CN109439623A - A method of preparing umbilical cord mesenchymal stem cells - Google Patents
A method of preparing umbilical cord mesenchymal stem cells Download PDFInfo
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- CN109439623A CN109439623A CN201811379568.3A CN201811379568A CN109439623A CN 109439623 A CN109439623 A CN 109439623A CN 201811379568 A CN201811379568 A CN 201811379568A CN 109439623 A CN109439623 A CN 109439623A
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Abstract
The present invention provides a kind of methods for preparing umbilical cord mesenchymal stem cells, comprising the following steps: S1, acquisition umbilical cord sample；S2, removal umbilical cord epithelial tissue, the magnificent Tong Shi glue of abundant exposed inner；S3, removal umbilical cord arteriovenous, produce magnificent Tong Shi glue；S4, the adherent processing of tissue, by magnificent Tong Shi sticker into Tissue Culture Dish；S5, CO is set2It is cultivated in incubator, obtains umbilical cord mesenchymal stem cells.With the above mentioned technical proposal, compared with prior art, the invention has the advantages that this method removes umbilical cord epithelium layer, sufficiently magnificent Tong Shi glue of the exposure rich in umbilical cord mesenchymal stem cells, no matter how to be pasted after being cut into small pieces, is all the fresh sticky magnificent Tong Shi glue surface of a wound, it is easy to operate, it is easily adherent, it is also beneficial to umbilical chord mesenchymal cells and climbs out of.
Description
Technical field
The present invention relates to cell biologies.
Specifically, being to be related to a kind of method for preparing umbilical cord mesenchymal stem cells.
Background technique
It is mesoblastic a kind of with height self-renewal capacity and more that umbilical cord mesenchymal stem cells are derived from mesoderm growing early stage
To the stem cell of differentiation potential, lipoblast, osteoblast, cardiac muscle cell, vascular endothelial cell and table can be broken up in vitro
Chrotoplast etc..Umbilical cord mesenchymal stem cells because materials are convenient, without advantages such as immunological rejections due to become the ideal seed of organizational project
Source.The mescenchymal stem cell form of in vitro culture is uniform, and it is anti-can to express a variety of surfaces for Parallel Growth or circinate growth
Original, such as high expression interstitial cell mark (CD44, CD90, CD29), does not express candidate stem cell mark (CD34, CD45) and people
Leukocyte antigen (HLA-DR) etc..
Currently, the separating and purifying technology of umbilical cord mesenchymal stem cells, which exists, is easily polluted by birth canal bacterium and separates for the first time time-consuming too
The problems such as long.Common umbilical cord transport liquid and cleaning solution are mostly to contain mycillin, the PBS buffer solution of the antibiotic such as gentamicin, by
More in the antibody-resistant bacterium of antibiotic, fungistatic effect is bad, under normal conditions can be antibacterial to improve by increasing the amount of antibiotic
Effect leads to abuse of antibiotics；Time-consuming for umbilical cord mesenchymal stem cells stationary culture separating primary cells, and tissue block is easily de-
It falls, it is thus proposed that with removal vascular wall face paste wall approach, operability is poor, and digestion method is with adherent method after first digesting, cell purity
It is low, and more or less have certain damage to cell.
Summary of the invention
It is an object of the invention to overcome the shortcoming of above-mentioned traditional technology, it is dry thin to provide a kind of umbilical cord mesenchyma for preparing
The method of born of the same parents, the umbilical cord that this method provides transport in liquid and cleaning solution and are added to antibacterial peptide, gymnema sylvestre and trehalose, and three is
It is again useful and harmless to human body to the pollution of cell to can effectively reduce bacterium and fungi for natural materials.In addition, umbilical cord epithelium is thin
Born of the same parents' layer removal, can sufficiently expose the magnificent Tong Shi glue for being rich in mescenchymal stem cell, tissue block is easier to adherent, and umbilical chord mesenchymal cells are climbed
Extracting rate is higher.
The purpose of the present invention is what is reached by following technical measures:
A method of preparing umbilical cord mesenchymal stem cells, comprising the following steps:
S1, acquisition umbilical cord sample；
S2, removal umbilical cord epithelial tissue, the magnificent Tong Shi glue of abundant exposed inner；
S3, removal umbilical cord arteriovenous, produce magnificent Tong Shi glue；
S4, the adherent processing of tissue, by magnificent Tong Shi sticker into Tissue Culture Dish；
S5, CO is set2It is cultivated in incubator, obtains umbilical cord mesenchymal stem cells.
