CN108865984A - A kind of preparation and extracting method of umbilical cord mesenchymal stem cells - Google Patents
A kind of preparation and extracting method of umbilical cord mesenchymal stem cells Download PDFInfo
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- CN108865984A CN108865984A CN201810650658.5A CN201810650658A CN108865984A CN 108865984 A CN108865984 A CN 108865984A CN 201810650658 A CN201810650658 A CN 201810650658A CN 108865984 A CN108865984 A CN 108865984A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a kind of preparation of umbilical cord mesenchymal stem cells and extracting methods, include the following steps, cleaning；Enzymatic hydrolysis takes out umbilical cord pulverization process, is handled using the clostridiopetidase A II that concentration is 0.1%, and enzymolysis time is 6~8 hours；Filtering, brings the distilled water of 5 times of volumes into umbilical cord enzymolysis liquid, carries out primary filtration using sieve, is subsequently poured into differential separator and further filters；Originally culture；Secondary culture；Activity determination；Cell cluster grouping after preferably is stored in heat insulation tank by freezen protective, and the temperature of heat insulation tank should be 4 DEG C, and the present invention relates to technical field of bioengineering.The preparation and extracting method of the umbilical cord mesenchymal stem cells, the cell there are gene defect can be effectively removed, reduce the damage to stem cell recipient, make temperature change in stem cell heating and cold storage procedure more gentle simultaneously, the temperature during cell extraction is avoided to change the problem of being easy to make cell inactivation, reducing the recovery rate of cell.
Description
Technical field
The present invention relates to technical field of bioengineering, the preparation of specially a kind of umbilical cord mesenchymal stem cells and extraction side
Method.
Background technique
Stem cell is a kind of multipotential cell with the of self-replication capacity, and under certain condition, it can be divided into more
Kind functioning cell, the stage of development according to locating for stem cell is divided into embryonic stem cell and adult stem cell, according to the hair of stem cell
Potential is educated to be divided into three classes:Myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell (specially energy stem cell), stem cell are that one kind is not filled
Differentiation, still immature cell have the potential function for regenerating various histoorgans and human body, and referred to as " general-purpose is thin for medical field
Born of the same parents ".But the cell that existing stem cell extracting method extracts is easy to influence the strong of recipient there may be gene defect
Health, while the temperature change during cell extraction is easy to make cell inactivation, reduces the recovery rate of cell.
Summary of the invention
（One）The technical issues of solution
In view of the deficiencies of the prior art, the present invention provides a kind of preparation of umbilical cord mesenchymal stem cells and extracting methods, solve
The cell that existing stem cell extracting method extracts is easy to influence the health of recipient, simultaneously there may be gene defect
The problem of temperature change during cell extraction is easy to make cell inactivation, reduces the recovery rate of cell.
（Two）Technical solution
In order to achieve the above object, the present invention is achieved by the following technical programs：A kind of preparation of umbilical cord mesenchymal stem cells
And extracting method, include the following steps,
（1）Cleaning, is rinsed well umbilical cord with distilled water, then with alcohol swab that umbilical cord surface wipes are clean；
（2）Enzymatic hydrolysis takes out umbilical cord pulverization process, is handled using the clostridiopetidase A II that concentration is 0.1%, and enzymolysis time is 6~8 hours；
（3）Filtering, brings the distilled water of 5 times of volumes into umbilical cord enzymolysis liquid, carries out primary filtration using sieve, is subsequently poured into difference
It is further filtered in fast seperator；
（4）Originally culture, filtered umbilical cord cells are inoculated into serum-free basal medium and are cultivated, and incubation time is
2 days；
（5）Secondary culture replaces serum free medium, and serum-free nutrient additive is added in serum free medium, periodically
Culture medium is replaced, until generating cell cluster；
（6）Activity determination takes part cell from different Place cell clusters respectively and is grouped label, and different groups of cell is distinguished
Carry out induction differentiation culture, the cell activity after detection differentiation, the poor cell cluster of knockout cell activity；
（7）Cell cluster grouping after preferably is stored in heat insulation tank by freezen protective, and the temperature of heat insulation tank should be 4 DEG C.
Preferably, centrifugation time of the enzymolysis liquid in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
Preferably, it should be cut into small pieces to be put into heat insulation tank before the umbilical cord enzymatic hydrolysis and refrigerate, the refrigeration duration is 2~6
Month.
