CN109628400A - A kind of separator and secondary culture method of neural stem cell - Google Patents
A kind of separator and secondary culture method of neural stem cell Download PDFInfo
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- CN109628400A CN109628400A CN201811588331.6A CN201811588331A CN109628400A CN 109628400 A CN109628400 A CN 109628400A CN 201811588331 A CN201811588331 A CN 201811588331A CN 109628400 A CN109628400 A CN 109628400A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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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
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a kind of separator of neural stem cell and secondary culture methods, including outer cylinder, feed inlet, cutting blade, filtering baffle, deflector, the first inlet tube, drain pipe, valve, digester, the second inlet tube, outlet tube, chassis, surge drum and filter membrane.The beneficial effects of the present invention are: cutting blade is in shape of threads, and cutting blade is provided with two layers in feed inlet, convenient for fat lump to be cut into the adipose tissue of fritter, first inlet tube is at the upward skewed bottom end for being placed in filtering baffle, and first inlet tube be formed by filter chamber to filtering baffle and outer cylinder and pour phosphate buffered saline solution, and it is discharged via outlet tube, the haemocyte on adipose tissue can be removed, second inlet tube ramp-like is placed in digester upper end in dipping down, convenient for digestive juice is injected in digester, filter membrane is provided with two layers, and the aperture of filter membrane is 30 μm, to be more easier, accurately neural stem cell is separated.
Description
Technical field
The present invention relates to a kind of cell culture processes, the separator of specially a kind of neural stem cell and secondary culture side
Method belongs to technical field of cell culture.
Background technique
Neural 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；Neural 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 neural stem cell purity that existing isolated neural 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 neural stem cell
Afterwards, neural 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 neural 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 separator and biography of a kind of neural stem cell to solve the above-mentioned problems
For cultural method.
The present invention is through the following technical solutions to achieve the above objectives: a kind of separator of neural stem cell, separation dress
It sets including outer cylinder, feed inlet, cutting blade, filtering baffle, deflector, the first inlet tube, drain pipe, valve, digester, second
Inlet tube, outlet tube, chassis, surge drum and filter membrane；The outer cylinder is placed in the top of surge drum, the top peace of the outer cylinder
It is equipped with feed inlet, the cutting blade is mounted in feed inlet, and is located at the bottom of feed inlet, and the filtering baffle setting is outside
In cylinder, and the filtering baffle is connected to the lower section of feed inlet, and the deflector tiltably-mounted is described in the outside of filtering baffle
First inlet tube is mounted on the side wall of outer cylinder, and the liquid feeding end of first inlet tube is located at filtering baffle and is formed with outer cylinder
Filter chamber in, the drain pipe is located at the top of deflector, and the bottom of filtering baffle, the digester is arranged in the valve
Card is placed in outer cylinder, and the digester is placed in filtering baffle and outer cylinder is formed by below filter chamber, second feed liquor
Pipe is mounted on the side wall of outer cylinder, and second inlet tube is located at the lower section of the first inlet tube, and second inlet tube
Liquid feeding end is arranged in digester, and the outlet tube is connected to the bottom of digester, and the chassis is located at the bottom of the separator
End, the surge drum is placed on chassis, and the surge drum is located at the underface of outlet tube, and the filter membrane horizontal setting exists
In surge drum.
Preferably, for the ease of fat lump to be cut into the adipose tissue of fritter, the cutting blade is in shape of threads, and is cut
Cutting blade is provided with two layers in feed inlet.
Preferably, in order to remove the haemocyte on adipose tissue, first inlet tube is at upward skewed placement
In the bottom end of filtering baffle, and the first inlet tube is formed by filter chamber that pour phosphate-buffered salt molten to filtering baffle with outer cylinder
Liquid, and be discharged via outlet tube.
Preferably, for the ease of by digestive juice inject digester in, while can by adipose tissue collagen and on
Skin tissue is digested, second inlet tube in dip down it is ramp-like be placed in digester upper end, and the second inlet tube is to digestion
Filled with the tissue digestion liquid being configured to by clostridiopetidase A and trypsase in pond.
Preferably, in order to improve the rate of recovery of working efficiency and neural stem cell, the filter membrane is provided with two layers, and mistake
The aperture of filter membrane is 30 μm.
A kind of secondary culture method of the separator based on neural stem cell, secondary culture method include 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 neural stem cell；
Step D: the neural stem cell of acquisition is inoculated into culture dish by Culture of neural stem cells, is purified through adherent screening method thin
Born of the same parents simultaneously carry out in vitro culture using serum free medium, obtain neural stem cell primary；
Step E: neural stem cell secondary culture, the neural 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 mind of secondary culture
Through stem cell
The beneficial effects of the present invention are: separator and secondary culture the method design of the neural stem cell are rationally, cutting blade
In shape of threads, and cutting blade is provided with two layers in feed inlet, convenient for fat lump to be cut into the adipose tissue of fritter, first
Inlet tube is at the upward skewed bottom end for being placed in filtering baffle, and the first inlet tube was formed by filtering baffle and outer cylinder
Phosphate buffered saline solution is poured in filter chamber, and is discharged via outlet tube, the adipose tissue shredded can be cleaned, and then remove
Remove the structural haemocyte of fat, the second inlet tube in dip down it is ramp-like be placed in digester upper end, and the second inlet tube is to disappearing
Change filled with the tissue digestion liquid being configured to by clostridiopetidase A and trypsase in pond, convenient for injecting digestive juice in digester, simultaneously
Can by adipose tissue collagen and epithelial tissue digest, suspension is made, filter membrane is provided with two layers, and filter
The aperture of film is 30 μm, separates, improves work efficiency and nerve cord to be more easier, accurately to neural stem cell
The rate of recovery of cell.