As an improvement: in step S1, the umbilical cord sample of acquisition is placed in transport liquid, and the transport liquid includes with the following group
Point:
Antibacterial peptide 5-20ug/ml；
Gymnema sylvestre 50-100ug/ml；
Trehalose 0.5-10mg/ml；
Solvent is the PBS of PH6.8-7.6.
Antibacterial peptide is a kind of basic polypeptide substance with antibacterial activity, has thermal stability, broad spectrum antibacterial, to bacterium
Have very strong lethal effect, to common drug resistance pathogen have same inhibiting effect, to fractionated viral, fungi, protozoon and
Cancer cell etc. also has killing effect, not only harmless, or even can improve immunity, accelerating wound healing process.
Gymnema sylvestre is a kind of green plants, wherein the gymnemic acid contained can effectively inhibit birth canal common bacteria Candida albicans
The mycelia of bacterium grows and its toxicity, can equally prevent the growth of the fungies such as Eurotium, and to staphylococcus aureus, change
The various bacterias such as shape bacillus and Escherichia coli also have obvious inhibitory effect.
Trehalose has magical protective effect to organism, severe in high temperature, high and cold, hyperosmosis and dry dehydration etc.
Unique protective film can be formed in cell surface under environmental condition, is effectively protected protein molecule invariance inactivation.Fortune is added
Unique protective film can be formed in cell surface in infusion and washing lotion, reduce the injury in transport and washing process to cell.
It is added to three of the above ingredient in umbilical cord transport liquid and cleaning solution, that is, can effectively reduce bacterium and fungi to cell
Pollution reduces the injury to cell, and can prevent abuse of antibiotics, can also be stored at room temperature, and useful and harmless to human body.
As an improvement: in step S1, before the umbilical cord sample of acquisition is placed in transport liquid, first the bleeding of the umbilicus in umbilical cord is arranged
Only, then by the sterile ligation in umbilical cord both ends.
As an improvement: in step S2, the method for removal umbilical cord epithelium is that umbilical cord is taken out in super-clean bench, with washing
Liquid cleaning, is put into equipped in 0.05%-2% Type I collagen enzyme and 0.01%-0.25% trypsase mixture slaking liquid, 37 DEG C of digestion 5-
30min takes out umbilical cord, is rinsed with PBS, then removes the epithelial tissue on umbilical cord surface, and PBS is cleaned.
As an improvement: in step S2, the umbilical cord after digestion process is immersed in during removal umbilical cord epithelial tissue
In sterile PBS, one layer of epithelial tissue of huatong plastic surface covering is struck off with steril cell scraping blade.
As an improvement: in step S3, the product of S2 is made to the segment of 1-2cm, then washed with PBS, removes umbilical cord
In two radicular arteries, umbilical cord is cut off along vein, scrapes off venous blood tube wall.
As an improvement: in step S3, before the product of S2 is cut into the segment of 1-2cm, first by umbilical cord tissue oedema, fill
It is removed at blood, breakage and ligation.
As an improvement: it is before magnificent Tong Shi sticker is into Tissue Culture Dish, magnificent Tong Shi glue is abundant with PBS in step S4
Washing, is then made 1-2mm3Size tissue block.
As an improvement: in step S4, Tissue Culture Dish is first infiltrated with culture solution, then tissue block is uniformly attached to
In culture dish, it is spaced 4-6mm, stands 5-30min, every piece of tissue plus a drop culture medium.
As an improvement: in step S5, set CO2In incubator, saturated humidity, 5%CO2, 37 DEG C are cultivated, every after 3-16h
Ware fluid infusion 5-8ml, the 3-5 days every ware fluid infusion 5-10ml, half amount changes liquid, the 6th day beginning microscopically observation cell within the 6-10 days
Clone situation need to abandon tissue block if cellular morphology is uniform and quantity meets predetermined value and change liquid entirely, if cell quantity do not meet it is predetermined
Value, need to continue half amount and change liquid to cell quantity to meet predetermined value, can abandon tissue block and change liquid entirely, change the liquid once within hereafter every 2-3 days,
Reach certain density harvest cell.
By adopting the above-described technical solution, compared with prior art, the invention has the advantages that
1, it is added to antibacterial peptide in the umbilical cord transport liquid and cleaning solution provided, antibacterial peptide has broad spectrum antibiotic activity, has to bacterium
Very strong lethal effect also has same inhibiting effect to common drug resistance pathogen, can room temperature storage, and it is nontoxic to the human body
Harmless, the antibiotic such as alternative mycillin, effectively inhibition bacterium reduce abuse of antibiotics.
