CN109234230A - A kind of primary separation method of skin mesenchymal stem cells - Google Patents
A kind of primary separation method of skin mesenchymal stem cells Download PDFInfo
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- CN109234230A CN109234230A CN201811156966.9A CN201811156966A CN109234230A CN 109234230 A CN109234230 A CN 109234230A CN 201811156966 A CN201811156966 A CN 201811156966A CN 109234230 A CN109234230 A CN 109234230A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to stem cell fields, disclose a kind of primary separation method of skin mesenchymal stem cells, after taking skin histology to pre-process, add Dispase II dispase digestion, blowing and beating tissue block after cleaning repeatedly separates its cell, it is centrifuged after filtering, obtains being inoculated in culture dish after cell precipitation is resuspended with Lonza complete medium and cultivate 60-90min；It takes not adherent cell suspension inoculation to cultivate into the coated culture plate of fine laminins, is passed on after cell confluency reaches 80%~90%.It will not be come to harm using the skin mesenchymal stem cells cell membrane that primary separation method of the present invention is separately cultured, cell culture is stablized, and will not introduce mycoplasma contamination, may separate out a large amount of skin mesenchymal stem cells, and cell viability is good, it being capable of subsequent stable culture.
Description
Technical field
The invention belongs to stem cell fields, and in particular to a kind of primary separation method of skin mesenchymal stem cells.
Background technique
Stem cell is a kind of primary fine with self-renewal capacity and in the case where being suitble to microenvironment with polyphyly differentiation potential
Born of the same parents.Different according to the precedence that occurs in ontogenetic process, stem cell can be divided into embryonic stem cell, multipotential stem cell and
Committed stem cell such as candidate stem cell, neural stem cell in various tissues etc..Obstructed currently, having using stem-cell therapy cardiac muscle
The Experimental report of plug, lupus erythematosus, rheumatoid arthritis, neurotrosis, muscular atrophy etc..Human stem cell is to be present in people
With a kind of super population of stem cells of polyphyly differentiation potential in body tissue, it can induce that be divided into Various Tissues thin in certain circumstances
Born of the same parents.In human stem cell, mescenchymal stem cell is widely present in the various tissues of whole body, especially marrow, fatty umbilical cord, skin
Skin and placenta.And the research of existing mescenchymal stem cell is primarily focused on and is obtained from marrow and umbilical cord, for being filled between skin
The research of matter stem cell is seldom.Since skin is the maximum histoorgan of volume in human body, and it is convenient to draw materials, so selection skin
Skin mescenchymal stem cell has bright prospects as the seed cell of cell therapy.
Skin progenitor cell plays an important role in the normal organization and cell homeostasis for maintaining skin and mucous membrane.
In the gene therapy of hereditary dermatosis and in terms of being damaged the reconstruction of skin and mucosal function and structure, skin progenitor cell has
Extremely important effect.Skin progenitor cell is had a wide range of applications by its own characteristic, tool, such as: organization engineering skin
Building, the application in wound repair, in the treatment of gene studies and genetic disease, the research of cell lineage etc..
The separation method of skin mesenchymal stem cells is that skin group is digested with the trypsase containing EDTA in the prior art
It knits, is inoculated in defined medium after filtering.Trypsase belongs to enzyme of proteolysis, and adopting said method dispersion tissue divides
Cell membrane can be made to be badly damaged from cell, it is unstable in cell culture, or even introduce mycoplasma contamination.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of efficient, economic, stable, convenient and fast skin mesenchymas to do carefully
The primary separation method of born of the same parents, to isolate a large amount of skin mesenchymal stem cells, and being capable of subsequent stable culture.
To achieve the purpose of the present invention, the present invention adopts the following technical scheme:
A kind of primary separation method of skin mesenchymal stem cells, includes the following steps:
A, add Dispase II dispase digestion after taking skin histology to pre-process, blowing and beating tissue block after cleaning repeatedly keeps it thin
Born of the same parents' separation, is centrifuged after filtering, obtains being inoculated in culture dish after cell precipitation is resuspended with Lonza complete medium and cultivate；
B, it takes not adherent cell suspension inoculation to cultivate into the coated culture plate of fine laminins, reaches to cell confluency
It is passed on after 80%~90%.
