CN104560868B - A kind of primary isolated culture method of fat stem cell - Google Patents
A kind of primary isolated culture method of fat stem cell Download PDFInfo
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Abstract
The invention discloses a kind of primary isolated culture methods of fat stem cell, comprise the following steps：1) adipose tissue is washed；2) mixing liquid of isometric type i collagen enzyme and trypsase is added in adipose tissue；3) FBS is added in, covers seal after bottle cap and shakes up, centrifugation；Supernatant is abandoned after centrifugation；4) precipitation is resuspended with the DMEM containing FBS, it is 1 to precipitate with the volume ratio of the DMEM containing FBS:1, form cell suspension after blowing and beating uniformly；By cell suspension inoculation into culture medium；5) the medium culture after inoculation；6) after cultivating, shift and continue to cultivate in cell incubator.The enzymatic hydrolysis condition explored of the present invention is stable, efficient, and adipose tissue is thoroughly digested, and what each component cells were maximized releases；On the premise of the activity of ADSCs is not influenced, the amount for the ADSCs that unit fat volume is obtained is stablized relatively.
Description
Technical field
The present invention relates to the isolated culture methods of stem cell, and in particular to a kind of primary side of being separately cultured of fat stem cell
Method.
Background technology
People source fat stem cell (Adipose-Derived Stem Cells, ADSCs) is in recent years from body fat group
Isolated a kind of stem cell with multi-lineage potential in knitting.ADSCs not only can using directed differentiation as fat, cartilage,
The various kinds of cell such as cardiac muscle cell；It is a variety of with biological activity that a variety of promotion angiogenesis factors, antiapoptotic factors etc. can also be secreted
Cell factor.In addition, ADSCs also has many advantages, such as that pain is less when materials are easy, amount to obtain is big, donor is drawn materials.At present
It is widely used in organizational project and regenerative medicine field.
The Isolation and culture of people source fat stem cell is but always a problem.All it is to digest in art methods
It is improved on condition and condition of culture.But still it is mostly cracked using Mechanical Method removal blood vessel, single enzyme digestion, using lysate
Red blood cell takes airflow classification stem cell, obtains the cell mass of purer ADSCs as far as possible；It is added in culture
The antibiotic such as green grass or young crops-streptomysin although playing certain antibacterial action, also cause cell different degrees of murder by poisoning.Entire fat
The process that is separately cultured of fat stem cell operates numerous and complicated mixed and disorderly, time and effort consuming, production cost multiplication.In addition, it is sent out after practical operation
Existing effect is not very preferable, and due to the use of more chemical reagent, vigor is relatively low after isolation, multiplication is fast by ADSCs
Degree is slow；And cellular morphology variation is larger.Adipose tissue is digested using low concentration, single enzyme solution, enzymolysis is not thorough
Fat stem cell is caused not released fully.Generally speaking, the separation of ADSCs and cultural method are all unsatisfactory.
The content of the invention
Existing in the prior art to solve the problems, such as, the present invention is for some problems occurred in practice process, in correlative link
It is had made some improvements on section, by repeatedly attempting, forms relatively perfect, a workable and time-consuming short people source fat
The cultural method of cell after the digestion separation system of fat stem cell and separation.The purpose of the present invention, it is intended to further optimization enzymolysis
Scheme simplifies operating process, isolates the fat stem cell of high activity, high-purity, can also further adapt to industrialized production.This
Invented technology is simple and efficient, and as a result has the advantages that stable, repeatability.In addition, having used for the present invention also creativeness is higher
Concentration mixed enzymolysis liquid, not only extracts fat stem cell to the greatest extent, while also assures the high vigor of fat stem cell.
Finally, due to which operating procedure greatly simplifies, pollution probability reduces, and on the premise of without using green grass or young crops-streptomysin, pollution rate is still
0%.
The object of the invention is achieved through the following technical solutions.
A kind of primary isolated culture method of fat stem cell, comprises the following steps：
1) adipose tissue, cleaning are extracted；
2) mixing liquid of isometric type i collagen enzyme and trypsase is added in adipose tissue；Wherein mixing liquid
The mass percent of middle type i collagen enzyme and trypsase is respectively 0.05%~0.3% and 0.01%~0.1%, abundant mixing
30~60min is digested afterwards；
3) FBS is added in, covers seal after bottle cap and shakes up, centrifugation；Supernatant is abandoned after centrifugation；
4) cell precipitation is resuspended with the DMEM containing FBS, the volume ratio of cell precipitation and the DMEM containing FBS are 1:1, it blows
Cell suspension is formed after beating uniformly；It will be in cell suspension inoculation to the DMEM in high glucose culture medium containing FBS；
5) media transfer after inoculation is cultivated into cell incubator；
6) after cultivating 24~72h, culture medium in ware is abandoned in suction, and the DMEM containing EGF and FBS is added after cleaning, shifts cell
Continue to cultivate in incubator.
