CN109504655A - A kind of method of the separation and secondary culture of fat stem cell - Google Patents
A kind of method of the separation and secondary culture of fat stem cell Download PDFInfo
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- CN109504655A CN109504655A CN201811560112.7A CN201811560112A CN109504655A CN 109504655 A CN109504655 A CN 109504655A CN 201811560112 A CN201811560112 A CN 201811560112A CN 109504655 A CN109504655 A CN 109504655A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- 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
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- 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
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Abstract
The invention discloses a kind of separation of fat stem cell and the method for secondary culture, including fat acquisition, obtains and prepare fritter adipose tissue, prepares fatty single cell suspension, immunological magnetic bead sorting, fluidic cell sorting, fat stem cell culture and fat stem cell secondary culture.Prepare the fatty fritter that fritter adipose tissue is 1-2m3 or so, and fatty fritter is digested using trypsase joint I type-II type mixing clostridiopetidase A time gradient digestion method, so that tissue digestion is more thorough, used incubator temperature is equipped with 37 DEG C, and the internal CO for being 5% filled with content when fat stem cell culture2Gas, meet environment and temperature needed for hematopoietic stem cell growth is bred, it can be improved the culture speed of fat stem cell, when primary cultured cell reaches 85% fusion, with 25% trypsase by attached cell digestion separation 3 ~ 5 minutes, and it is inoculated with according to the ratio of 1:3, the cell density after guaranteeing passage inoculation.
Description
Technical field
The present invention relates to a kind of cell culture processes, the separation of specially a kind of fat stem cell and the side of secondary culture
Method belongs to technical field of cell culture.
Background technique
Fat stem cell is the stem cell that the one kind obtained from adipose tissue has multi-lineage potential, in specific induction item
It can be divided into osteoblast, chondroblast and fat cell etc. under part, be that a group is similar to having for mesenchymal stem cell
Differentiation capability cell is regenerated, does not express the hematopoietic cells surface antigen such as CD34, CD45, therefore will not break up in the physiological state
As haemocyte and vascular endothelial cell；Fat stem cell human leucocyte HLA-DR expression is negative, therefore in migration process just
Allosome rejection will not be generated, to be the excellent cell kind of a progress heteroplastic transplantation.
And the fat stem cell purity that existing isolated fat stem cell obtains is not high, the growth and cell due to cell it
Between signal traffic it is related, the not high stem cell of purity is more easy to aging, often in vitro through 5-6 secondary culture fat stem cell
Afterwards, fat stem cell occurs as soon as aging, and when in addition cultivating cell inoculation into culture bottle, the culture solution used saves less steady
Fixed, cell proliferation rate is slower, and fat stem cell in vitro culture is difficult to the problem of maintaining self-renewing and multi-lineage potential, pole
The earth limits comparison and repetition between domestic and international result of study.
Summary of the invention
The object of the invention is that providing the separation and passage training of a kind of fat stem cell to solve the above-mentioned problems
Feeding method.
The present invention through the following technical solutions to achieve the above objectives: a kind of fat stem cell separation and secondary culture
Method, comprising the following steps:
Step A: fat acquisition screens the donor of medical fitness, uses suction strength for the hydrodynamic force liposuction technique of 300mmHg,
After donor local anaesthesia, mouth is operated at liposuction and injects inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction,
Adipose tissue is sucked into receiver, and carries out freezen protective；
Step B: obtaining and prepares fritter adipose tissue, the fatty stewing process that will acquire, the fat after making inflation fluid and screening
According to its specific gravity difference natural separation, adipose tissue is obtained by Screening Network, and adipose tissue is sucked by syringe and is injected
Device obtains fritter adipose tissue particle；
Step C: fatty single cell suspension is prepared, takes fritter adipose tissue particle to be digested with cell separation agent, increase serum
Digestion is terminated, by centrifugation layering after breaing up, removes supernatant；Cell is resuspended with buffer, filter screen obtains single cell suspension；
Step D: the immunomagnetic beads prepared are added to single cell suspension, remove adipose tissue medium vessels by immunological magnetic bead sorting
The Lin+ cell of base portion obtains Lin- cell mass；
Step E: fluidic cell sorting is enriched with CD271+Sca-1+ cell from the Lin- cell mass obtained, and it is dry thin to obtain fat
Born of the same parents；
Step F: the fat stem cell of acquisition is inoculated into culture dish by fat stem cell culture, is purified through adherent screening method thin
Born of the same parents simultaneously carry out in vitro culture using serum free medium, obtain fat stem cell primary；
Step G: fat stem cell secondary culture, the fat stem cell renewed vaccination primary that culture is obtained to multiple culture dishes
In, adherent screening method purifying cells are carried out again and in vitro culture is carried out using serum free medium, obtain the rouge of secondary culture
Fat stem cell.
