CN106939296A - A kind of isolated culture method of dermal mesenchymal stem cells - Google Patents

A kind of isolated culture method of dermal mesenchymal stem cells Download PDF

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CN106939296A
CN106939296A CN201710114065.2A CN201710114065A CN106939296A CN 106939296 A CN106939296 A CN 106939296A CN 201710114065 A CN201710114065 A CN 201710114065A CN 106939296 A CN106939296 A CN 106939296A
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culture method
corium
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stem cells
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CN106939296B (en
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贾延辉
张曦
杨世杰
邓怡
王筱琪
王瑞
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Third Military Medical University TMMU
Second Affiliated Hospital of TMMU
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/0668—Mesenchymal stem cells from other natural sources
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Abstract

The present invention relates to technical field of cell culture, more particularly to a kind of isolated culture method of dermal mesenchymal stem cells.The isolated culture method comprises the following steps：Skin histology is pre-processed, digested using neutral proteinase II, digestion product 1 is obtained；The digestion product 1, separation epidermis and corium are taken, the corium is processed into fritter, digested using clostridiopetidase A IV, digestion product 2 is obtained；Indigested tissue and digestive juice in the digestion product 2 are removed, gained cell is added in culture medium and carries out cell culture.Damage of the isolated culture method of the present invention to dermal mesenchymal stem cells is small, and the dermal mesenchymal stem cells motility rate and purity of acquisition are higher.

Description

A kind of isolated culture method of dermal mesenchymal stem cells
Technical field
The present invention relates to technical field of cell culture, more particularly to a kind of side of being separately cultured of dermal mesenchymal stem cells Method.
Background technology
Skin is organ maximum on vertebrate body, and it has many functions, including body heat regulation, conduction tactile, Protect the body from the invasion and attack and injury in the external world.Skin mainly has two layers, epidermis and corium.Epidermis is made up of keratinocyte Multilayer scaly epithelium, corium is by two layers of mastoid process layer and lamina reticularis.Corium can not regenerate, and be repaired after defect by fibr tissue, this Many functions of skin, the individual quality of life of influence will be lost.The research of skin tissue engineering carries for the treatment of such patient Supply to wish.
Skin tissue engineering has 3 important factors：Seed cell, timbering material, and both combinations.Mesenchyma is done Cell is used as seed cell, the effect with hematopoiesis support and immunological regulation, the incidence and the death rate that reduce GVHD, Ke Yiti The success rate of high HSCT.There are many scholars to grind the seed cell of skin tissue engineering both at home and abroad Study carefully.Research more predominantly epidermal stem cells and mesenchymal stem cells MSCs.From 2000, Canadian Studies person Toma etc. from Dermal mesenchymal stem cells (DMSCs) are isolated in childhood and adult rat skin, this cell division multiplication capacity is strong, has Many differentiation potentials, are a kind of newfound multipotential stem cells from corium, have just started the research boom to DMSCs.
In recent years, domestic and foreign scholars are separated to DMSCs, purified, the research of amplification in vitro has carried out continuous exploration.Such as poplar What the sub- winter was reported in 2008《Rat dermal mescenchymal stem cell is separately cultured》In to disclose a kind of corium mesenchyma dry thin Born of the same parents' is separately cultured amplification method, with 0.25% trypsinization epidermis and corium, then with 0.1% I type glue Protoenzyme digestion obtains single cell suspension, goes supernatant to add appropriate L-DMEM complete mediums after centrifugation, is put into 24 orifice plates, is placed in 37 DEG C, 5%CO2Incubator culture.
Disclosure/notification number provides a kind of separation method of corium stem cell for CN102498205A patent of invention, by group Knit piece to be dispersed in the DMEM culture mediums containing 0.1% trypsase and 0.2% clostridiopetidase A, digest skin histology, centrifuge Afterwards, cell is resuspended in DMEM culture mediums.
