CN107593686A - A kind of cryopreservation methods of people's navel mescenchymal stem cell - Google Patents
A kind of cryopreservation methods of people's navel mescenchymal stem cell Download PDFInfo
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- CN107593686A CN107593686A CN201710866761.9A CN201710866761A CN107593686A CN 107593686 A CN107593686 A CN 107593686A CN 201710866761 A CN201710866761 A CN 201710866761A CN 107593686 A CN107593686 A CN 107593686A
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Abstract
The present invention relates to technical field of cell biology, is related to a kind of cryopreservation methods of people's navel mescenchymal stem cell, the Rapid-Freezing Method method of especially a kind of improvement, and this method can improve the anabiosis rate of cell in the case where ensureing that cellular morphology characteristic is constant.This method inserts the cryopreservation tube equipped with people's navel mescenchymal stem cell in the freezing storing box containing isopropanol, place into the thick foam boxs of 2cm~4cm, then it is suspended in liquid nitrogen vapor, 4~6 can as a child directly take out cryopreservation tube, immerse progress low temperature in liquid nitrogen and preserve for a long time.In this method, the use of isopropanol and foam box can make cell during freezing, and reduce the formation of ice crystal, reduce the injury that cell is subject to during freezing.Using this method freeze-stored cell, even if freezing 5 years, cell recovery rate is still up to 95%~99%.The advantages of this method has technique simple compared with routinely using programmed cooling method freeze-stored cell, and the instrument and equipment being related to is less, time-consuming shorter, and cell recovery rate is high, present invention is suitably applied to the preservation of the prolonged cold of people's navel mescenchymal stem cell.
Description
Technical field
The present invention relates to technical field of cell biology, is related to cell freezing method, relates in particular to people's navel mesenchyma and does
The cryopreservation methods of cell.
Background technology
Mescenchymal stem cell（MSC）It is current laboratory and the most Adult multipotent stem cells of clinical research, in Hematopoietic Stem
There is important application in cell transplantation, graft versus host disease(GVH disease) etc..Derived from bone marrow MSC can be divided into according to source（BMSC）, fat
Source MSC and umbilical cord source MSC（hUCMSCs).Most study is the most widely BMSC, but due to when forefathers' bone at present
Marrow source BMSC levels are extremely low, and have that viral infection rate is higher, cell proliferation and differentiation potential decline with the increase at age and
The operation that materials are an invasion, have wound, easily pollute is punctured, limitation is brought to its wide clinical application.Research find from
The hUCMSCs isolated in people's umbilical cord, BMSC is superior in terms of cell content, multiplication capacity, repeatedly passes on amplification and remain to protect
Vigorous function is held, is not expressed or low expression immunological rejection mark of correlation, immunogenicity is lower than BMSC, and convenient material drawing, no ethics
Dispute is learned, is presently considered to be ideal seed cell.The deep-bed drying of people's navel mescenchymal stem cell is realized, to whole group
Weaver's journey, clinical treatment etc. are all significant.
Research shows, cell is placed in into Cord blood in -196 DEG C of liquid nitrogen using Cryopreservation Technology, cell can be made temporarily to take off
Cell characteristics are saved from growth conditions, are used to test after recovery cell is carried out when needing.It is currently used
Cell preservation method enters line program cooling, Ran Houzhuan for cell cryopreservation tube is sequentially placed into 4 DEG C, -20 DEG C and -80 DEG C of environment
Move to progress prolonged cold preservation in -196 DEG C of liquid nitrogen.Such a store method technique is more, required instrument and equipment also compared with
It is more, and time interval is longer, and the formation of ice crystal can not be avoided during freezing to the infringement of cell, substantial amounts of actual biological cell
Learn experiment and statistics shows, the cell frozen in this way survival rate after recovery typically 50%~80%, survives
Rate is than relatively low.
The content of the invention
To solve the deficiencies in the prior art, the present invention provides a kind of new side of freezing for people's navel mescenchymal stem cell
Method, the method can reduce the formation of people's navel mesenchyma stem cell suspension ice crystal during freezing, and reduce cellular damage；Simultaneously
It can simplify and freeze technique, reduce the use of instrument, shortening freezes the time, improves cell recovery rate.It is a feature of the present invention that
It includes following cryopreservation step.
（1）Take the logarithm people's navel mescenchymal stem cell in growth period, be put into centrifuge tube, under the conditions of 4 DEG C, with 1200rpm's
Centrifugation 5 minutes, discards supernatant, and sedimentation cell is mixed into individual cells suspension with culture medium.
（2）Prepare cells frozen storing liquid：DMSO, Dextran40 are configured to freezing protective agent by a certain percentage, guarantor will be frozen
Shield agent is put into 4 DEG C of refrigerator precooling 30min.
（3）By a certain percentage, freezing protective agent is slowly added in cell suspension, side edged slowly shakes suspension, by 1.2
ML/ pipes are sub-packed in cryopreservation tube.