It is preferred that step (2) digestion is carried out in 37 DEG C of constant temperature gas bath shaking table, speed is shaken as 100~250r/min.
It is preferred that the rotating speed of step (3) described centrifugation is 1000~1500rpm, centrifugation time is 3~10min.
It is preferred that the percentage by volume of the FBS is 10%, the concentration of the EGF is 10ng/mL.
It is preferred that the optimal enzymolysis scheme of the present invention：The mixed enzyme solution (two of isometric Type I collagen enzyme and trypsase enzyme is used
Person's mass percent is respectively 0.25%, 0.05%), and enzymolysis time 50min, to shake speed be 200 turns.
The present invention is compared with advantage possessed by the prior art and advantageous effect：
1. the enzymatic hydrolysis condition explored of the present invention is stable, efficient, adipose tissue is thoroughly digested, and each component cells are by most
That changes greatly releases；On the premise of the activity of ADSCs is not influenced, the amount for the ADSCs that unit fat volume is obtained is opposite
Stablize.
2. method provided by the invention is simple, operation is easy, for the of less demanding of instrument and equipment, common super-clean bench,
Low speed centrifuge and shaking table can be met the requirements, and are more conducive to large-scale production and prepared.
3. the present invention avoids the problem of erythrocyte cracked liquid remains without using erythrocyte cracked liquid.
4. efficient, to take short, extraction cell viability high, for example, using 0.075% Type I collagen enzyme, 37 DEG C,
The enzymatic hydrolysis condition of 100R, adipose tissue enzymolysis are not thorough, and ADSCs losses are larger；The present invention is using I clostridiopetidase A and trypsase
Mixed enzyme solution, adipose tissue reach maximization in the short time by the acquisition rate of thorough enzymolysis, ADSCs.In addition, separate
ADSCs after the high inoculation of the vigor of ADSCs can be grown after three days is fused to 80%-90%.
Description of the drawings
Situation adherent after being fat stem cell inoculation 1d Fig. 1；
Fig. 2 compares situation for fat stem cell vigor；
Fig. 3 is fat stem cell form comparison diagram；
Fig. 4 is two kinds of enzymolysis separated fat stem cell skeletonization adipogenic induction Experimental comparisons of scheme.
Specific embodiment
Embodiment 1
1) adipose tissue containing tissue fluid is stood, 10mL pipettes are inhaled and abandoned down after adipose tissue and liquid natural layering
Square liquid；Isometric PBS is drawn with 50mL syringes to be added in adipose tissue, rocks fatty group repeatedly after covering bottle cap
It knits, stands 5min；Solution (being repeated once) below adipose tissue is abandoned with the suction of 25mL pipettes.
2) disposable pipette moves to adipose tissue in 50mL centrifuge tubes；Add in isometric type i collagen enzyme and pancreas egg
White enzyme mixation (mass percent concentration is respectively 0.25%, 0.05%), the bottle that turns upside down after sealing rocks, fully mixed
It is even.It is transferred in 37 DEG C of constant temperature gas bath shaking tables, 200R digestion 50min
3) often pipe adds in 1mLFBS, covers seal after bottle cap and shakes up, 1500rpm/min, centrifugation 5min；Supernatant is abandoned after centrifugation
Liquid.
4) according to fatty volume：The DMEM=1 of 10%FBS:ADSCs is resuspended with the DMEM containing 10%FBS in 1 ratio
Precipitation forms cell suspension after blowing and beating uniformly；Suspension is seeded in the DMEM in high glucose culture medium containing 10%FBS.
5) ADSCs after inoculation is transferred to 5%CO2, 37 DEG C, cultivate in the cell incubator that saturated humidity is 95%.
6) after cultivating 48h, culture medium in ware is abandoned in suction, adds in 10mL/ wares PBS cleaning (being repeated 2 times).Add 10%FBS's
DMEM, 10ng/mLEGF are shifted and are continued to cultivate in cell incubator.
Embodiment 2
1) adipose tissue containing tissue fluid is stood, 10mL pipettes are inhaled and abandoned down after adipose tissue and liquid natural layering
Square liquid；Isometric PBS is drawn with 50mL syringes to be added in adipose tissue, rocks fatty group repeatedly after covering bottle cap
It knits, stands 5min；Solution (being repeated once) below adipose tissue is abandoned with the suction of 25mL pipettes.
