condition,
Step 8: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.21%, Eddy diffusion cell in cell precipitation.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard operating procedure aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculation culture 48h, at the dexamethasone containing 8mmol/L, in the nutrient solution of the β-phospho-glycerol sodium of 2.16g/L and the 2-phosphoric acid xitix of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA nutrient solution of the trypsinase-0.02% with 0.25% carries out the 1st time and goes down to posterity; With 1.3 × 10
4/ cm
2cell density inoculation, be cultured to P2 generation.
Step 2: P2 is fully immersed in the salt solution of 0.85% ~ 0.9% for Cord blood group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the perviousness protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumin of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1% ~ 2.3%, continue to vibrate 30 ~ 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed solution that obtains in step 2 under vacuum conditions, in direct plunge into Liquid Nitrogen, be cooled to-196 DEG C rapidly according to the rate of temperature fall of average 600 DEG C/min;
Step 4: preserve 24h stored in liquid nitrogen container.
Take out frozen storing liquid and carry out recovery cultivation by aforementioned method for resuscitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell cultured continuously of individual recovery 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after anabiosis rate=cultivation
5× appreciation rate), obtain the anabiosis rate of freeze-stored cell; Simultaneously by 3 × 10
5individual P2, for stem cell cultured continuously 6 days, calculates the total cellular score after cultivation, according to formula with blood counting chamber: total cellular score ÷ (3 × 10 after appreciation rate=cultivation
5).
Experimental result: recording anabiosis rate is 97.4%
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. a freezing and storing umbilical hemocytoblast method for resuscitation for high anabiosis rate, is characterized in that comprising the following steps:
Step one: taken out from liquid nitrogen by frozen storing liquid, drops in LNG the 1min that heats up;
Step 2: taken out from LNG by frozen storing liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 ~ 3.9 DEG C according to the temperature rise rate of average 560 DEG C/min;
Step 3: frozen storing liquid is poured in the salt solution of 0.9%, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the human serum albumin of the not fibrinogen of 17% is added, the gamma globulin of 3%, the monose of 0.8% ~ 1% under infrasound oscillating condition, the non-essential amino acid of 0.8% ~ 1.2%, the R-glutamine of 0.08% ~ 0.12%, the beta-mercaptoethanol of 0.03% ~ 0.04%, the pH of regulation system is 7.6 ~ 7.9, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30 ~ 45s under infrasound condition;
Step 4: by the frozen storing liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant liquor, adds the cell growth factor of 0.21% ~ 0.23%, Eddy diffusion cell in cell precipitation.
2. the freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate as claimed in claim 1, is characterized in that: described infrasonic frequency is 8 ~ 13Hz.
3. the freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate as claimed in claim 1, is characterized in that: described cell growth factor is Urogastron, Prostatropin and vascular endothelial growth factor.
4. the freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate as claimed in claim 3, it is characterized in that: the concentration of described Urogastron is 50 ~ 75 μ g/L, the concentration of described Prostatropin is 15 ~ 20 μ g/L, and the concentration of described vascular endothelial growth factor is 4 ~ 6 μ g/L.
5. the freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate as claimed in claim 1, is characterized in that: described monose is the mixing solutions of otan and L-Glycerose.
6. the freezing and storing umbilical hemocytoblast method for resuscitation of a kind of high anabiosis rate as claimed in claim 5, is characterized in that: described monose comprises the otan of 0.3% ~ 0.5% and the L-Glycerose of 0.5% ~ 0.7%.
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| CN102228016A (en) * | 2011-04-18 | 2011-11-02 | 上海安集协康生物技术有限公司 | Cryopreservation and resuscitation method of neural stem cells |
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| CN102228016A (en) * | 2011-04-18 | 2011-11-02 | 上海安集协康生物技术有限公司 | Cryopreservation and resuscitation method of neural stem cells |
| CN104004708A (en) * | 2014-06-16 | 2014-08-27 | 内蒙古大学 | Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells |
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