（4）Cryopreservation tube is placed in freezing storing box, freezing storing box bottom adds isopropanol, makes cryopreservation tube lower submerged in isopropanol
In.Freezing storing box is placed in foam box, is suspended in the vapor film of liquid nitrogen, after a period of time, removes foam box and freezing storing box, will
Cryopreservation tube moves into progress profound hypothermia in liquid nitrogen and preserved for a long time.
In the inventive method, during cryopreserved human navel mescenchymal stem cell, culture medium is used by cell is resuspended after centrifugation
AIM-V culture mediums.
In the inventive method, during cryopreserved human navel mescenchymal stem cell, the preparation of cells frozen storing liquid according to DMSO with
Dextran40 is 3:5~7 ratio is prepared.
In the inventive method, during cryopreserved human navel mescenchymal stem cell, the ratio of cell suspension and freezing protective agent is 11:3
~6.
In the inventive method, during cryopreserved human navel mescenchymal stem cell, the cell cryopreservation box in foam box need to be suspended in midair
The 4-6 hours in liquid nitrogen vapor.
In the inventive method, during cryopreserved human navel mescenchymal stem cell, the thickness of foam box should be between 2cm-4cm.
As a result show, people's navel mesenchymal stem cell cryopreserving method provided by the invention, due to isopropyl outside cryopreservation tube be present
The use of the protection of alcohol and foam box, especially foam box so that people's navel mescenchymal stem cell in cryopreservation tube is in liquid nitrogen vapor
In layer, the possibility of ice crystal formation can be reduced, reduces injury of the cell during freezing.Using such a cryopreservation methods, not only
Help to reduce people navel mesenchymal stem cell cryopreserving technique, shorten the cell cryopreservation time, and cell after recovery can be significantly improved
Vigor, after testing, the vigor of recovery descendant's navel mescenchymal stem cell are up to 95%-99%.
The method of the present invention is applied to the Cord blood of people's navel mescenchymal stem cell, and the method is not only advantageous to omission and frozen
Step, simplify technique, reduce the use of some Cryo Equipment instruments, improve the anabiosis rate of cell, and tracked by 5 years, the party
The use of method will not cause the change of people's navel mescenchymal stem cell characteristic, and the prolonged cold available for people's navel mescenchymal stem cell is protected
Deposit.
Brief description of the drawings
Fig. 1 is using cell freezing method provided by the invention and the anabiosis rate after regular growth freezing preservation method freeze-stored cell.
Embodiment
In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.
Embodiment one.
（1）Take the logarithm people's navel mescenchymal stem cell in growth period, be put into centrifuge tube, under the conditions of 4 DEG C, with 1200rpm's
Centrifugation 5 minutes, discards supernatant, and sedimentation cell is mixed into individual cells suspension with AIM-V culture mediums.
（2）Prepare cells frozen storing liquid：DMSO, Dextran40 are pressed 3:6 ratio is configured to freezing protective agent, will freeze
Protective agent is put into 4 DEG C of refrigerator precooling 30min.
（3）By 11:5 ratio, freezing protective agent is slowly added in cell suspension, and side edged slowly shakes suspension, presses
1.2 mL/ pipes are sub-packed in cryopreservation tube.
（4）Cryopreservation tube is placed in freezing storing box, freezing storing box bottom adds isopropanol, makes cryopreservation tube lower submerged in isopropanol
In.Freezing storing box is placed in foam box, is suspended in the vapor film of liquid nitrogen, after 4h, removes foam box and freezing storing box, by cryopreservation tube
Move into progress profound hypothermia in liquid nitrogen to preserve for a long time, preserve 5 years.
Embodiment two.
（1）Take the logarithm people's navel mescenchymal stem cell in growth period, be put into centrifuge tube, under the conditions of 4 DEG C, with 1200rpm's
Centrifugation 5 minutes, discards supernatant, and sedimentation cell is mixed into individual cells suspension with AIM-V culture mediums.
（2）Prepare cells frozen storing liquid：DMSO, Dextran40 are pressed 3:7 ratio is configured to freezing protective agent, will freeze
Protective agent is put into 4 DEG C of refrigerator precooling 30min.
（3）By 11:4 ratio, freezing protective agent is slowly added in cell suspension, and side edged slowly shakes suspension, presses
1.2 mL/ pipes are sub-packed in cryopreservation tube.
（4）Cryopreservation tube is placed in freezing storing box, freezing storing box bottom adds isopropanol, makes cryopreservation tube lower submerged in isopropanol
In.Freezing storing box is placed in foam box, is suspended in the vapor film of liquid nitrogen, after 6h, removes foam box and freezing storing box, by cryopreservation tube
Move into progress profound hypothermia in liquid nitrogen to preserve for a long time, preserve 5 years.
Embodiment three.