2, it is added to gymnema sylvestre in umbilical cord transport liquid and cleaning solution, it is normal that the birth canals such as Candida albicans can be effectively suppressed in gymnema sylvestre
See fungi, the contamination probability of fungi in umbilical cord transport and preparation process is effectively reduced, and useful and harmless to human body and cell.
3, it is added to trehalose in the umbilical cord transport liquid and cleaning solution provided, trehalose can be formed in albumen and cell surface
Unique protective film reduces the injury in umbilical cord transport and washing process to cell, and useful and harmless to human body and cell.
4, this method removes umbilical cord epithelium layer, sufficiently magnificent Tong Shi glue of the exposure rich in umbilical cord mesenchymal stem cells,
No matter how to be pasted after being cut into small pieces, is all the fresh sticky magnificent Tong Shi glue surface of a wound, it is easy to operate, it is easily adherent, it is also beneficial to umbilical cord
Mesenchymal cell climbs out of.
Specific embodiment
Embodiment 1: a method of preparing umbilical cord mesenchymal stem cells, comprising the following steps:
S1, acquisition umbilical cord sample, empty bleeding of the umbilicus, and by the sterile ligation in umbilical cord both ends, the umbilical cord sample of acquisition is placed in transport liquid, will
Transport bottle equipped with umbilical cord, which is placed in 4 DEG C of constant temperature transport cases, is transported to laboratory, separates isolated time about 3h before stem cell.
The transport liquid includes following components:
Antibacterial peptide 5ug/ml；
Gymnema sylvestre 50ug/ml；
Trehalose 0.5mg/ml；
Solvent is the PBS solution of PH6.8.
S2, removal umbilical cord epithelial tissue: umbilical cord is taken out in super-clean bench, is cleaned in sterilized petri dishes with cleaning solution, is put into
Equipped in 0.05% Type I collagen enzyme and 0.01% trypsase mixture slaking liquid, 37 DEG C of digestion 30min take out umbilical cord, and use is sterile
Then PBS buffer solution repeated flushing strikes off the epithelial tissue on umbilical cord surface, the magnificent Tong Shi of abundant exposed inner with cell scapes
Umbilical cord tissue is immersed in PBS by glue, removal umbilical cord epithelial tissue in the process, is finally rinsed epithelial tissue with sterile PBS dry
Only.
S3, removal umbilical cord arteriovenous: by the ligation of umbilical cord tissue both ends, oedema, hyperemia, breakage remove, be then made
The segment of 1cm, then washed 2 times with PBS, with two radicular arteries in ophthalmic tweezers removal umbilical cord, with eye scissors along vein by umbilical cord
It cuts off, scrapes off venous blood tube wall with ophthalmic tweezers, produce magnificent Tong Shi glue.
S4, the adherent processing of tissue: using mescenchymal stem cell culture medium infiltrating cells culture dish, by magnificent Tong Shi glue with sterile
PBS washing, is then cut into 1-2mm3Magnificent Tong Shi glue tissue block is uniformly attached to Tissue Culture Dish with ophthalmic tweezers by size tissue block
In, tissue block and tissue block gap 4mm stand 30min, every piece of 1 drop culture medium of tissue drop.
S5, later period culture, harvest cell: setting CO2It is cultivated in incubator, saturated humidity, 5%CO2, 37 DEG C, every ware is mended after 3h
Liquid 5ml, the 3rd day every ware fluid infusion 10ml, observation determination is pollution-free, and the 7th day half amount changes liquid, and first observed is to there is cell to climb out of, and the
10 days microscopically observation cell clone situations averagely have 8.8 clones, the tissues of 1.8 pieces of floatings in each Tissue Culture Dish
Block, cellular morphology is uniform and clone is larger, abandons tissue block and changes liquid entirely, and the 13rd day harvest cell counts, and average every ware obtains P0
For cell 5.3 × 106 It is a.
Embodiment 2: a method of preparing umbilical cord mesenchymal stem cells, comprising the following steps:
S1, acquisition umbilical cord sample, empty bleeding of the umbilicus, and by the sterile ligation in umbilical cord both ends, the umbilical cord sample of acquisition is placed in transport liquid, will
Transport bottle equipped with umbilical cord, which is placed in 4 DEG C of constant temperature transport cases, is transported to laboratory, separates isolated time about 2h before stem cell.