Disclosure/notification number discloses a kind of separation method of human dermis stem cell for CN104403989A patent of invention, This method comprises the following steps：(1) skin histology of in vitro people is handled with Dispase II；(2) by dermal partial and Skin portion is torn, and carries out digestion process with type i collagen enzyme working solution；(3) blown repeatedly at digestion with type i collagen enzyme working solution Tissue fluid after reason, filtering；(4) filtrate is centrifuged, precipitation and supernatant is taken respectively；(5) will be through heavy obtained by step (4) Forming sediment, it is resuspended to carry out, and obtains tissue fluid 1；(6) it will be centrifuged through the supernatant obtained by step (4), abandon supernatant, take precipitation, to institute State precipitation progress resuspended, obtain tissue fluid 2；(7) tissue fluid 1 and tissue fluid 2 are mixed, obtained tissue fluid 3；(8) to tissue Cell in liquid 3 is screened, and obtains human dermis stem cell.
But the separation method of existing dermal cell is larger to cellular damage in separation process.Therefore, it is badly in need of providing one Plant to the less dermal mesenchymal stem cells separation method of cellular damage.
The content of the invention
In view of this, the invention provides a kind of isolated culture method of dermal mesenchymal stem cells.What this method was obtained The purity and motility rate of dermal mesenchymal stem cells are higher.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of isolated culture method of dermal mesenchymal stem cells, it is characterised in that including following step Suddenly：
Skin histology is pre-processed, digested using neutral proteinase II, digestion product 1 is obtained；
The digestion product 1, separation epidermis and corium are taken, the corium is processed into fritter, is carried out using clostridiopetidase A IV Digestion, obtains digestion product 2；
Indigested tissue and digestive juice in the digestion product 2 are removed, gained cell is added in culture medium and carried out carefully Born of the same parents cultivate.
Preferably, in terms of g/mL, neutral proteinase II digestion concentration is 0.2%~0.3%.
Preferably, in terms of g/mL, neutral proteinase II digestion concentration is 0.25%.
Preferably, clostridiopetidase A IV mass percent concentration is 0.05%~0.15%.
Preferably, clostridiopetidase A IV mass percent concentration is 0.1%.
Preferably, using the temperature that neutral proteinase II is digested for 0~10 DEG C, the time is 10~20h.
Preferably, use the temperature that neutral proteinase II is digested for 4 DEG C, the time is 12h.
Preferably, using the temperature that clostridiopetidase A IV is digested for 35~38 DEG C, CO2Concentration is 5%, the time be 1~ 3h。
Preferably, the temperature that clostridiopetidase A IV is digested is used for 37 DEG C, CO2Concentration is 5%, and the time is 2h.
Preferably, during being digested using clostridiopetidase A IV, every 10~12min concussions once.
In the embodiment that the present invention is provided, during being digested using clostridiopetidase A IV, one is shaken every 15min It is secondary.
Preferably, culture medium is the DMEM high glucose mediums containing 10% (mass fraction) FBS and 5 μ g/500mL BFGF.
Preferably, the condition of cell culture is 5%CO2、37℃。
Preferably, gained cell is added in culture medium after cell culture also include：Liquid is changed after cell attachment, Cell fusion degree reach 70% after Secondary Culture.
Preferably, corium be processed into fritter being：Corium is cut into (0.05~0.15) cm × (0.05~0.15) cm Fritter.
In the embodiment that the present invention is provided, corium is processed into fritter is：Corium is cut into the small of 0.1cm × 0.1cm Block.
Preferably, being by skin histology progress pretreatment：Skin histology is cleaned, fat and connective group is removed Knit, be processed into (0.45~0.55) cm × (0.15~0.25) cm tissue block.
In the embodiment that the present invention is provided, cleaning is using added with dual anti-PBS solution.
The invention provides a kind of isolated culture method of dermal mesenchymal stem cells.The isolated culture method includes as follows Step：Skin histology is pre-processed, digested using neutral proteinase II, digestion product 1 is obtained；Take the digestion production Thing 1, separation epidermis and corium, are processed into fritter by the corium, are digested using clostridiopetidase A IV, obtain digestion product 2；Go Except indigested tissue and digestive juice in the digestion product 2, gained cell is added in culture medium and carries out cell culture.This hair It is bright one of at least to have the advantages that：
1st, damage of the isolated culture method of the present invention to dermal mesenchymal stem cells is small, the dermal mesenchymal stem cells of acquisition Motility rate is up to 95.3%；
2nd, the purity for the dermal mesenchymal stem cells that isolated culture method of the present invention is obtained is high, up to 94%；
3rd, in isolated culture method of the present invention, dermal mesenchymal stem cells derive from dermal tissue, skin histology materials side Just, it is easy to obtain；
4th, isolated culture method step of the present invention is simple, is easy to operation.
