CN105394028A - Method for cryopreserving umbilical cord blood stem cells with low cryo-damage - Google Patents
Method for cryopreserving umbilical cord blood stem cells with low cryo-damage Download PDFInfo
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- CN105394028A CN105394028A CN201510777329.3A CN201510777329A CN105394028A CN 105394028 A CN105394028 A CN 105394028A CN 201510777329 A CN201510777329 A CN 201510777329A CN 105394028 A CN105394028 A CN 105394028A
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Abstract
The invention discloses a method for cryopreserving umbilical cord blood stem cells with low cryo-damage, and belongs to the field of stem cell cryoprservation. The method comprises the following four steps: extracting bone marrow, and purifying and separating single karyocytes; inoculating and culturing primary cells for 48 hours, and culturing for 8 days at 37 DEG C in a culture solution comprising 8mmol/L of dexamethasone, 2.16g/L of beta-glycerol phosphate sodium and 37.5mg/L of ascorbic acid-2-phosphate in an incubator having 5% CO2; conducting first generation with a culture solution consisting of 0.25 percent of trypsin and 0.02 percent of EDTA; inoculating with a cell density of 1.3*10<4> /cm<2>, culturing to P2 generation and the like. The method has the characteristics of low toxic injury and low protectant osmotic injury on cryopreserved cells in the freezing process, and can be used for preventing mechanical injury caused by ice crystal and improving the thawing rate of cryopreserved cells.
Description
Technical field
The present invention relates to a kind of stem cell cryopreserving method, particularly a kind of cord blood stem cell cryopreservation methods of low freezing injury.
Background technology
Cord blood stem cell builds the important seed cell of tissue-engineered bone, and the deep-bed drying realizing cord blood stem cell has great importance to bone tissue engineer.Glass frozen preservation cell and protectant solution thereof is crossed with enough fast rate of temperature fall be as cold as its glass transition temperature; and be cured as glassy state the method for preserving for a long time at low temperatures with this glassy 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; there is cytotoxicity in DMSO, may cause thus cause cytotoxic damage in refrigerating process, protectant permeability damage; form ice crystal simultaneously and cause mechanical damage, these freezing injuries 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 damage freeze-stored cell low cytotoxicity in refrigerating process; low protectant permeability damage; avoid the formation of ice crystal simultaneously and cause mechanical damage, improve the cord blood stem cell cryopreservation methods of a kind of low freezing injury of freeze-stored cell anabiosis rate.
The technical solution used in the present invention is as follows:
The cord blood stem cell cryopreservation methods of a kind of low freezing injury of the present invention, comprises following step:
Step one: extract Cord blood, purification & isolation goes out mononuclearcell, after primary cell inoculated and cultured 48h, at the dexamethasone containing 8mmol/L, in the culture fluid of the β-phosphoglycerin sodium of 2.16g/L and the 2-phosphoric acid ascorbic acid of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA culture fluid of the trypsase-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.
Easily sustain damage when cell dispersal becomes single and apoptosis, owing to have employed technique scheme, preserve again after stem cell clone is agglomerating, the damage suffered by cell can be reduced, improve the survival rate of cell, thus improve anabiosis rate.
Step 2: P2 is fully immersed in the salt solution of 0.85% ~ 0.9% for cord blood stem cell group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the permeability protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumins 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;
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 mechanical damage to cell in cryopreserving liquid, therefore controls temperature at 3.2 ~ 3.9 DEG C.
Prior art adopts the DMSO of 20% as protection liquid usually; owing to have employed technique scheme; reduce the content of permeability protection liquid, thus reduce protectant cytotoxicity, reduce the cytotoxic damage that protectant causes freeze-stored cell in refrigerating process.
Monose not only can provide certain nutrition for cellular metabolism, as impermeability protectant, can ensure to reduce permeability protectant content, can not impact frozen effect simultaneously.