The transport liquid includes following components:
Antibacterial peptide 10ug/ml；
Gymnema sylvestre 80ug/ml；
Trehalose 5mg/ml；
Solvent is the PBS solution of PH7.2.
S2, removal umbilical cord epithelial tissue: umbilical cord is taken out in super-clean bench, is cleaned in sterilized petri dishes with cleaning solution, is put into
Equipped in 1% Type I collagen enzyme and 0.1% trypsase mixture slaking liquid, 37 DEG C of digestion 15min take out umbilical cord, slow with sterile PBS
Then fliud flushing repeated flushing strikes off the epithelial tissue on umbilical cord surface, the magnificent Tong Shi glue of abundant exposed inner, removal with cell scapes
Umbilical cord tissue is immersed in PBS during umbilical cord epithelial tissue, is finally rinsed well epithelial tissue with sterile PBS.
S3, removal umbilical cord arteriovenous: by the ligation of umbilical cord tissue both ends, oedema, hyperemia, breakage remove, be then cut into
The segment of 1.5cm or so, then washed 2 times with PBS, two radicular arteries in umbilical cord are removed with ophthalmic tweezers, with eye scissors along vein
Umbilical cord is cut off, scrapes off venous blood tube wall with ophthalmic tweezers, produces magnificent Tong Shi glue.
S4, the adherent processing of tissue: using mescenchymal stem cell culture medium infiltrating cells culture dish, by magnificent Tong Shi glue with sterile
PBS washing, is then cut into 1-2mm3Magnificent Tong Shi glue tissue block is uniformly attached to Tissue Culture Dish with ophthalmic tweezers by size tissue block
In, tissue block and tissue block gap 5mm stand 15min, every piece of 1 drop culture medium of tissue drop.
S5, later period culture, harvest cell: setting CO2It is cultivated in incubator, saturated humidity, 5%CO2, 37 DEG C, every ware is mended after 6h
Liquid 6ml, the 4th day every ware fluid infusion 8ml, observation determination is pollution-free, and the 6th day half amount changes liquid, and first observed is to there is cell to climb out of, and the 9th
Its microscopic observation cell clone situation averagely has 9.7 clones, the tissue blocks of 1.5 pieces of floatings, cell in each Tissue Culture Dish
Form is uniform and clone is larger, abandons tissue block and changes liquid entirely, and the 12nd day harvest cell counts, and average every ware obtains P0For cell 6.8
×106 It is a.
Embodiment 3: a method of preparing umbilical cord mesenchymal stem cells, comprising the following steps:
S1, acquisition umbilical cord sample, empty bleeding of the umbilicus, and by the sterile ligation in umbilical cord both ends, the umbilical cord sample of acquisition is placed in transport liquid, will
Transport bottle equipped with umbilical cord, which is placed in 4 DEG C of constant temperature transport cases, is transported to laboratory, separates isolated time about 4h before stem cell.
The transport liquid includes following components:
Antibacterial peptide 20ug/ml；
Gymnema sylvestre 100ug/ml；
Trehalose 10mg/ml；
Solvent is the PBS solution of PH7.6.
S2, removal umbilical cord epithelial tissue: umbilical cord is taken out in super-clean bench, is cleaned in sterilized petri dishes with cleaning solution, is put into
Equipped in 2% Type I collagen enzyme and 0.25% trypsase mixture slaking liquid, 37 DEG C of digestion 5min take out umbilical cord, slow with sterile PBS
Then fliud flushing repeated flushing strikes off the epithelial tissue on umbilical cord surface, the magnificent Tong Shi glue of abundant exposed inner, removal with cell scapes
Umbilical cord tissue is immersed in PBS during umbilical cord epithelial tissue, is finally rinsed well epithelial tissue with sterile PBS.
S3, removal umbilical cord arteriovenous: by the ligation of umbilical cord tissue both ends, oedema, hyperemia, breakage remove, be then made
The segment of 2cm, then washed 2 times with PBS, with two radicular arteries in ophthalmic tweezers removal umbilical cord, with eye scissors along vein by umbilical cord
It cuts off, scrapes off venous blood tube wall with ophthalmic tweezers, produce magnificent Tong Shi glue.
S4, the adherent processing of tissue: using mescenchymal stem cell culture medium infiltrating cells culture dish, by magnificent Tong Shi glue with sterile
PBS washing, is then cut into 1-2mm3Magnificent Tong Shi glue tissue block is uniformly attached to Tissue Culture Dish with ophthalmic tweezers by size tissue block
In, tissue block and tissue block gap 5mm stand 5min, every piece of 1 drop culture medium of tissue drop.