Preferably, the heat insulation tank includes tank body, and the inner wall of the tank body is fixedly connected with insulating layer, the insulating layer
Inner wall two sides are fixedly connected to electric heating piece, and inner canister is fixedly connected on the inside of the electric heating piece, and the inside of the inner canister opens up
There is water-bath pipeline, one end of the water-bath pipeline runs through inner canister and extends to the top of inner canister, the top installation of the tank body
There is insulation cover, top one end of the insulation cover offers blasthole compatible with water-bath pipeline.
Preferably, the side of the tank body offers automatically controlled slot, and the inner wall bottom of the automatically controlled slot is fixedly connected with power supply
Case is fixedly connected with controller at the top of the power supply box, and it is electrical to pass through conducting wire between the power supply box, controller and electric heating piece
It connects, is fixedly connected with baffle between the two sides of the automatically controlled slot, the side of the baffle offers air hole.
Preferably, the bottom end of the tank body offers water outlet, the water outlet through tank body and inner canister and with
The connection of water-bath pipeline, the inside of the water outlet are equipped with electrically-controlled valve, and the electrically-controlled valve is electrically connected by conducting wire and controller.
Preferably, the side of the blasthole offers seal groove, and the inner wall of the seal groove is far from the side of blasthole
Tilted block is fixedly connected with by spring, and the inner wall of the tilted block and seal groove is slidably connected, and the bottom of the tilted block is solid
Surely it is connected with sealing block.
Preferably, handle is fixedly connected at the top of the insulation cover.
Preferably, bracket is slidably connected between the inner wall of the inner canister.
Preferably, the top both ends of the bracket are fixedly connected to handle.
（Three）Beneficial effect
The present invention provides a kind of preparation of umbilical cord mesenchymal stem cells and extracting methods.Has following beneficial effect：
（1）, the umbilical cord mesenchymal stem cells preparation and extracting method, should be cut into small pieces before being digested by umbilical cord and be put into heat insulation tank
Middle refrigeration, refrigeration duration are 2~6 months, and filtered umbilical cord cells are inoculated into serum-free basal medium and are carried out
Culture, incubation time are 2 days, replace serum free medium, and serum-free nutrient additive is added in serum free medium, fixed
Phase replaces culture medium, until generating cell cluster, takes part cell respectively from different Place cell clusters and is grouped label, will be different
The cell of group carries out induction differentiation culture, the cell activity after detection differentiation, the poor cell cluster of knockout cell activity, energy respectively
The cell there are gene defect is enough effectively removed, the damage to stem cell recipient is reduced.
（2）, the umbilical cord mesenchymal stem cells preparation and extracting method, by will preferably after cell cluster grouping be stored in
In heat insulation tank, should be cut into small pieces to be put into heat insulation tank and refrigerates before umbilical cord enzymatic hydrolysis, the heat insulation tank includes tank body, the tank body it is interior
Wall is fixedly connected with insulating layer, and the inner wall two sides of the insulating layer are fixedly connected to electric heating piece, and the inside of the electric heating piece is solid
Surely it is connected with inner canister, the inside of the inner canister offers water-bath pipeline, and one end of the water-bath pipeline through inner canister and extends
To the top of inner canister, insulation cover is installed at the top of the tank body, top one end of the insulation cover offers and water-bath pipeline
It is passed through hot water and ice water when compatible blasthole, heating and refrigeration into water-bath pipeline respectively, temperature passes when electric heating piece heats
It is delivered to the buffering that inner canister also passes through water flow in water-bath pipeline, keeps temperature change in stem cell heating and cold storage procedure more gentle,
The temperature during cell extraction is avoided to change the problem of being easy to make cell inactivation, reducing the recovery rate of cell.
Detailed description of the invention
Fig. 1 is schematic structural diagram of the device of the invention；
Fig. 2 is seal groove structure schematic diagram of the present invention；
Fig. 3 is the automatically controlled slot structure schematic diagram of the present invention；
In figure：1- tank body, 2- insulating layer, 3- electric heating piece, 4- inner canister, 5- water-bath pipeline, 6- insulation cover, 7- blasthole, 8- are automatically controlled
Slot, 9- power supply box, 10- controller, 11- baffle, 12- air hole, 13- water outlet, 14- electrically-controlled valve, 15- seal groove, 16- bullet
Spring, 17- tilted block, 18- sealing block, 19- handle, 20- bracket, 21- handle.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Fig. 1-3 is please referred to, the present invention provides a kind of technical solution：A kind of preparation of umbilical cord mesenchymal stem cells and extraction side
Method, it is characterised in that：Include the following steps,
（1）Cleaning, is rinsed well umbilical cord with distilled water, then with alcohol swab that umbilical cord surface wipes are clean；
（2）Enzymatic hydrolysis takes out umbilical cord pulverization process, is handled using the clostridiopetidase A II that concentration is 0.1%, and enzymolysis time is 6~8 hours；
（3）Filtering, brings the distilled water of 5 times of volumes into umbilical cord enzymolysis liquid, carries out primary filtration using sieve, is subsequently poured into difference
It is further filtered in fast seperator；
（4）Originally culture, filtered umbilical cord cells are inoculated into serum-free basal medium and are cultivated, and incubation time is
2 days；
（5）Secondary culture replaces serum free medium, and serum-free nutrient additive is added in serum free medium, periodically
Culture medium is replaced, until generating cell cluster；
（6）Activity determination takes part cell from different Place cell clusters respectively and is grouped label, and different groups of cell is distinguished
Induction differentiation culture, the cell activity after detection differentiation are carried out, the poor cell cluster of knockout cell activity can be effectively removed and be deposited
In the cell of gene defect, the damage to stem cell recipient is reduced；
（7）Cell cluster grouping after preferably is stored in heat insulation tank by freezen protective, and the temperature of heat insulation tank should be 4 DEG C.