Brief description of the drawings
Fig. 1 shows the cellular morphology figure of the isolated dermal mesenchymal stem cells of embodiment 1；Wherein, 1-1,1-3 are respectively The cellular morphology of culture 5d after 100 times, 200 times of amplification, 1-2,1-4 are respectively to amplify the culture the after 100 times, 200 times 14d cellular morphology；
Fig. 2 shows the streaming qualification figure of the isolated dermal mesenchymal stem cells of embodiment 1；2-1~2-4 shows respectively CD14/CD20/CD3/CD45, CD90, CD105, CD73 expression.
Embodiment
The invention discloses a kind of isolated culture method of dermal mesenchymal stem cells, those skilled in the art can use for reference Present disclosure, is suitably modified technological parameter realization.In particular, all similar replacements and change are to this area skill It is it will be apparent that they are considered as being included in the present invention for art personnel.The method of the present invention and application by compared with Good embodiment is described, and related personnel substantially can be not departing from present invention, in spirit and scope to as described herein Methods and applications are modified or suitably change is with combining, to realize and apply the technology of the present invention.
The invention provides a kind of isolated culture method of dermal mesenchymal stem cells, it is characterised in that including following step Suddenly：
Skin histology is pre-processed, digested using neutral proteinase II, digestion product 1 is obtained；
The digestion product 1, separation epidermis and corium are taken, the corium is processed into fritter, is carried out using clostridiopetidase A IV Digestion, obtains digestion product 2；
Indigested tissue and digestive juice in the digestion product 2 are removed, gained cell is added in culture medium and carried out carefully Born of the same parents cultivate.
In the specific embodiment that the present invention is provided, the isolated culture method of dermal mesenchymal stem cells includes following step Suddenly：
1st, fresh prepuce tissues are cleaned with containing dual anti-PBS solution, cut off subepidermal fat and connective tissue.So Prepuce tissues are cut to 0.5 × 0.2cm strip afterwards, culture dish is put into, and it is (neutral to add 0.2%~0.3%dispaseII Proteinase II) solution, it is put into 4 DEG C of digested overnights of refrigerator.
2nd, the tissue of digested overnight, separation epidermis and corium are taken out.The corium of separation is cut into 0.1 × 0.1cm fritter, It is put into culture dish, adds 0.05%~0.15%collagenaseIV (clostridiopetidase A IV) digestive juice, culture dish is put into CO2Incubate 37 DEG C of digestion 2h of case, every 15min concussions once.
3rd, the dermal tissue of digestion is taken out, the filtering of 100 mesh cell sieves removes indigested tissue, collects digestive juice, adds DMEM high glucose mediums, centrifuge 1000rpm, 5min.
4th, centrifuge tube is taken out, supernatant is abandoned, DMEM high glucose medium re-suspended cells are added.
5th, DMEM high glucose mediums are added in blake bottle, and add cell.Blake bottle is put into CO2Incubator, 37 DEG C, 5% CO2Under the conditions of cultivate.
6th, change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
Material therefor and reagent can be by markets in the isolated culture method for the dermal mesenchymal stem cells that the present invention is provided Buy.Wherein, digestive ferment is purchased from sigma companies.
With reference to embodiment, the present invention is expanded on further：
The dermal mesenchymal stem cells of embodiment 1 (DMSCs) are separately cultured
1st, dual anti-addition PBS solution, fresh people's prepuce tissues are cleaned three times with PBS solution, cut off with eye scissors Subepidermal fat and connective tissue.Then prepuce tissues are cut to 0.5 × 0.2cm strip, 100mm culture dish is put into, And 10mL 0.25%dispaseII (neutral proteinase II) solution is added, it is put into 4 DEG C of digested overnights of refrigerator.