Rho inhibitor can suppress the kinase whose cellular activity of Rho, thus the cellular activities such as the restructuring of T suppression cell skeleton, cell adhesion and movement, division of cytoplasm and gene expression, improve frozen effect.
Physiological saline reduces protectant osmotic pressure, reduces and causes protectant permeability to damage in frozen process, thus improve anabiosis rate.
Although carry out the frozen survival rate that improve cell again by agglomerating for cell clone; but clone's agglomerate limits clone-internal cell and is exposed to cryoprotector; therefore under infrasound oscillating condition; cryoprotector can be spread apart rapidly; go deep into clone-internal cell, thus the cell of clone agglomerate inside is also exposed in cryoprotector.
Step 3: by the mixed liquor 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;
Owing to have employed technique scheme, under vacuum conditions, intercept air heat exchange, improve freezing efficiency, can lower the temperature rapidly, thus avoid in refrigerating process, occur little ice crystal, make the freezing liquid comprising stem cell directly form vitreum, improve frozen efficiency.
Step 4: preserve stored in liquid nitrogen container.
Owing to have employed technique scheme, under liquid nitrogen temperature, vigor and the metabolism of the interior various enzyme of cell, tissue are very low, almost nil, and statement is in so-called " stagnation " state.Cell frozen at this temperature, can preserve in theory indefinite duration.
The cord blood stem cell cryopreservation methods of a kind of low freezing injury 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 cryopreserving liquid environment, improve protectant osmotic effect.
The cord blood stem cell cryopreservation methods of a kind of low freezing injury of the present invention, described permeability protection liquid is the mixed solution of DMSO and polyethylene glycol.
Owing to have employed technique scheme; polyethylene glycol also can as permeability protectant; the effect of polyethylene glycol is a little less than DMSO; but cytotoxicity is well below DMSO; therefore the mixed solution both adopting; although DMSO effect is slightly poor than using, cytotoxicity is less than use DMSO, and anabiosis rate is a little more than being used alone DMSO generally.
The cord blood stem cell cryopreservation methods of a kind of low freezing injury of the present invention, described permeability protection liquid is the DMSO of 43% and the polyethylene glycol of 57%.
A kind of freezing protective agent for glass frozen preservation mesenchymal stem cells MSCs of the present invention, the concentration of described DMSO is 0.34 ~ 0.37g/mL.
A kind of freezing protective agent for glass frozen preservation mesenchymal stem cells MSCs of the present invention, the concentration of described polyethylene glycol is 0.53 ~ 0.55g/mL.
The cord blood stem cell cryopreservation methods of a kind of low freezing injury of the present invention, described monose is the mixed solution of glucose and fructose.
The cord blood stem cell cryopreservation methods of a kind of low freezing injury of the present invention, described monose comprises the glucose of 0.3% ~ 0.5% and the fructose of 0.5% ~ 0.7%.
The cord blood stem cell cryopreservation methods of a kind of low freezing injury of the present invention, described Rho inhibitor is one or more in you of Y-27632, ground you and hydroxyl method Soviet Union of method Soviet Union ground.
Owing to have employed technique scheme, above-mentioned three kinds of Rho inhibitor all can be good at suppressing Rho kinases movable, three parallel relatively on the impact of anabiosis rate without too much influence.
In sum, owing to have employed technique scheme, the beneficial effects of the utility model are:
1, the protectant content of cytotoxicity in protectant is reduced; reduce the cytotoxic damage that protectant causes freeze-stored cell in refrigerating process; suppress the kinase whose cellular activity of Rho; thus the cellular activities such as the restructuring of T suppression cell skeleton, cell adhesion and movement, division of cytoplasm and gene expression, improve frozen anabiosis rate on the whole.
2, preserve again after stem cell clone is agglomerating, the damage suffered by cell can be reduced, improve the survival rate of cell, avoid in cryopreserving liquid, form little ice crystal, thus avoid causing mechanical damage to cell, thus improve frozen anabiosis rate.