Centrifugation time of the enzymolysis liquid in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
It should be cut into small pieces to be put into heat insulation tank before umbilical cord enzymatic hydrolysis and refrigerate, the refrigeration duration is 2~6 months.
Heat insulation tank includes tank body 1, and the inner wall of tank body 1 is fixedly connected with insulating layer 2, and the inner wall two sides of insulating layer 2 are fixed
It is connected with electric heating piece 3, the inside of electric heating piece 3 is fixedly connected with inner canister 4, and the inside of inner canister 4 offers water-bath pipeline 5, water-bath pipe
The one end in road 5 runs through inner canister 4 and extends to the top of inner canister 4, and the top of tank body 1 is equipped with insulation cover 6, the top of insulation cover 6
Portion one end is passed through hot water into water-bath pipeline 5 respectively when offering blasthole 7 compatible with water-bath pipeline 5, heating and refrigeration
And ice water, electric heating piece 3 heat when temperature be transmitted to the buffering that inner canister 4 also passes through water flow in water-bath pipeline 5, make stem cell heating and
Temperature change is more gentle in cold storage procedure, avoids the temperature change during cell extraction from being easy to make cell inactivation, reduces thin
The problem of recovery rate of born of the same parents.
The side of tank body 1 offers automatically controlled slot 8, and the inner wall bottom of automatically controlled slot 8 is fixedly connected with power supply box 9, power supply box 9
Top is fixedly connected with controller 10, is electrically connected between power supply box 9, controller 10 and electric heating piece 3 by conducting wire, automatically controlled slot 8
Two sides between be fixedly connected with baffle 11, the side of baffle 11 offers air hole 12.
The bottom end of tank body 1 offers water outlet 13, water outlet 13 through tank body 1 and inner canister 4 and with water-bath pipeline 5
Connection, the inside of water outlet 13 are equipped with electrically-controlled valve 14, and electrically-controlled valve 14 is electrically connected by conducting wire and controller 10.
The side of blasthole 7 offers seal groove 15, and the inner wall of seal groove 15 passes through spring 16 far from the side of blasthole 7
It is fixedly connected with tilted block 17, tilted block 17 and the inner wall of seal groove 15 are slidably connected, and the bottom of tilted block 17 is fixedly connected with
Sealing block 18.
The top of insulation cover 6 is fixedly connected with handle 19.
Bracket 20 is slidably connected between the inner wall of inner canister 4.
The top both ends of bracket 20 are fixedly connected to handle 21.
When work, umbilical cord is rinsed well with distilled water, it is then with alcohol swab that umbilical cord surface wipes are clean, take out umbilical cord
Pulverization process is handled using the clostridiopetidase A II that concentration is 0.1%, and enzymolysis time is 6~8 hours, brings 5 into umbilical cord enzymolysis liquid
The distilled water of times volume carries out primary filtration using sieve, is subsequently poured into differential separator and further filters, will be filtered
Umbilical cord cells are inoculated into serum-free basal medium and are cultivated, and incubation time is 2 days, replacement serum free medium, and
Serum-free nutrient additive is added in serum free medium, regularly replaces culture medium, until cell cluster is generated, it is thin from different location
Part cell is taken in born of the same parents' cluster respectively and is grouped label, different groups of cell is carried out to induction differentiation culture respectively, after detection differentiation
Cell activity, the poor cell cluster of knockout cell activity, will preferably after cell cluster grouping be stored in heat insulation tank, heat insulation tank
Temperature should be 4 DEG C.