2nd, the tissue of digested overnight is taken out, epidermis and corium are separated with ophthalmic tweezers.The corium of separation is cut into eye scissors 0.1 × 0.1cm fritter, is put into 35mm culture dish, adds 3mL 0.1%collagenaseIV (clostridiopetidase A IV) digestion Liquid, culture dish is put into CO237 DEG C of digestion 2h of incubator, every 15min concussions once.
3rd, the dermal tissue of digestion is taken out, the filtering of 100 mesh cell sieves removes indigested tissue, collects digestive juice, adds Isometric DMEM high glucose mediums.Centrifuge 1000rpm, 5min.
4th, centrifuge tube is taken out, supernatant is abandoned, 1mL DMEM high glucose medium re-suspended cells are added.
5th, 5mL DMEM high glucose mediums are added in T25 blake bottles, and add cell.Blake bottle is put into CO2Incubator, 37 DEG C, 5%CO2Under the conditions of cultivate.
6th, change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
The P0 of above-mentioned acquisition is shown in Fig. 1 for cellular morphology figure.The streaming qualification figure of above-mentioned cell is shown in Fig. 2.As a result show, obtain Cell be dermal mesenchymal stem cells.
The dermal mesenchymal stem cells of embodiment 2 (DMSCs) are separately cultured
1st, dual anti-addition PBS solution, fresh prepuce tissues are cleaned three times with PBS solution, cut off very with eye scissors Subcutaneous fat and connective tissue.Then prepuce tissues are cut to 0.5 × 0.2cm strip, 100mm culture dish is put into, and 10mL 0.20%dispaseII (neutral proteinase II) solution is added, 4 DEG C of digested overnights of refrigerator are put into.
2nd, the tissue of digested overnight is taken out, epidermis and corium are separated with ophthalmic tweezers.The corium of separation is cut into eye scissors 0.1 × 0.1cm fritter, is put into 35mm culture dish, and the 0.15%collagenaseIV (clostridiopetidase A IV) for adding 3mL disappears Change liquid, culture dish is put into CO237 DEG C of digestion 2h of incubator, every 15min concussions once.
3rd, the dermal tissue of digestion is taken out, the filtering of 100 mesh cell sieves removes indigested tissue, collects digestive juice, adds Isometric DMEM high glucose mediums.Centrifuge 1000rpm, 5min.
4th, centrifuge tube is taken out, supernatant is abandoned, 1mL DMEM high glucose medium re-suspended cells are added.
5th, 5mL DMEM high glucose mediums are added in T25 blake bottles, and add cell.Blake bottle is put into CO2Incubator, 37 DEG C, 5%CO2Under the conditions of cultivate.
6th, change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
The cellular morphology that the embodiment is obtained is close with embodiment 1 with streaming qualification result, shows that the cell obtained is true Skin mescenchymal stem cell.
The dermal mesenchymal stem cells of embodiment 3 (DMSCs) are separately cultured
1st, dual anti-addition PBS solution, fresh prepuce tissues are cleaned three times with PBS solution, cut off very with eye scissors Subcutaneous fat and connective tissue.Then prepuce tissues are cut to 0.5 × 0.2cm strip, 100mm culture dish is put into, and 10mL 0.30%dispaseII (neutral proteinase II) solution is added, 4 DEG C of digested overnights of refrigerator are put into.
2nd, the tissue of digested overnight is taken out, epidermis and corium are separated with ophthalmic tweezers.The corium of separation is cut into eye scissors 0.1 × 0.1cm fritter, is put into 35mm culture dish, and the 0.05%collagenaseIV (clostridiopetidase A IV) for adding 3mL disappears Change liquid, culture dish is put into CO237 DEG C of digestion 2h of incubator, every 15min concussions once.
3rd, the dermal tissue of digestion is taken out, the filtering of 100 mesh cell sieves removes indigested tissue, collects digestive juice, adds Isometric DMEM high glucose mediums.Centrifuge 1000rpm, 5min.
4th, centrifuge tube is taken out, supernatant is abandoned, 1mL DMEM high glucose medium re-suspended cells are added.