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 utility model 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 cord blood stem cell cryopreservation methods of low freezing injury, comprises following step:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculated and cultured 48h, at the dexamethasone containing 8mmol/L, in the culture fluid of the β-phosphoglycerin sodium of 2.16g/L and the 2-phosphoric acid ascorbic acid of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA culture fluid of the trypsase-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 for cord blood stem cell group in the mixed solution of 0.6%NaCl and 67.9% deionized water, holding temperature is under the condition of 3.2 DEG C, in infrasound frequency of oscillation be add under the condition of 8Hz 3.4% DMSO, the polyethylene glycol of 5.2%, the glucose of 0.3%, the fructose of 0.5%, the human serum albumins of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1%, wherein Rho inhibitor is Y-27632, the concentration of DMSO is 0.34g/mL, the concentration of polyethylene glycol is 0.53g/mL, continue to vibrate 30s under infrasound condition, add vetrifying solution,
Step 3: by the mixed liquor 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 stored in liquid nitrogen container.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard practice instructions aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
After the frozen stem cell 24h of above-mentioned four steps,
Step 5: taken out from liquid nitrogen by cryopreserving liquid, drops in LNG the 1min that heats up;
Step 6: taken out from LNG by cryopreserving liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 ~ 3.9 DEG C according to the heating rate of average 560 DEG C/min;
Step 7: cryopreserving 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 albumins of the not fibrinogen of 17% is added under infrasound (frequency is 8 ~ 13Hz) oscillating condition, the gamma globulin of 3%, the monose of 0.8% ~ 1%, the dispensable amino acid of 0.8% ~ 1.2%, the Glu of 0.08% ~ 0.12%, the beta-mercaptoethanol of 0.03% ~ 0.04%, the pH of regulation system is 6.8 ~ 7.6, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30 ~ 45s under infrasound condition;
Step 8: by the cryopreserving liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant, adds the Porcine HGF of 0.21% ~ 0.23%, Eddy diffusion cell in cell precipitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell continuous culture 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 continuous culture 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 97.6%(Fig. 1 is not frozen stem cell, and figure below is the stem cell recovered after frozen 24h)
Embodiment 2
A cord blood stem cell cryopreservation methods for low freezing injury, comprises following step:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculated and cultured 48h, at the dexamethasone containing 8mmol/L, in the culture fluid of the β-phosphoglycerin sodium of 2.16g/L and the 2-phosphoric acid ascorbic acid of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA culture fluid of the trypsase-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 for cord blood stem cell group in the mixed solution of 0.6%NaCl and 67.9% deionized water, holding temperature is under the condition of 3.9 DEG C, in infrasound frequency of oscillation be add under the condition of 13Hz 3.2% DMSO, the polyethylene glycol of 5%, the glucose of 0.5%, the fructose of 0.7%, the human serum albumins of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.3%, wherein Rho inhibitor is Y-27632, the mixture of method Soviet Union ground that and hydroxyl method Soviet Union you 1:1:1 of ground, the concentration of DMSO is 0.37g/mL, the concentration of polyethylene glycol is 0.55g/mL, continue to vibrate 45s under infrasound condition, add vetrifying solution,
Step 3: by the mixed liquor 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 stored in liquid nitrogen container.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard practice instructions aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
After the frozen stem cell 24h of above-mentioned four steps,
Step 5: taken out from liquid nitrogen by cryopreserving liquid, drops in LNG the 1min that heats up;
Step 6: taken out from LNG by cryopreserving liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 ~ 3.9 DEG C according to the heating rate of average 560 DEG C/min;