Separation method of the present invention first pre-processes skin histology.
Wherein, the pretreatment is that skin histology is cut into 3-5mm rectangle, is cleaned with sodium chloride injection and is placed on 75%
It is cleaned in ethyl alcohol, then is cut into fine tissue block after being cleaned with sodium chloride injection.
In some embodiments, for the skin histology of fresh acquisition, it is also necessary to containing dual anti-physiological saline repeatedly
It rinses for several times, then uses 75% alcohol rinse, be finally stored in save containing dual anti-4 DEG C of glucose solution and be used for subsequent pre- place
Reason.Wherein dual anti-is mycillin mixed liquor.In Pen .- Strep solution (100 ×), the content of penicillin is 10000U/
Ml, the content of streptomysin are 10mg/ml.Dual anti-concentration is preferably 1 in physiological saline of the present invention and glucose solution
×, i.e., the working concentration of penicillin is 100U/ml, and the working concentration of streptomysin is 0.1mg/ml.
Separation method of the present invention digests skin histology using Dispase II dispase.Dispase II points
Enzyme, i.e. neutral proteinase are dissipated, is a kind of nonspecific metalloproteinases, which will not injure cell membrane, and because its source
In bacterium, therefore mycoplasma or any animal virus will not be introduced；The difference of temperature, PH, serum composition etc. is to Dispase II points
The influence very little of enzyme is dissipated, enzyme activity after diluting substantially reduces, and facilitates subsequent cell culture.
Wherein, preferably, the concentration of Dispase II dispase described in step A is 0.5U/ml-2.5U/ml.Some
In embodiment, the concentration of the Dispase II dispase is 2U/ml.
Preferably, digestion described in step A is 37 DEG C of digestion 1h.
Preferably, cleaning described in step A is that sodium chloride injection cleans.
Filtering removal impurity after separation method separation cell of the present invention.Preferably, being filtered into 100 μ described in step A
The disposable cell screen clothes filtering of m.
Preferably, centrifugation described in step A is that 400g is centrifuged 10min.
Preferably, culture described in step A is 37 DEG C, CO2Cultivate 60-90min.
Preferably, the inoculum density of cell suspension not adherent described in step B is 5 × 104/ml。
Preferably, the concentration for being coated with the fine laminins of culture plate described in step B is 10-50 μ g/ml.In some implementations
In scheme, the concentration for being coated with the fine laminins of culture plate is 20 μ g/ml.
Preferably, method for coating described in step B is 37 DEG C of incubation 30-60min.
Preferably, culture described in step B is 37 DEG C, 5%CO2It is cultivated under concentration.
It should be spaced 72h preferably, cultivating described in step B and changing liquid for the first time, change a not good liquor within every 1-3 days later.
As shown from the above technical solution, the present invention provides a kind of primary separation methods of skin mesenchymal stem cells.Benefit
It will not be come to harm with the skin mesenchymal stem cells cell membrane that primary separation method of the present invention is separately cultured, cell culture
Stablize, and mycoplasma contamination will not be introduced.Experiment shows the skin mesenchyma that primary separation method of the present invention is separately cultured
Stem cell may separate out a large amount of skin mesenchymal stem cells, and cell viability is good, being capable of subsequent stable culture.
Specific embodiment
The invention discloses a kind of primary separation methods of skin mesenchymal stem cells.Those skilled in the art can use for reference
Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.Method and product of the invention by compared with
Good embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to as described herein
Method is modified or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal
Road purchase obtains.Wherein Dispase II dispase is purchased from Sigma company；Lonza complete medium is purchased from Switzerland Long Sha company；
Fine laminins are purchased from Corning company.