5th, 5mL DMEM high glucose mediums are added in T25 blake bottles, and add cell.Blake bottle is put into CO2Incubator, 37 DEG C, 5%CO2Under the conditions of cultivate.
6th, change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
The cellular morphology that the embodiment is obtained is close with embodiment 1 with streaming qualification result, shows that the cell obtained is true Skin mescenchymal stem cell.
Comparative example 1
Reported with reference to the Yang Ya winters in 2008《Rat dermal mescenchymal stem cell is separately cultured》Disclosed in corium The digestive ferment species being separately cultured in amplification method and digestion method of mescenchymal stem cell are carried out to dermal mesenchymal stem cells It is separately cultured, is specially：
1st, dual anti-addition PBS solution, fresh prepuce tissues are cleaned three times with PBS solution, cut off very with eye scissors Subcutaneous fat and connective tissue.Then prepuce tissues are cut to 0.5 × 0.2cm strip, 100mm culture dish is put into, and 10mL 0.25% trypsin solution is added, 37 DEG C of digestion 4h are put into.
2nd, the tissue of digestion is taken out, epidermis and corium are separated with ophthalmic tweezers.The corium of separation is cut into 0.1 with eye scissors × 0.1cm fritter, is put into 35mm culture dish, adds 3mL 0.1%I Collagenase Type digestive juices, and 4 DEG C of digestion are stayed overnight.
3rd, the dermal tissue of digestion is taken out, the filtering of 100 mesh cell sieves removes indigested tissue, collects digestive juice, adds Isometric DMEM high glucose mediums.Centrifuge 1000rpm, 5min.
4th, centrifuge tube is taken out, supernatant is abandoned, 1mL DMEM high glucose medium re-suspended cells are added.
5th, 5mL DMEM high glucose mediums are added in T25 blake bottles, and add cell.Blake bottle is put into CO2Incubator, 37 DEG C, 5%CO2Under the conditions of cultivate.
6th, change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
Comparative example 2
It is that digestive ferment species disclosed in CN104403989A and digestion method are dry to corium mesenchyma with reference to disclosure/notification number Cell is separately cultured, and is specially：
1st, dual anti-addition PBS solution, fresh prepuce tissues are cleaned three times with PBS solution, cut off very with eye scissors Subcutaneous fat and connective tissue.Then prepuce tissues are cut to 0.5 × 0.2cm strip, 100mm culture dish is put into, and 10mL 0.25%dispaseII (neutral proteinase II) solution is added, 4 DEG C of digested overnights of refrigerator are put into.
2nd, the tissue of digested overnight is taken out, epidermis and corium are separated with ophthalmic tweezers.The corium of separation is cut into eye scissors 0.1 × 0.1cm fritter, is put into 35mm culture dish, adds 3mL 0.1%I Collagenase Type digestive juices, and culture dish is put into CO237 DEG C of digestion 2h of incubator, every 15min concussions once.
3rd, the dermal tissue of digestion is taken out, the filtering of 100 mesh cell sieves removes indigested tissue, collects digestive juice, adds Isometric DMEM high glucose mediums.Centrifuge 1000rpm, 5min.
4th, centrifuge tube is taken out, supernatant is abandoned, 1mL DMEM high glucose medium re-suspended cells are added.
5th, 5mL DMEM high glucose mediums are added in T25 blake bottles, and add cell.Blake bottle is put into CO2Incubator, 37 DEG C, 5%CO2Under the conditions of cultivate.
6th, change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
Comparative example 3
Win with reference to state is worn《Fetal membrane derived mesenchymal stem cell promotes the analysis of burn-healing》Middle digestion enzyme method is to corium Mescenchymal stem cell is separately cultured, and is specially：
1st, dual anti-addition PBS solution, fresh prepuce tissues are cleaned three times with PBS solution, cut off very with eye scissors Subcutaneous fat and connective tissue.Then prepuce tissues are cut to 0.5 × 0.2cm strip, 100mm culture dish is put into, and 10mL 0.1%dispaseII (neutral proteinase II) solution and 10mL 0.4% clostridiopetidase A IV are added, 4 DEG C of mistakes of refrigerator are put into Night digests.