Step 7: cryopreserving 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 albumins of the not fibrinogen of 17% is added under infrasound (frequency is 8 ~ 13Hz) oscillating condition, the gamma globulin of 3%, the monose of 0.8% ~ 1%, the dispensable amino acid of 0.8% ~ 1.2%, the Glu of 0.08% ~ 0.12%, the beta-mercaptoethanol of 0.03% ~ 0.04%, the pH of regulation system is 6.8 ~ 7.6, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30 ~ 45s under infrasound condition;
Step 8: by the cryopreserving liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant, adds the Porcine HGF of 0.21% ~ 0.23%, Eddy diffusion cell in cell precipitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell continuous culture 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 continuous culture 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 cord blood stem cell cryopreservation methods for low freezing injury, comprises following step:
Step one: bone marrow extraction, purification & isolation goes out mononuclearcell, after primary cell inoculated and cultured 48h, at the dexamethasone containing 8mmol/L, in the culture fluid of the β-phosphoglycerin sodium of 2.16g/L and the 2-phosphoric acid ascorbic acid of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA culture fluid of the trypsase-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 for cord blood stem cell group in the mixed solution of 0.6%NaCl and 67.9% deionized water, holding temperature is under the condition of 3.5 DEG C, in infrasound frequency of oscillation be add under the condition of 10Hz 8.4% permeability protection liquid, the glucose of 0.35%, the fructose of 0.55%, the human serum albumins of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.2%, wherein permeability protection liquid is the DMSO of 43% and the polyethylene glycol of 57%, the concentration of DMSO is 0.36g/mL, the concentration of polyethylene glycol is 0.54g/mL, Rho inhibitor is you and hydroxyl method Soviet Union your mixed solution of ground of method Soviet Union ground, continue to vibrate 45s under infrasound condition, add vetrifying solution,
Step 3: by the mixed liquor 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 stored in liquid nitrogen container.
Experiment frozen material: according to the Cord blood of U.S.'s Cord Blood Bank standard practice instructions aseptic collection health, term birth puerpera, process in 24h after gathering.
Experimental technique:
After the frozen stem cell 24h of above-mentioned four steps,
Step 5: taken out from liquid nitrogen by cryopreserving liquid, drops in LNG the 1min that heats up;
Step 6: taken out from LNG by cryopreserving liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 ~ 3.9 DEG C according to the heating rate of average 560 DEG C/min;
Step 7: cryopreserving 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 albumins of the not fibrinogen of 17% is added under infrasound (frequency is 8 ~ 13Hz) oscillating condition, the gamma globulin of 3%, the monose of 0.8% ~ 1%, the dispensable amino acid of 0.8% ~ 1.2%, the Glu of 0.08% ~ 0.12%, the beta-mercaptoethanol of 0.03% ~ 0.04%, the pH of regulation system is 6.8 ~ 7.6, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30 ~ 45s under infrasound condition;
Step 8: by the cryopreserving liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant, adds the Porcine HGF of 0.21% ~ 0.23%, Eddy diffusion cell in cell precipitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell continuous culture 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 continuous culture 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%
Embodiment 4
A stem cell cryopreserving method for low freezing injury, comprises following step:
Step one: extract Cord blood, purification & isolation goes out mononuclearcell, after primary cell inoculated and cultured 48h, at the dexamethasone containing 8mmol/L, in the culture fluid of the β-phosphoglycerin sodium of 2.16g/L and the 2-phosphoric acid ascorbic acid of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA culture fluid of the trypsase-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 for cord blood stem cell group in the mixed solution of 0.6%NaCl and 67.9% deionized water, holding temperature is under the condition of 3.5 DEG C, in infrasound frequency of oscillation be add under the condition of 10Hz 8.5% permeability protection liquid, the monose of 1%, the human serum albumins of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.1%, all the other components are physiological saline, Rho inhibitor is the mixture of Y-27632, method Soviet Union ground that and hydroxyl method Soviet Union you 1:1:1 of ground, continue to vibrate 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed liquor 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 stored in liquid nitrogen container.