The primary separation of embodiment 1, skin mesenchymal stem cells
1. 20 μ g/ml fibre laminins FN 2ml is taken to spread into 10cm culture plate, 37 DEG C of incubation 30-60min are spare；
2. take skin histology about (30mm × 5mm), with containing 1 × dual anti-physiological saline repeated flushing for several times, then with 75%
Alcohol rinse is primary, is finally stored in containing in dual anti-glucose solution, 4 DEG C of preservations are taken back laboratory and operated in for 24 hours；
3. skin histology is cut into 3~5mm rectangle, cleaned, is placed in 75% ethyl alcohol clear with sodium chloride injection
It washes, is finally cleaned one time with sodium chloride injection again；
4. rectangle is cut into fine tissue block, it is placed in 37 DEG C of digestion 1h of Dispase II dispase of 2U/ml；
Tissue block is taken out after 5.1h, is cleaned one time with sodium chloride injection；
6. piping and druming tissue block separates its cell repeatedly, crosses 100 μm of disposable cell screen clothes and remove impurity；
7. filtered cell 400g, 10min are centrifuged；
8. obtaining the resuspension of cell precipitation Lonza complete medium to be inoculated in 10cm ware, it is put into 37 DEG C of carbon dioxide trainings
Case culture 60-90min is supported to remove fibroblast；
9. taking not adherent cell suspension to count, with 5 × 104The density of/ml is inoculated in the culture plate for being coated with FN；
Liquid is changed after 10.72h for the first time, changes within every 2 days later liquid, direct cell is long to convergence degree 80%-90% passage.
The primary separation of embodiment 2, skin mesenchymal stem cells
1. 10 μ g/ml fibre laminins FN 2ml is taken to spread into 10cm culture plate, 37 DEG C of incubation 30-60min are spare；
2. take skin histology about (30mm × 5mm), with containing 1 × dual anti-physiological saline repeated flushing for several times, then with 75%
Alcohol rinse is primary, is finally stored in containing in dual anti-glucose solution, 4 DEG C of preservations are taken back laboratory and operated in for 24 hours；
3. skin histology is cut into 3~5mm rectangle, cleaned, is placed in 75% ethyl alcohol clear with sodium chloride injection
It washes, is finally cleaned one time with sodium chloride injection again；
4. rectangle is cut into fine tissue block, it is placed in 37 DEG C of digestion 1h of Dispase II dispase of 0.5U/ml；
Tissue block is taken out after 5.1h, is cleaned one time with sodium chloride injection；
6. piping and druming tissue block separates its cell repeatedly, crosses 100 μm of disposable cell screen clothes and remove impurity；
7. filtered cell 400g, 10min are centrifuged；
8. obtaining the resuspension of cell precipitation Lonza complete medium to be inoculated in 10cm ware, it is put into 37 DEG C of carbon dioxide trainings
Case culture 60-90min is supported to remove fibroblast；
9. taking not adherent cell suspension to count, with 5 × 104The density of/ml is inoculated in the culture plate for being coated with FN；
Liquid is changed after 10.72h for the first time, changes within every 2 days later liquid, direct cell is long to convergence degree 80%-90% passage.