2nd, the dermal tissue of digestion is taken out, the filtering of 100 mesh cell sieves removes indigested tissue, collects digestive juice, adds Isometric DMEM high glucose mediums.Centrifuge 1000rpm, 5min.
3rd, centrifuge tube is taken out, supernatant is abandoned, 1mL DMEM high glucose medium re-suspended cells are added.
4th, 5mL DMEM high glucose mediums are added in T25 blake bottles, and add cell.Blake bottle is put into CO2Incubator, 37 DEG C, 5%CO2Under the conditions of cultivate.
5th, change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
The Cell viability of test example 1 and purity detecting experiment
When cell fusion degree reaches 70% in example 1-3 to be performed, comparative example 1-3, detection living cells quantity, Cell viability, Purity.Cell viability detection method is Trypan Blue；Cell purity detection method is flow cytometry.Result of the test is shown in Table 1, 2。
The quantity and viability examination of the dermal mesenchymal stem cells of table 1
The purity of the dermal mesenchymal stem cells of table 2
Embodiment of the present invention 1-3 dermal mesenchymal stem cells isolated culture method is obtained it can be seen from the data of table 1 Living cells quantity is more, and average cell motility rate is significantly higher than comparative example 1-3 up to 95.3%.
As can be seen from Table 2, the dermal mesenchymal stem cells average purity that the method applied in the present invention is extracted Up to 94%, it is significantly higher than comparative example 1-3.
It can be seen that, dermal mesenchymal stem cells are separately cultured using the embodiment 1-3 methods provided, cell acquisition rate Height, Cell viability and purity are higher, are better than prior art.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (10)

1. a kind of isolated culture method of dermal mesenchymal stem cells, it is characterised in that comprise the following steps：
Skin histology is pre-processed, digested using neutral proteinase II, digestion product 1 is obtained；
The digestion product 1, separation epidermis and corium are taken, the corium is processed into fritter, digested using clostridiopetidase A IV, Obtain digestion product 2；
Indigested tissue and digestive juice in the digestion product 2 are removed, gained cell is added cell training is carried out in culture medium Support.
2. isolated culture method according to claim 1, it is characterised in that in terms of g/mL, the neutral proteinase II's It is 0.2%~0.3% to digest concentration.
3. isolated culture method according to claim 1 or 2, it is characterised in that in terms of g/mL, the clostridiopetidase A IV's disappears It is 0.05%~0.15% to change concentration.
4. isolated culture method according to any one of claim 1 to 3, it is characterised in that the use neutral protein The temperature that enzyme II is digested is 0~10 DEG C, and the time is 10~20h.
5. isolated culture method according to any one of claim 1 to 4, it is characterised in that the use clostridiopetidase A IV The temperature digested is 35~38 DEG C, CO2Concentration is 5%, and the time is 1~3h.
6. isolated culture method according to any one of claim 1 to 5, it is characterised in that the culture medium be containing 10%FBS and 5 μ g/500mL BFGF DMEM high glucose mediums.
7. isolated culture method according to any one of claim 1 to 5, it is characterised in that the bar of the cell culture Part is 5%CO2、37℃。
8. isolated culture method according to any one of claim 1 to 5, it is characterised in that described to add gained cell Enter in culture medium also includes after progress cell culture：Change liquid after cell attachment, cell fusion degree reach 70% after Secondary Culture.
9. isolated culture method according to any one of claim 1 to 5, it is characterised in that described to be processed into corium Fritter is：Corium is cut into (0.05~0.15) cm × (0.05~0.15) cm fritter.
10. isolated culture method according to any one of claim 1 to 5, it is characterised in that described to enter skin histology Row pretreatment is：Skin histology is cleaned, fat and connective tissue is removed, is processed into (0.45~0.55) cm × (0.15 ~0.25) cm tissue block.
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陈甫寰等: "表皮干细胞向汗腺细胞分化的表型改变及其通路机制研究", 《中国修复重建外科杂志》 *

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Publication number Priority date Publication date Assignee Title
CN109234230A (en) * 2018-09-30 2019-01-18 广州赛莱拉干细胞科技股份有限公司 A kind of primary separation method of skin mesenchymal stem cells

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