2) disposable pipette moves to adipose tissue in 50mL centrifuge tubes；Add in isometric type i collagen enzyme and pancreas egg
White enzyme mixation (mass percent concentration is respectively 0.05%, 0.1%), the bottle that turns upside down after sealing rocks, abundant mixing.
It is transferred in 37 DEG C of constant temperature gas bath shaking tables, 100R digestion 30min
3) often pipe adds in 1mLFBS, covers seal after bottle cap and shakes up, 1000rpm/min, centrifugation 3min；Supernatant is abandoned after centrifugation
Liquid.
4) according to fatty volume：The DMEM=1 of 10%FBS:ADSCs is resuspended with the DMEM containing 10%FBS in 1 ratio
Precipitation forms cell suspension after blowing and beating uniformly；Suspension is seeded in the DMEM in high glucose culture medium containing 10%FBS.
5) ADSCs after inoculation is transferred to 5%CO2, 37 DEG C, cultivate in the cell incubator that saturated humidity is 95%.
6) after culture for 24 hours, culture medium in ware is abandoned in suction, adds in 10mL/ wares PBS cleaning (being repeated 2 times).Add 10%FBS's
DMEM, 10ng/mLEGF are shifted and are continued to cultivate in cell incubator.
Embodiment 3
1) adipose tissue containing tissue fluid is stood, 10mL pipettes are inhaled and abandoned down after adipose tissue and liquid natural layering
Square liquid；Isometric PBS is drawn with 50mL syringes to be added in adipose tissue, rocks fatty group repeatedly after covering bottle cap
It knits, stands 5min；Solution (being repeated once) below adipose tissue is abandoned with the suction of 25mL pipettes.
2) disposable pipette moves to adipose tissue in 50mL centrifuge tubes；Add in isometric type i collagen enzyme and pancreas egg
White enzyme mixation (mass percent concentration is respectively 0.3%, 0.01%), the bottle that turns upside down after sealing rocks, abundant mixing.
It is transferred in 37 DEG C of constant temperature gas bath shaking tables, 250R digestion 60min
3) often pipe adds in 1mLFBS, covers seal after bottle cap and shakes up, 1500rpm/min, centrifugation 10min；It is abandoned after centrifugation
Clear liquid.
4) according to fatty volume：The DMEM=1 of 10%FBS:ADSCs is resuspended with the DMEM containing 10%FBS in 1 ratio
Precipitation forms cell suspension after blowing and beating uniformly；Suspension is seeded in the DMEM in high glucose culture medium containing 10%FBS.
5) ADSCs after inoculation is transferred to 5%CO2, 37 DEG C, cultivate in the cell incubator that saturated humidity is 95%.
6) after cultivating 72h, culture medium in ware is abandoned in suction, adds in 10mL/ wares PBS cleaning (being repeated 2 times).Add 10%FBS's
DMEM, 10ng/mLEGF are shifted and are continued to cultivate in cell incubator.
Reference examples 1
Remaining condition is identical, using type i collagen enzyme in 0.075% Type I collagen enzymes extraction embodiment 1 and trypsase
Mixed liquor.
Table 1
| 0.075% Type I collagen enzyme | Mixed liquor | |
| The quantity of ADSCs | 0.86×107cell | 2.89×107cell |
| Total cell quantity | 2.21×107cell | 3.79×107cell |
| The percentage composition of cell | 39.11% | 74.6% |
Embodiment 1 separated with two kinds of enzymolysis schemes in reference examples 1 after 1 institute of fat stem cell content balance such as Fig. 1 and table
Show (in units of the fatty volume of 10mL), Figure 1A is feelings adherent after the fat stem cell inoculation 1d of 0.075% Type I collagen enzyme
Condition, B are situation adherent after the fat stem cell inoculation 1d of mixed enzyme.
Two kinds enzymolysis schemes separation after fat stem cell vigor to shown in such as table 2 and Fig. 2 (with the fatty volume of 10mL
For unit) A is the cell viability of 0.075% Type I collagen enzyme in Fig. 2, B is the cell viability of mixed enzyme solution, trypan blue staining
(living cells is refused to contaminate, and dead cell is dyed to blueness).
Table 2
| 0.075% Type I collagen enzyme | Mixed enzyme solution | |
| Living cells quantity | 0.94×107cell | 3.32×107cell |
| Total cell quantity | 2.21×107cell | 3.79×107cell |
| Living cells (%) | 42.63% | 92.59% |
Fat stem cell form comparison after 4 two kinds of enzymolysis scheme separation of embodiment
Preceding two width is that 1 mixed enzyme solution of embodiment digests the cell come in Fig. 3, and rear two width is 0.075% Type I collagen enzyme
The cell come is digested, P0-4x-3d, that is, primitive cell culture was amplified to observed at 40 times under the microscope to the 3rd day
Cellular morphology, P0-10x-3d, that is, primitive cell culture was amplified to observed thin at 100 times under the microscope to the 3rd day
Born of the same parents' form.