Detailed description of the invention
Fig. 1 is the schematic diagram of the section structure of the present invention；
Fig. 2 is schematic diagram of the three-dimensional structure.
In figure: 1, outer cylinder, 2, feed inlet, 3, cutting blade, 4, filtering baffle, 5, deflector, the 6, first inlet tube, 7, row
Liquid pipe, 8, valve, 9, digester, the 10, second inlet tube, 11, outlet tube, 12, chassis, 13, surge drum and 14, filter membrane.
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.
Please refer to Fig. 1 ~ 2, a kind of separator and secondary culture method of neural stem cell, separator include outer cylinder 1,
Feed inlet 2, cutting blade 3, filtering baffle 4, deflector 5, the first inlet tube 6, drain pipe 7, valve 8, digester 9, second into
Liquid pipe 10, outlet tube 11, chassis 12, surge drum 13 and filter membrane 14；The outer cylinder 1 is placed in the top of surge drum 13, described
The top of outer cylinder 1 is mounted with feed inlet 2, and the cutting blade 3 is mounted in feed inlet 2, and is located at the bottom of feed inlet 2, institute
It states filtering baffle 4 to be arranged in outer cylinder 1, and the filtering baffle 4 is connected to the lower section of feed inlet 2, the inclination of deflector 5 peace
Set in the outside of filtering baffle 4, first inlet tube 6 is mounted on the side wall of outer cylinder 1, and first inlet tube 6 into
Liquid end is located at filtering baffle 4 and outer cylinder 1 is formed by filter chamber, and the drain pipe 7 is located at the top of deflector 5, the valve
The bottom of filtering baffle 4 is arranged in door 8, and the card of digester 9 is placed in outer cylinder 1, and the digester 9 is placed in filtering baffle
4 are formed by below filter chamber with outer cylinder 1, and second inlet tube 10 is mounted on the side wall of outer cylinder 1, and second feed liquor
Pipe 10 is located at the lower section of the first inlet tube 6, and the liquid feeding end of second inlet tube 10 is arranged in digester 9, the liquid out
Pipe 11 is connected to the bottom of digester 9, and the chassis 12 is located at the bottom end of the separator, and the surge drum 13 is placed in chassis
On 12, and the surge drum 13 is located at the underface of outlet tube 11, and 14 horizontal setting of filter membrane is in surge drum 13.
The cutting blade 3 is in shape of threads, and cutting blade 3 is provided with two layers in feed inlet 2, convenient for cutting fat lump
Be cut into the adipose tissue of fritter, first inlet tube 6 at the upward skewed bottom end for being placed in filtering baffle 4, and first into
Liquid pipe 6 is formed by filter chamber to filtering baffle 4 and outer cylinder 1 and pours phosphate buffered saline solution, and is discharged via outlet tube 11,
The adipose tissue shredded can be cleaned, so remove adipose tissue on haemocyte, second inlet tube 10 be in
Under it is skewed be placed in 9 upper end of digester, and the second inlet tube 10 into digester 9 filled with being prepared by clostridiopetidase A and trypsase
At tissue digestion liquid while can be by the collagen and epithelium group in adipose tissue convenient for injecting digestive juice in digester 9
It knits and is digested, suspension is made, the filter membrane 14 is provided with two layers, and the aperture of filter membrane 14 is 30 μm, so as to more
Easily and accurately neural stem cell is separated, improves work efficiency the rate of recovery with neural stem cell.
A kind of secondary culture method of the separator based on neural stem cell, secondary culture method include 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 neural stem cell；
Step D: the neural stem cell of acquisition is inoculated into culture dish by Culture of neural stem cells, is purified through adherent screening method thin
Born of the same parents simultaneously carry out in vitro culture using serum free medium, obtain neural stem cell primary；
Step E: neural stem cell secondary culture, the neural 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 mind of secondary culture
Through stem cell.