Preferably, in order to improve the separative efficiency of fat stem cell, in the step B, preparing fritter adipose tissue is 1-
The fatty fritter of 2m3 or so, and using trypsase joint I type-II type mixing clostridiopetidase A time gradient digestion method to fat
Fritter is digested.
Preferably, in order to avoid the vigor of cell after sorting from being affected, in the step E, fluidic cell sorting
Marker used in method is Lin, CD271 and Sca-1.
Preferably, in order to improve the culture speed of fat stem cell, in the step F and step G, fat stem cell
Used incubator temperature is equipped with 37 DEG C, and the internal CO2 gas for being 5% filled with content when culture.
Preferably, in order to guarantee the cell density after passage inoculation, in the step G, primary cultured cell reaches 85% and melts
When conjunction, with 25% trypsase by attached cell digestion separation 3 ~ 5 minutes, and it is inoculated with according to the ratio of 1:3.
Preferably, acquired in the step G in order to the cell activity of the maintenance fat stem cell of long period
The fat stem cell of secondary culture is frozen in the environment of being placed on -80 DEG C.
The beneficial effects of the present invention are: the separation of the fat stem cell and the method design of secondary culture are reasonable, step B
In, the fatty fritter that fritter adipose tissue is 1-2m3 or so is prepared, and I type-II type mixing collagen is combined using trypsase
Enzyme time gradient digestion method digests fatty fritter, so that tissue digestion is more thorough, greatly improves point of fat stem cell
From efficiency, in step E, marker used in fluidic cell separating method is Lin, CD271 and Sca-1, is had to fat stem cell
There is the higher rate of recovery, while faster than traditional separation velocity, and then the vigor of cell is affected after can be avoided sorting,
In step F and step G, when fat stem cell culture, used incubator temperature was equipped with 37 DEG C, and internal filled with content is 5%
CO2 gas, meet environment and temperature needed for hematopoietic stem cell growth is bred, and then can be improved the culture speed of fat stem cell
It spends, in step G, when primary cultured cell reaches 85% fusion, with 25% trypsase by attached cell digestion separation 3 ~ 5 minutes,
And it is inoculated with according to the ratio of 1:3, the cell density after guaranteeing passage inoculation, and then improve the speed of secondary culture, step G
In, the fat stem cell of acquired secondary culture is frozen in the environment of being placed on -80 DEG C, is capable of the maintenance rouge of long period
The cell activity of fat stem cell avoids the proliferation or differentiation capability that influence fat stem cell.
Detailed description of the invention
Fig. 1 is schematic structural view of the invention.
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.
Referring to Fig. 1, a kind of method of the separation and secondary culture of fat stem cell, comprising the following steps:
Step A: fat acquisition screens the donor of medical fitness, uses suction strength for the hydrodynamic force liposuction technique of 300mmHg,
After donor local anaesthesia, mouth is operated at liposuction and injects inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction,
Adipose tissue is sucked into receiver, and carries out freezen protective；
Step B: obtaining and prepares fritter adipose tissue, the fatty stewing process that will acquire, the fat after making inflation fluid and screening
According to its specific gravity difference natural separation, adipose tissue is obtained by Screening Network, and adipose tissue is sucked by syringe and is injected
Device obtains fritter adipose tissue particle；
Step C: fatty single cell suspension is prepared, takes fritter adipose tissue particle to be digested with cell separation agent, increase serum
Digestion is terminated, by centrifugation layering after breaing up, removes supernatant；Cell is resuspended with buffer, filter screen obtains single cell suspension；
Step D: the immunomagnetic beads prepared are added to single cell suspension, remove adipose tissue medium vessels by immunological magnetic bead sorting
The Lin+ cell of base portion obtains Lin- cell mass；
Step E: fluidic cell sorting is enriched with CD271+Sca-1+ cell from the Lin- cell mass obtained, and it is dry thin to obtain fat
Born of the same parents；
Step F: the fat stem cell of acquisition is inoculated into culture dish by fat stem cell culture, is purified through adherent screening method thin
Born of the same parents simultaneously carry out in vitro culture using serum free medium, obtain fat stem cell primary；
Step G: fat stem cell secondary culture, the fat stem cell renewed vaccination primary that culture is obtained to multiple culture dishes
In, adherent screening method purifying cells are carried out again and in vitro culture is carried out using serum free medium, obtain the rouge of secondary culture
Fat stem cell.