Experiment frozen material: cord blood stem cell
Experimental technique:
After the frozen stem cell 24h of above-mentioned four steps,
Step 5: taken out from liquid nitrogen by cryopreserving liquid, drops in LNG the 1min that heats up;
Step 6: taken out from LNG by cryopreserving liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 ~ 3.9 DEG C according to the heating rate of average 560 DEG C/min;
Step 7: cryopreserving 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 albumins of the not fibrinogen of 17% is added under infrasound (frequency is 8 ~ 13Hz) oscillating condition, the gamma globulin of 3%, the monose of 0.8% ~ 1%, the dispensable amino acid of 0.8% ~ 1.2%, the Glu of 0.08% ~ 0.12%, the beta-mercaptoethanol of 0.03% ~ 0.04%, the pH of regulation system is 6.8 ~ 7.6, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30 ~ 45s under infrasound condition;
Step 8: by the cryopreserving liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant, adds the Porcine HGF of 0.21% ~ 0.23%, Eddy diffusion cell in cell precipitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell continuous culture 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 continuous culture 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 94.3%
Embodiment 5
A stem cell cryopreserving method for low freezing injury, comprises following step:
Step one: extract Cord blood, purification & isolation goes out mononuclearcell, after primary cell inoculated and cultured 48h, at the dexamethasone containing 8mmol/L, in the culture fluid of the β-phosphoglycerin sodium of 2.16g/L and the 2-phosphoric acid ascorbic acid of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA culture fluid of the trypsase-0.02% with 0.25% carries out the 1st time and goes down to posterity; Inoculate with the cell density of 1.3 × 104/cm2, be cultured to P2 generation;
Step 2: P2 is fully immersed in the permeability protection liquid of 8.4% for cord blood stem cell group, the monose of 0.9%, the human serum albumins of the not fibrinogen of 17%, the gamma globulin of 3%, the Rho inhibitor of 2.3%, all the other components are physiological saline, and Rho inhibitor is the mixture of Y-27632 and hydroxyl method Soviet Union you 2:1 of ground, continue to vibrate 45s under infrasound condition, add vetrifying solution;
Step 3: by the mixed liquor 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 stored in liquid nitrogen container.
Experiment frozen material: cord blood stem cell
Experimental technique:
After the frozen stem cell 24h of above-mentioned four steps,
Step 5: taken out from liquid nitrogen by cryopreserving liquid, drops in LNG the 1min that heats up;
Step 6: taken out from LNG by cryopreserving liquid, drops in ice bath under vacuum conditions, is cooled to rapidly 3.2 ~ 3.9 DEG C according to the heating rate of average 560 DEG C/min;
Step 7: cryopreserving 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 albumins of the not fibrinogen of 17% is added under infrasound (frequency is 8 ~ 13Hz) oscillating condition, the gamma globulin of 3%, the monose of 0.8% ~ 1%, the dispensable amino acid of 0.8% ~ 1.2%, the Glu of 0.08% ~ 0.12%, the beta-mercaptoethanol of 0.03% ~ 0.04%, the pH of regulation system is 6.8 ~ 7.6, add the hydroxyethyl piperazine second thiosulfonic acid of 1.5%, continue to vibrate 30 ~ 45s under infrasound condition;
Step 8: by the cryopreserving liquid centrifugal 5min under the condition of 400r/min after dilution, removes supernatant, adds the Porcine HGF of 0.21% ~ 0.23%, Eddy diffusion cell in cell precipitation.