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 separator of neural stem cell, it is characterised in that: separator includes outer cylinder (1), feed inlet (2), cutting
Blade (3), filtering baffle (4), deflector (5), the first inlet tube (6), drain pipe (7), valve (8), digester (9), second
Inlet tube (10), outlet tube (11), chassis (12), surge drum (13) and filter membrane (14)；The outer cylinder (1) is placed in surge drum
(13) top of top, the outer cylinder (1) is mounted with feed inlet (2), and the cutting blade (3) is mounted in feed inlet (2),
And it is located at the bottom of feed inlet (2), filtering baffle (4) setting is in outer cylinder (1), and the filtering baffle (4) is connected to
The lower section of feed inlet (2), deflector (5) tiltably-mounted is in the outside of filtering baffle (4), the first inlet tube (6) peace
On the side wall of outer cylinder (1), and the liquid feeding end of first inlet tube (6) is located at filtering baffle (4) and is formed with outer cylinder (1)
Filter chamber in, the drain pipe (7) is located at the top of deflector (5), bottom of valve (8) setting in filtering baffle (4)
Portion, digester (9) card is placed in outer cylinder (1), and the digester (9) is placed in filtering baffle (4) and outer cylinder (1) institute shape
At filter chamber below, second inlet tube (10) is mounted on the side wall of outer cylinder (1), and second inlet tube (10) position
In the lower section of the first inlet tube (6), and the liquid feeding end setting of second inlet tube (10) is interior in digester (9), the liquid out
Pipe (11) is connected to the bottom of digester (9), and the chassis (12) is located at the bottom end of the separator, surge drum (13) peace
It is placed on chassis (12), and the surge drum (13) is located at the underface of outlet tube (11), filter membrane (14) horizontal setting
In surge drum (13).
2. a kind of separator of neural stem cell according to claim 1, it is characterised in that: the cutting blade (3)
In shape of threads, and cutting blade (3) is provided with two layers in feed inlet (2).
3. a kind of separator of neural stem cell according to claim 1, it is characterised in that: first inlet tube
(6) at the upward skewed bottom end for being placed in filtering baffle (4), and the first inlet tube (6) is to filtering baffle (4) and outer cylinder (1)
It is formed by filter chamber and pours phosphate buffered saline solution, and be discharged via outlet tube (11).
4. a kind of separator of neural stem cell according to claim 1, it is characterised in that: second inlet tube
(10) in dip down it is ramp-like be placed in digester (9) upper end, and the second inlet tube (10) into digester (9) filled with by clostridiopetidase A
The tissue digestion liquid being configured to trypsase.
5. a kind of separator of neural stem cell according to claim 1, it is characterised in that: the filter membrane (14)
It is provided with two layers, and the aperture of filter membrane (14) is 30 μm.
6. a kind of secondary culture method of the separator based on neural stem cell, it is characterised in that: its secondary culture method packet
Include 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 neural stem cell；
Step D: the neural stem cell of acquisition is inoculated into culture dish by Culture of neural stem cells, is purified through adherent screening method thin
Born of the same parents simultaneously carry out in vitro culture using serum free medium, obtain neural stem cell primary；
Step E: neural stem cell secondary culture, the neural 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 mind of secondary culture
Through stem cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811588331.6A CN109628400A (en) | 2018-12-25 | 2018-12-25 | A kind of separator and secondary culture method of neural stem cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811588331.6A CN109628400A (en) | 2018-12-25 | 2018-12-25 | A kind of separator and secondary culture method of neural stem cell |
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| Publication Number | Publication Date |
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| CN109628400A true CN109628400A (en) | 2019-04-16 |
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| CN201811588331.6A Withdrawn CN109628400A (en) | 2018-12-25 | 2018-12-25 | A kind of separator and secondary culture method of neural stem cell |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690537A (en) * | 2020-07-03 | 2020-09-22 | 山东万能干细胞生物技术有限公司 | Stem cell preparation system |
| CN111849762A (en) * | 2020-07-29 | 2020-10-30 | 湖北明德健康科技有限公司 | Stem cell extraction element |
| CN112029709A (en) * | 2020-09-11 | 2020-12-04 | 孙震颖 | Culture method for induced differentiation of stem cells |
| CN112852734A (en) * | 2021-02-24 | 2021-05-28 | 河南省银丰生物工程技术有限公司 | Method for separating and identifying human peripheral blood hematopoietic stem cells |
-
2018
- 2018-12-25 CN CN201811588331.6A patent/CN109628400A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690537A (en) * | 2020-07-03 | 2020-09-22 | 山东万能干细胞生物技术有限公司 | Stem cell preparation system |
| CN111690537B (en) * | 2020-07-03 | 2023-08-01 | 山东万能干细胞生物技术有限公司 | Stem cell preparation system |
| CN111849762A (en) * | 2020-07-29 | 2020-10-30 | 湖北明德健康科技有限公司 | Stem cell extraction element |
| CN111849762B (en) * | 2020-07-29 | 2021-06-29 | 湖北明德健康科技有限公司 | Stem cell extraction element |
| CN112029709A (en) * | 2020-09-11 | 2020-12-04 | 孙震颖 | Culture method for induced differentiation of stem cells |
| CN112852734A (en) * | 2021-02-24 | 2021-05-28 | 河南省银丰生物工程技术有限公司 | Method for separating and identifying human peripheral blood hematopoietic stem cells |
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