It should be noted that, in this document, relational terms such as first and second and the like are used merely to a reality
Body or operation are distinguished with another entity or operation, are deposited without necessarily requiring or implying between these entities or operation
In any actual relationship or order or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to
Non-exclusive inclusion, so that the process, method, article or equipment including a series of elements is not only wanted including those
Element, but also including other elements that are not explicitly listed, or further include for this process, method, article or equipment
Intrinsic element.In the absence of more restrictions.By sentence " element limited including one ..., it is not excluded that
There is also other identical elements in the process, method, article or apparatus that includes the element ".
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (10)
1. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells, it is characterised in that：Include the following steps,
（1）Cleaning, is rinsed well umbilical cord with distilled water, then with alcohol swab that umbilical cord surface wipes are clean；
（2）Enzymatic hydrolysis takes out umbilical cord pulverization process, is handled using the clostridiopetidase A II that concentration is 0.1%, and enzymolysis time is 6~8 hours；
（3）Filtering, brings the distilled water of 5 times of volumes into umbilical cord enzymolysis liquid, carries out primary filtration using sieve, is subsequently poured into difference
It is further filtered in fast seperator；
（4）Originally culture, filtered umbilical cord cells are inoculated into serum-free basal medium and are cultivated, and incubation time is
2 days；
（5）Secondary culture replaces serum free medium, and serum-free nutrient additive is added in serum free medium, periodically
Culture medium is replaced, until generating cell cluster；
（6）Activity determination takes part cell from different Place cell clusters respectively and is grouped label, and different groups of cell is distinguished
Carry out induction differentiation culture, the cell activity after detection differentiation, the poor cell cluster of knockout cell activity；
（7）Cell cluster grouping after preferably is stored in heat insulation tank by freezen protective, and the temperature of heat insulation tank should be 4 DEG C.
2. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：It is described
Centrifugation time of the enzymolysis liquid in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
3. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：It is described
It should be cut into small pieces to be put into heat insulation tank before umbilical cord enzymatic hydrolysis and refrigerate, the refrigeration duration is 2~6 months.
4. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 1, it is characterised in that：It is described
Heat insulation tank includes tank body（1）, the tank body（1）Inner wall be fixedly connected with insulating layer（2）, the insulating layer（2）Inner wall two
Side is fixedly connected to electric heating piece（3）, the electric heating piece（3）Inside be fixedly connected with inner canister（4）, the inner canister（4）It is interior
Portion offers water-bath pipeline（5）, the water-bath pipeline（5）One end run through inner canister（4）And extend to inner canister（4）Top,
The tank body（1）Top insulation cover is installed（6）, the insulation cover（6）Top one end offer and water-bath pipeline（5）Phase
The blasthole of adaptation（7）.
5. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 4, it is characterised in that：It is described
Tank body（1）Side offer automatically controlled slot（8）, the automatically controlled slot（8）Inner wall bottom be fixedly connected with power supply box（9）, described
Power supply box（9）Top be fixedly connected with controller（10）, the power supply box（9）, controller（10）And electric heating piece（3）Between lead to
Cross conducting wire electric connection, the automatically controlled slot（8）Two sides between be fixedly connected with baffle（11）, the baffle（11）Side open
Equipped with air hole（12）.
6. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 5, it is characterised in that：It is described
Tank body（1）Bottom end offer water outlet（13）, the water outlet（13）Through tank body（1）And inner canister（4）And with water
Bathe pipeline（5）Connection, the water outlet（13）Inside electrically-controlled valve is installed（14）, the electrically-controlled valve（14）Pass through conducting wire and control
Device processed（10）It is electrically connected.
7. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 4, it is characterised in that：It is described
Blasthole（7）Side offer seal groove（15）, the seal groove（15）Inner wall far from blasthole（7）Side pass through bullet
Spring（16）It is fixedly connected with tilted block（17）, the tilted block（17）With seal groove（15）Inner wall be slidably connected, the inclination
Block（17）Bottom be fixedly connected with sealing block（18）.
8. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 4, it is characterised in that：It is described
Insulation cover（6）Top be fixedly connected with handle（19）.
9. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 4, it is characterised in that：It is described
Inner canister（4）Inner wall between slidably connect bracket（20）.
10. the preparation and extracting method of a kind of umbilical cord mesenchymal stem cells according to claim 9, it is characterised in that：Institute
State bracket（20）Top both ends be fixedly connected to handle（21）.
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Cited By (1)
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| CN112546073A (en) * | 2020-12-21 | 2021-03-26 | 吉林省熙达生物科技有限公司 | Mesenchymal stem cell preparation for treating osteoarthritis and preparation method and application thereof |
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