Measure the anabiosis rate of cell: by 3 × 10
5the frozen stem cell continuous culture 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 continuous culture 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 94.9%
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 (9)
1. a cord blood stem cell cryopreservation methods for low freezing injury, is characterized in that comprising following step:
Step one: extract Cord blood, purification & isolation goes out mononuclearcell, after primary cell inoculated and cultured 48h, at the dexamethasone containing 8mmol/L, in the culture fluid of the β-phosphoglycerin sodium of 2.16g/L and the 2-phosphoric acid ascorbic acid of 37.5mg/L, 5%CO
2incubator in, cultivate 8d at the temperature of 37 DEG C; The EDTA culture fluid of the trypsase-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 stem cell group, holding temperature is under the condition of 3.2 ~ 3.9 DEG C, the permeability protection liquid of 8.2% ~ 8.6% is added under infrasound oscillating condition, the monose of 0.8% ~ 1%, the human serum albumins 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 liquor 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 stored in liquid nitrogen container.
2. the cord blood stem cell cryopreservation methods of a kind of low freezing injury as claimed in claim 1, is characterized in that: described infrasonic frequency is 8 ~ 13Hz.
3. the cord blood stem cell cryopreservation methods of a kind of low freezing injury as claimed in claim 1, is characterized in that: described permeability protection liquid is the mixed solution of DMSO and polyethylene glycol.
4. the cord blood stem cell cryopreservation methods of a kind of low freezing injury as described in claim 1 or 3, is characterized in that: described permeability protection liquid is the DMSO of 43% and the polyethylene glycol of 57%.
5. a kind of freezing protective agent for glass frozen preservation mesenchymal stem cells MSCs as claimed in claim 4, is characterized in that: the concentration of described DMSO is 0.34 ~ 0.37g/mL.
6. a kind of freezing protective agent for glass frozen preservation mesenchymal stem cells MSCs as claimed in claim 4, is characterized in that: the concentration of described polyethylene glycol is 0.53 ~ 0.55g/mL.
7. the cord blood stem cell cryopreservation methods of a kind of low freezing injury as claimed in claim 1, is characterized in that: described monose is the mixed solution of glucose and fructose.
8. the cord blood stem cell cryopreservation methods of a kind of low freezing injury as claimed in claim 5, is characterized in that: described monose comprises the glucose of 0.3% ~ 0.5% and the fructose of 0.5% ~ 0.7%.
9. the cord blood stem cell cryopreservation methods of a kind of low freezing injury as claimed in claim 5, is characterized in that: described Rho inhibitor is one or more in you of Y-27632, ground you and hydroxyl method Soviet Union of method Soviet Union ground.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510777329.3A CN105394028A (en) | 2015-11-16 | 2015-11-16 | Method for cryopreserving umbilical cord blood stem cells with low cryo-damage |
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| CN107467014A (en) * | 2017-10-12 | 2017-12-15 | 北京臻惠康生物科技有限公司 | A kind of method that umbilical cord tissue freezes, recovered |
| CN108094404A (en) * | 2017-12-20 | 2018-06-01 | 北京臻惠康生物科技有限公司 | A kind of improved mesenchyme stem cell protection solution and application thereof |
| CN109152800A (en) * | 2016-08-01 | 2019-01-04 | 胚胎发育私人有限公司 | Skin care formulation |
| CN112391342A (en) * | 2020-12-07 | 2021-02-23 | 山东省齐鲁干细胞工程有限公司 | Efficient recovery method for umbilical cord blood stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109152800A (en) * | 2016-08-01 | 2019-01-04 | 胚胎发育私人有限公司 | Skin care formulation |
| CN107467014A (en) * | 2017-10-12 | 2017-12-15 | 北京臻惠康生物科技有限公司 | A kind of method that umbilical cord tissue freezes, recovered |
| CN108094404A (en) * | 2017-12-20 | 2018-06-01 | 北京臻惠康生物科技有限公司 | A kind of improved mesenchyme stem cell protection solution and application thereof |
| CN108094404B (en) * | 2017-12-20 | 2021-01-15 | 北京唐颐惠康生物医学技术有限公司 | Improved mesenchymal stem cell protection solution and application thereof |
| CN112391342A (en) * | 2020-12-07 | 2021-02-23 | 山东省齐鲁干细胞工程有限公司 | Efficient recovery method for umbilical cord blood stem cells |
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