（1）Take the logarithm people's navel mescenchymal stem cell in growth period, be put into centrifuge tube, under the conditions of 4 DEG C, with 1200rpm's
Centrifugation 5 minutes, discards supernatant, and sedimentation cell is mixed into individual cells suspension with AIM-V culture mediums.
（2）Prepare cells frozen storing liquid：DMSO, Dextran40 are pressed 3:5 ratio is configured to freezing protective agent, will freeze
Protective agent is put into 4 DEG C of refrigerator precooling 30min.
（3）By 11:7 ratio, freezing protective agent is slowly added in cell suspension, and side edged slowly shakes suspension, presses
1.2 mL/ pipes are sub-packed in cryopreservation tube.
（4）Cryopreservation tube is placed in freezing storing box, freezing storing box bottom adds isopropanol, makes cryopreservation tube lower submerged in isopropanol
In.Freezing storing box is placed in foam box, is suspended in the vapor film of liquid nitrogen, after 5h, removes foam box and freezing storing box, by cryopreservation tube
Move into progress profound hypothermia in liquid nitrogen to preserve for a long time, preserve 5 years.
People's navel mesenchymal stem cell cryopreserving in above-described embodiment is recovered after 5 years, using the blue dyeing of desk tray and blood
Ball count plate counts the viable count frozen before and after 5 years respectively, calculates cell recovery rate.Compare to be cooled using conventional program
Anabiosis rate after the recovery of method freeze-stored cell.Acquired results are shown in accompanying drawing 1.
People's navel mesenchymal stem cell cryopreserving method institute using 1-3 of the embodiment of the present invention is can be seen that from the result of accompanying drawing 1
The people's navel mescenchymal stem cell recovery culture 12 frozen was as a child observed, it is seen that 90% or so cell attachment, cellular morphology are plentiful
Well, sharpness of border, it is adherent good, it is evenly distributed, living cells is more.
Using carried out after conventional method cryopreserved human navel mescenchymal stem cell recovery culture 12 hours after observe, it is seen that blake bottle
In possess that more cell fragment, most cell boundaries are unintelligible, and form is irregular, and cell is not full enough, and attached cell is less,
About 70% or so.
Above-mentioned experimental result explanation：A kind of cryopreservation methods of people's navel mescenchymal stem cell provided by the invention, can be effective
Reduction freezes damage of the technique to people's navel mescenchymal stem cell, improves cell recovery rate.Such a method is applied to people's navel mesenchyma
The Cryopreserved of stem cell.
It should be noted that the invention is not limited in any way for above-described embodiment, it is all to use equivalent substitution or equivalent change
The technical scheme that the mode changed is obtained, all falls within protection scope of the present invention.
Claims (6)
1. a kind of cryopreservation methods of people's navel mescenchymal stem cell, it is characterised in that it comprises the following steps：
（1）Take the logarithm people's navel mescenchymal stem cell in growth period, be put into centrifuge tube, under the conditions of 4 DEG C, with 1200rpm speed
Centrifugation 5 minutes, discards supernatant, sedimentation cell is mixed into individual cells suspension with culture medium；
（2）Prepare cells frozen storing liquid：DMSO, Dextran40 are configured to freezing protective agent by a certain percentage, by freezing protective agent
It is put into 4 DEG C of refrigerator precooling 30min；
（3）By a certain percentage, freezing protective agent is slowly added in cell suspension, side edged slowly shakes suspension, by 1.2 mL/
Pipe is sub-packed in cryopreservation tube；
（4）Cryopreservation tube is placed in freezing storing box, freezing storing box bottom adds isopropanol, makes cryopreservation tube lower submerged in isopropanol；
Freezing storing box is placed in foam box, is suspended in the vapor film of liquid nitrogen, after a period of time, removes foam box and freezing storing box, will be frozen
Progress profound hypothermia in pipe immigration liquid nitrogen is deposited to preserve for a long time.
2. according to the step described in a kind of cryopreservation methods of people's navel mescenchymal stem cell of claim 1（1）In, sedimentation cell is resuspended
Culture medium used is AIM-V culture mediums.
3. according to the step described in a kind of cryopreservation methods of people's navel mescenchymal stem cell of claim 1（2）In, cells frozen storing liquid
It is 3 to prepare according to DMSO and Dextran40:5~7 ratio is prepared.
4. according to the step described in a kind of cryopreservation methods of people's navel mescenchymal stem cell of claim 1（3）In, cell suspension is with freezing
Protectant ratio is deposited as 11:3~6.
5. according to the step described in a kind of cryopreservation methods of people's navel mescenchymal stem cell of claim 1（4）In, need that foam will be mounted in
Cell cryopreservation box in box is suspended to 4-6 hours in liquid nitrogen vapor.
6. according to the step described in a kind of cryopreservation methods of people's navel mescenchymal stem cell of claim 1（4）In, the thickness of foam box
Should be between 2cm-4cm.
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