S5, later period culture, harvest cell: setting CO2It is cultivated in incubator, saturated humidity, 5%CO2, 37 DEG C, every ware is mended after 16h
Liquid 8ml, the 5th day every ware fluid infusion 6ml, observation determination is pollution-free, and the 8th day half amount changes liquid, and first observed is to there is cell to climb out of, and the 11st
Its microscopic observation cell clone situation averagely has 7.3 clones, the tissue blocks of 1.3 pieces of floatings, cell in each Tissue Culture Dish
Form is uniform and clone is larger, abandons tissue block and changes liquid entirely, and the 14th day harvest cell counts, and average every ware obtains P0For cell 4.6
×106 It is a.
Embodiment 4: the method that traditional tissue block adherent method prepares umbilical cord mesenchymal stem cells, comprising the following steps:
S1, acquisition umbilical cord sample: by the sterile ligation in umbilical cord both ends, the umbilical cord sample of acquisition is placed in transport liquid, and umbilical cord will be housed
Transport bottle be placed in 4 DEG C of constant temperature transport cases and be transported to laboratory, separate isolated time about 4.5h before stem cell, the transport liquid packet
Containing dual anti-PBS.
S2, removal arteriovenous: umbilical cord is taken out in super-clean bench, is cleaned in sterilized petri dishes with cleaning solution, 2cm is then cut into
Segment, then washed 4 times with PBS, with two radicular arteries in ophthalmic tweezers removal umbilical cord, with eye scissors along vein by umbilical cord scissors
It opens, scrapes off venous blood tube wall with ophthalmic tweezers.
S3, the adherent processing of tissue: tissue block is washed with sterile PBS, is then cut into 1-2mm3Size tissue block, uses ophthalmology
Tissue block is uniformly attached in Tissue Culture Dish by tweezer, is spaced 5mm, every piece of 1 drop culture medium of tissue drop.
S4, later period culture, harvest cell: setting CO2It is cultivated in incubator, saturated humidity, 5%CO2, 37 DEG C, every ware after 12h
Fluid infusion 8ml, the 4th day every ware fluid infusion 6ml, observation determination is pollution-free, and the 8th day half amount changes liquid, and first observed is to there is cell to climb out of, and the
12 days microscopically observation cell clone situations averagely have 5.3 clones, the tissues of 4.3 pieces of floatings in each Tissue Culture Dish
Block, cellular morphology is uniform and clone is larger, abandons tissue block and changes liquid entirely, and the 16th day harvest cell counts, and average every ware obtains P0Generation
Cell 1.8 × 106 It is a.
Embodiment 5: the method that adherent method prepares umbilical cord mesenchymal stem cells after tradition first digests, comprising the following steps:
S1, acquisition umbilical cord sample: by the sterile ligation in umbilical cord both ends, the umbilical cord sample of acquisition is placed in transport liquid, and umbilical cord will be housed
Transport bottle be placed in 4 DEG C of constant temperature transport cases and be transported to laboratory, separate isolated time about 3h before stem cell, the transport liquid includes
Dual anti-PBS.
S2, removal umbilical cord arteriovenous: umbilical cord is taken out in super-clean bench, is cleaned in sterilized petri dishes with cleaning solution, is then cut
It is washed 4 times at the segment of 2cm, then with PBS, with two radicular arteries in ophthalmic tweezers removal umbilical cord, with eye scissors along vein by navel
Band is cut off, and scrapes off venous blood tube wall with ophthalmic tweezers.
S3, tissue digestion processing: tissue block is washed with sterile PBS, is then cut into 2-3mm3Size tissue block is put into dress
Have in 1 % Type I collagen enzyme and 1% neutral proteinase mixture slaking liquid, 37 DEG C of digestion 60min, take out umbilical cord tissue block, use is sterile
PBS buffer solution repeated flushing,
Postdigestive tissue block: being uniformly attached in Tissue Culture Dish by S4, tissue block adherent with ophthalmic tweezers, is spaced 5mm, every piece of group
Knit 1 drop culture medium of drop.
S5, later period culture, harvest cell: setting CO2It is cultivated in incubator, saturated humidity, 5%CO2, 37 DEG C, every ware after 12h
Fluid infusion 8ml, the 4th day every ware fluid infusion 6ml, observation determination is pollution-free, and the 8th day half amount changes liquid, and the 10th day first observed is to there is cell
It climbs out of, the 14th day microscopic observation cell clone situation, there is 7 clones, the tissue of 1.3 pieces of floatings in average each Tissue Culture Dish
Block, cellular morphology is uniform and clone is larger, abandons tissue block and changes liquid entirely, and the 17th day harvest cell counts, and average every ware obtains P0Generation
Cell 4.1 × 106 It is a.