In the step B, the fatty fritter that fritter adipose tissue is 1-2m3 or so is prepared, and I is combined using trypsase
Type-II type mixing clostridiopetidase A time gradient digestion method digests fatty fritter, so that tissue digestion is more thorough, mentions significantly
The separative efficiency of high-fat stem cell, in the step E, marker used in fluidic cell separating method be Lin, CD271 and
Sca-1, to the fat stem cell rate of recovery with higher, and meanwhile it is faster than traditional separation velocity, and then can be avoided sorting
The vigor of cell is affected afterwards, and in the step F and step G, when fat stem cell culture, used incubator temperature was set
There are 37 DEG C, and the internal CO2 gas for being 5% filled with content, meets environment and temperature needed for hematopoietic stem cell growth is bred, in turn
It can be improved the culture speed of fat stem cell, in the step G, when primary cultured cell reaches 85% fusion, with 25% pancreas egg
Attached cell digestion separation 3 ~ 5 minutes are inoculated with by white enzyme according to the ratio of 1:3, and the cell after guaranteeing passage inoculation is close
It spends, and then the speed of raising secondary culture, in the step G, the fat stem cell of acquired secondary culture is placed on -80 DEG C
It is frozen under environment, is capable of the cell activity of the maintenance fat stem cell of long period, avoids the increasing for influencing fat stem cell
It grows or differentiation capability.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (6)
1. a kind of method of the separation and secondary culture of fat stem cell, it is characterised in that: the following steps are included:
Step A: fat acquisition screens the donor of medical fitness, uses suction strength for the hydrodynamic force liposuction technique of 300mmHg,
After donor local anaesthesia, mouth is operated at liposuction and injects inflation fluid, and under hydrodynamic promotion, carry out lower negative pressure liposuction,
Adipose tissue is sucked into receiver, and carries out freezen protective；
Step B: obtaining and prepares fritter adipose tissue, the fatty stewing process that will acquire, the fat after making inflation fluid and screening
According to its specific gravity difference natural separation, adipose tissue is obtained by Screening Network, and adipose tissue is sucked by syringe and is injected
Device obtains fritter adipose tissue particle；
Step C: fatty single cell suspension is prepared, takes fritter adipose tissue particle to be digested with cell separation agent, increase serum
Digestion is terminated, by centrifugation layering after breaing up, removes supernatant；Cell is resuspended with buffer, filter screen obtains single cell suspension；
Step D: the immunomagnetic beads prepared are added to single cell suspension, remove adipose tissue medium vessels by immunological magnetic bead sorting
The Lin+ cell of base portion obtains Lin- cell mass；
Step E: fluidic cell sorting is enriched with CD271+Sca-1+ cell from the Lin- cell mass obtained, and it is dry thin to obtain fat
Born of the same parents；
Step F: the fat stem cell of acquisition is inoculated into culture dish by fat stem cell culture, is purified through adherent screening method thin
Born of the same parents simultaneously carry out in vitro culture using serum free medium, obtain fat stem cell primary；
Step G: fat stem cell secondary culture, the fat stem cell renewed vaccination primary that culture is obtained to multiple culture dishes
In, adherent screening method purifying cells are carried out again and in vitro culture is carried out using serum free medium, obtain the rouge of secondary culture
Fat stem cell.
2. the method for the separation and secondary culture of a kind of fat stem cell according to claim 1, it is characterised in that: described
In step B, the fatty fritter that fritter adipose tissue is 1-2m3 or so is prepared, and mixed using trypsase joint I type-II type
Clostridiopetidase A time gradient digestion method is closed to digest fatty fritter.
3. the method for the separation and secondary culture of a kind of fat stem cell according to claim 1, it is characterised in that: described
In step E, marker used in fluidic cell separating method is Lin, CD271 and Sca-1.
4. the method for the separation and secondary culture of a kind of fat stem cell according to claim 1, it is characterised in that: described
In step F and step G, when fat stem cell culture, used incubator temperature was equipped with 37 DEG C, and internal filled with content is 5%
CO2 gas.
5. the method for the separation and secondary culture of a kind of fat stem cell according to claim 1, it is characterised in that: described
In step G, when primary cultured cell reaches 85% fusion, with 25% trypsase by attached cell digestion separation 3 ~ 5 minutes, and press
It is inoculated with according to the ratio of 1:3.
6. the method for the separation and secondary culture of a kind of fat stem cell according to claim 1, it is characterised in that: described
In step G, the fat stem cell of acquired secondary culture is frozen in the environment of being placed on -80 DEG C.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811560112.7A CN109504655A (en) | 2018-12-20 | 2018-12-20 | A kind of method of the separation and secondary culture of fat stem cell |
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| CN201811560112.7A CN109504655A (en) | 2018-12-20 | 2018-12-20 | A kind of method of the separation and secondary culture of fat stem cell |
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| CN109504655A true CN109504655A (en) | 2019-03-22 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862960A (en) * | 2019-11-12 | 2020-03-06 | 武汉济源高科技有限公司 | Novel adipose-derived stem cell culture method |
| 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 |
-
2018
- 2018-12-20 CN CN201811560112.7A patent/CN109504655A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862960A (en) * | 2019-11-12 | 2020-03-06 | 武汉济源高科技有限公司 | Novel adipose-derived stem cell culture method |
| 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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Application publication date: 20190322 |