The primary separation of embodiment 3, skin mesenchymal stem cells
1. 50 μ g/ml fibre laminins FN 2ml is taken to spread into 10cm culture plate, 37 DEG C of incubation 30-60min are spare；
2. take skin histology about (30mm × 5mm), with containing 1 × dual anti-physiological saline repeated flushing for several times, then with 75%
Alcohol rinse is primary, is finally stored in containing in dual anti-glucose solution, 4 DEG C of preservations are taken back laboratory and operated in for 24 hours；
3. skin histology is cut into 3~5mm rectangle, cleaned, is placed in 75% ethyl alcohol clear with sodium chloride injection
It washes, is finally cleaned one time with sodium chloride injection again；
4. rectangle is cut into fine tissue block, it is placed in 37 DEG C of digestion 1h of Dispase II dispase of 2.5U/ml；
Tissue block is taken out after 5.1h, is cleaned one time with sodium chloride injection；
6. piping and druming tissue block separates its cell repeatedly, crosses 100 μm of disposable cell screen clothes and remove impurity；
7. filtered cell 400g, 10min are centrifuged；
8. obtaining the resuspension of cell precipitation Lonza complete medium to be inoculated in 10cm ware, it is put into 37 DEG C of carbon dioxide trainings
Case culture 60-90min is supported to remove fibroblast；
9. taking not adherent cell suspension to count, with 5 × 104The density of/ml is inoculated in the culture plate for being coated with FN；
Liquid is changed after 10.72h for the first time, changes within every 2 days later liquid, direct cell is long to convergence degree 80%-90% passage.Comparative example
1, the primary separation of skin mesenchymal stem cells
1. take skin histology about (30mm × 5mm), with containing 1 × dual anti-physiological saline repeated flushing for several times, then with 75%
Alcohol rinse is primary, is finally stored in containing in dual anti-glucose solution, 4 DEG C of preservations are taken back laboratory and operated in for 24 hours；
2. skin histology is cut into 3~5mm rectangle, cleaned, is placed in 75% ethyl alcohol clear with sodium chloride injection
It washes, is finally cleaned one time with sodium chloride injection again；
3. rectangle is cut into fine tissue block, it is placed in 0.1% trypsase, 37 DEG C of digestion 1h；
Tissue block is taken out after 4.1h, is cleaned one time with sodium chloride injection；
5. piping and druming tissue block separates its cell repeatedly, crosses 100 μm of disposable cell screen clothes and remove impurity；
6. filtered cell 400g, 10min are centrifuged；
7. obtaining cell precipitation Lonza complete medium with 5 × 104The density of/ml is inoculated in 10cm ware, is put into 37
DEG C carbon dioxide incubator culture；
Liquid is changed after 8.72h for the first time, changes within every 2 days later liquid, direct cell is long to convergence degree 80%-90% passage.Comparative example
2, the primary separation of skin mesenchymal stem cells
1. take skin histology about (30mm × 5mm), with containing 1 × dual anti-physiological saline repeated flushing for several times, then with 75%
Alcohol rinse is primary, is finally stored in containing in dual anti-glucose solution, 4 DEG C of preservations are taken back laboratory and operated in for 24 hours；
2. skin histology is cut into 3~5mm rectangle, cleaned, is placed in 75% ethyl alcohol clear with sodium chloride injection
It washes, is finally cleaned one time with sodium chloride injection again；
3. rectangle is cut into fine tissue block, it is placed in 0.1% trypsase, 37 DEG C of digestion 1h；
Tissue block is taken out after 4.1h, is cleaned one time with sodium chloride injection；
5. piping and druming tissue block separates its cell repeatedly, crosses 100 μm of disposable cell screen clothes and remove impurity；
6. filtered cell 400g, 10min are centrifuged；
7. obtaining the resuspension of cell precipitation Lonza complete medium to be inoculated in 10cm ware, it is put into 37 DEG C of carbon dioxide trainings
Case culture 60-90min is supported to remove fibroblast；
8. taking not adherent cell suspension to count, with 5 × 104The density of/ml is inoculated in 10cm culture dish；
Liquid is changed after 9.72h for the first time, changes within every 2 days later liquid, direct cell is long to convergence degree 80%-90% passage.Test example,
Cell Proliferation and Cell viability compare
Compare cell Proliferation and Cell viability after being respectively adopted method culture 7 days of embodiment 1-3 and comparative example 1-2,
It the results are shown in Table 1.