Comparison between four case study on implementation results:
As can be seen from the above table, the method provided by the invention for preparing umbilical cord mesenchymal stem cells, tissue block is easily adherent, and cell is climbed
The time is more early out, and obtained number of cell clones is more, and the primary cell amount of harvest is larger.
Several embodiments of the invention are described in detail above, but the content is only preferable implementation of the invention
Example, should not be considered as limiting the scope of the invention.It is all according to all the changes and improvements made by the present patent application range
Deng should all fall within the scope of the patent of the present invention.
Claims (10)
1. a kind of method for preparing umbilical cord mesenchymal stem cells, which comprises the following steps:
S1, acquisition umbilical cord sample；
S2, removal umbilical cord epithelial tissue, the magnificent Tong Shi glue of abundant exposed inner；
S3, removal umbilical cord arteriovenous, produce magnificent Tong Shi glue；
S4, the adherent processing of tissue, huatong plastic is attached in Tissue Culture Dish；
S5, CO is set2It is cultivated in incubator, obtains umbilical cord mesenchymal stem cells.
2. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S1, acquisition
Umbilical cord sample be placed in transport liquid in, the transport liquid includes following components:
Antibacterial peptide 5-20ug/ml；
Gymnema sylvestre 50-100ug/ml；
Trehalose 0.5-10mg/ml；
Solvent is the PBS of PH6.8-7.6.
3. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S1, acquisition
Umbilical cord sample be placed in transport liquid before, first by umbilical cord bleeding of the umbilicus empty, then by the sterile ligation in umbilical cord both ends.
4. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S2, removal
The method of umbilical cord epithelium is that umbilical cord is taken out in super-clean bench, is cleaned with cleaning solution, be put into equipped with 0.05%-2% Type I collagen enzyme and
In 0.01%-0.25% trypsase mixture slaking liquid, 37 DEG C of digestion 5-30min take out umbilical cord, are rinsed with PBS, then remove navel
The epithelial tissue of belt surface, PBS are cleaned.
5. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S2, removal
The umbilical cord after digestion process is immersed in sterile PBS during umbilical cord epithelial tissue, strikes off huatong plastic with steril cell scraping blade
One layer of epithelial tissue of surface covering.
6. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S3, by S2
Product be cut into the segment of 1-2cm, then washed with PBS, remove two radicular arteries in umbilical cord, umbilical cord is cut off along vein, is scraped
Remove venous blood tube wall.
7. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S3, by S2
Product the segment of 1-2cm is made before, first will at umbilical cord tissue oedema, hyperemia, breakage and ligation remove.
8. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S4, Hua Tong
Before family name's sticker is into Tissue Culture Dish, magnificent Tong Shi glue is sufficiently washed with PBS, is then cut into 1-2mm3Size tissue block.
9. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S4, cell
Culture dish is first infiltrated with culture solution, then tissue block is uniformly attached in culture dish, is spaced 4-6mm, stands 5-30min, often
Block tissue plus a drop culture medium.
10. the method according to claim 1 for preparing umbilical cord mesenchymal stem cells, it is characterised in that: in step S5, set CO2
In incubator, saturated humidity, 5%CO2, every ware fluid infusion 5-8ml, the 3-5 days every ware fluid infusion 5-10ml, the after 37 DEG C of culture 3-16h
6-10 days half amounts change liquid, the 6th day beginning microscopically observation cell clone situation, if cellular morphology is uniform and quantity meet it is predetermined
Value, tissue block need to be abandoned and change liquid entirely, if cell quantity does not meet predetermined value, need to continue half amount change liquid to cell quantity meet it is predetermined
Value, can abandon tissue block and change liquid entirely, change the liquid once within hereafter every 2-3 days, reach certain density harvest cell.
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| CN109234226A (en) * | 2018-10-15 | 2019-01-18 | 潍坊市康华生物技术有限公司 | Stem cell terminates liquid and preparation method thereof and stem cell propagating method with trypsase |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234226A (en) * | 2018-10-15 | 2019-01-18 | 潍坊市康华生物技术有限公司 | Stem cell terminates liquid and preparation method thereof and stem cell propagating method with trypsase |
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