Cell Proliferation and Cell viability after the culture of table 17 days
As seen from the above table, primary separation method described in embodiment 1-3 can obtain more cells, and skin obtained by this method
Mescenchymal stem cell possesses higher cell viability.And comparative example 1 is without differential velocity adherent 60-90min after cell dissociation, gained
Mescenchymal stem cell number is zero.Differential velocity adherent 60-90min is necessary after showing cell dissociation, this step utilizes different cells
Characteristic different (the adherent time is different) is that foundation can effectively remove remaining heteroproteose cell.And utilize other enzymes such as trypsase
It is larger to the damage of cell, so that Cell viability is bad, cannot preferably be proliferated.
Claims (10)
1. a kind of primary separation method of skin mesenchymal stem cells, which comprises the steps of:
A, add Dispase II dispase digestion after taking skin histology to pre-process, blowing and beating tissue block after cleaning repeatedly makes its cell point
From, it is centrifuged after filtering, obtains being inoculated in culture dish after cell precipitation is resuspended with Lonza complete medium and cultivate, progress differential
It is adherent；
B, it takes not adherent cell suspension inoculation to cultivate into the coated culture plate of fine laminins, reaches 80% to cell confluency
It is passed on after~90%.
2. primary separation method according to claim 1, which is characterized in that pretreatment described in step A is that skin histology is transported
4 DEG C are stored in when defeated, containing in dual anti-glucose solution, skin histology is cut into 3-5mm rectangle, is cleaned with sodium chloride injection
It is placed in 75% ethyl alcohol and cleans, then be cut into fine tissue block after being cleaned with sodium chloride injection.
3. primary separation method according to claim 1, which is characterized in that DispaseII dispase is dense described in step A
Degree is 0.5U/ml-2.5U/ml.
4. primary separation method according to claim 1, which is characterized in that digestion described in step A is 37 DEG C of digestion 1h.
5. primary separation method according to claim 1, which is characterized in that cleaning described in step A is sodium chloride injection
Cleaning.
6. primary separation method according to claim 1, which is characterized in that be filtered into described in step A 100 μm disposable thin
Born of the same parents' the screen to filtrate.
7. primary separation method according to claim 1, which is characterized in that centrifugation described in step A is that 400g is centrifuged
10min。
8. primary separation method according to claim 1, which is characterized in that differential velocity adherent described in step A is 37 DEG C, 5%
CO2Cultivate 60-90min.
9. primary separation method according to claim 1, which is characterized in that not adherent cell suspension described in step B
Inoculum density is 5 × 104/ml。
10. primary separation method according to claim 1, which is characterized in that the fibre for being coated with culture plate described in step B is adhered
The concentration of albumen is 10-50 μ g/ml；The method for coating is 37 DEG C of incubation 30-60min；The culture is 37 DEG C, 5%CO2It is dense
The lower culture of degree；Liquid is changed in the culture for the first time should be spaced 72h, change a not good liquor within every 1-3 days later.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110241073A (en) * | 2019-07-15 | 2019-09-17 | 朱家源 | The method of quick separating extraction epidermal stem cells |
| CN112920995A (en) * | 2021-03-31 | 2021-06-08 | 赵峻岭 | Mesenchymal stem cell culture serum refueling bag and application thereof |
| CN114557336A (en) * | 2021-11-25 | 2022-05-31 | 中国人民解放军空军军医大学 | Tissue treatment fluid capable of improving primary tissue isolation quantity and activity and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110241073A (en) * | 2019-07-15 | 2019-09-17 | 朱家源 | The method of quick separating extraction epidermal stem cells |
| CN112920995A (en) * | 2021-03-31 | 2021-06-08 | 赵峻岭 | Mesenchymal stem cell culture serum refueling bag and application thereof |
| CN114557336A (en) * | 2021-11-25 | 2022-05-31 | 中国人民解放军空军军医大学 | Tissue treatment fluid capable of improving primary tissue isolation quantity and activity and preparation method and application thereof |
| CN114557336B (en) * | 2021-11-25 | 2023-05-05 | 中国人民解放军空军军医大学 | Tissue treatment fluid capable of improving primary separation quantity and activity of tissues as well as preparation method and application thereof |
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