From the four width figures of Fig. 3 as it can be seen that mixed enzyme solution digests, the cell proliferation rate come is fast, and cell is i.e. reachable at 3 days
To 80% or so fusion rate.Fat stem cell cell is shuttle-type form, fresh rare to branched cell.0.075% Type I collagen
Enzyme digests the fat stem cell growth rate come significantly lower than the former, and the cell confluency of 3 days only has 40% or so, and thin
Born of the same parents' form is mostly irregular, branched.
Embodiment 5
Two kinds of enzymolysis separated fat stem cell skeletonization adipogenic induction Experimental comparisons of scheme
It was found from the picture of Fig. 4, A, B and C are the cells obtained after 1 mixed enzyme of embodiment enzymolysis, and A is control
Group, do not tested by skeletonization adipogenic induction, B are that adipogenic induction is tested, C is osteogenic induction experiment.
D, tri- width figure of E, F is the cell that " 0.075% Type I collagen enzyme " enzymolysis scheme is obtained.Wherein D is control group, is not had
Have by the experiment of skeletonization adipogenic induction.E is adipogenic induction experiment.F is osteogenic induction experiment, as seen from the figure, mixed enzyme
In skeletonization is tested into fat induction, good differentiation capability is presented in acquired fat stem cell.It is tested in adipogenic induction
In, after oil red dyes, it is seen that the adipocyte that stem cell is differentiated to form contains apparent fat drips；In osteogenic induction experiment,
There are apparent bony structures after alizarin red colouring in fat stem cell；This illustrates that the fat acquired in the enzymolysis scheme is done carefully
Born of the same parents still maintain good dryness.But the fat stem cell extracted in 0.075% Type I collagen enzyme, in skeletonization into fat
In Induction experiments, it is inferior to the former into fat and osteogenic ability.It was found from experimental result, mixed enzyme solution does fat stem cell
Property does not influence.
Claims (3)
1. the primary isolated culture method of a kind of fat stem cell, which is characterized in that comprise the following steps：
1) adipose tissue, cleaning are obtained；
2) mixing liquid of isometric type i collagen enzyme and trypsase is added in adipose tissue；I types wherein in mixing liquid
The mass percent of clostridiopetidase A and trypsase is respectively 0.05%~0.3% and 0.01%~0.1%, is digested after abundant mixing
30~60min；
3) FBS is added in, covers seal after bottle cap and shakes up, centrifugation；Supernatant is abandoned after centrifugation；
4) cell precipitation is resuspended with the DMEM containing FBS, the volume ratio of cell precipitation and the DMEM containing FBS are 1:1, piping and druming is equal
Cell suspension is formed after even；It will be in cell suspension inoculation to the DMEM in high glucose culture medium containing FBS；
5) media transfer after inoculation is cultivated into cell incubator；
6) after cultivating 24~72h, culture medium in ware is abandoned in suction, and the DMEM containing EGF and FBS is added after cleaning, shifts cell culture
Continue to cultivate in case；
Wherein, the step 4) and 6) in DMEM in FBS percentage by volume for 10%, and in the step 6) DMEM
The concentration of EGF is 10ng/mL.
2. primary isolated culture method according to claim 1, which is characterized in that step (2) digestion is at 37 DEG C
Constant temperature gas bath shaking table in carry out, shake speed as 100~250r/min.
3. primary isolated culture method according to claim 1, which is characterized in that the rotating speed of step (3) described centrifugation is
1000~1500rpm, centrifugation time are 3~10min.
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| US20180298342A1 (en) * | 2015-10-08 | 2018-10-18 | Kaohsiung Medical University | Composition for rapidly separating adipose tissue-derived stromal cells |
| CN106754685A (en) * | 2017-01-17 | 2017-05-31 | 四川华皓生物科技有限公司 | A kind of construction method of Human fat mesenchymal stem cell bank |
| CN106957818A (en) * | 2017-05-03 | 2017-07-18 | 泰州市数康生物科技有限公司 | A kind of method for efficiently promoting fat stem cell to breed and its kit |
| CN108315297B (en) * | 2018-02-26 | 2022-04-05 | 福建省银丰干细胞工程有限公司 | Method for separating and purifying adipose-derived stem cells from adipose tissues |
| CN108728411B (en) * | 2018-06-26 | 2021-06-25 | 吉林省太阳鸟再生医学工程有限责任公司 | Isolated culture method of adipose-derived stem cells |
| CN112779213A (en) * | 2021-02-04 | 2021-05-11 | 河南科技大学 | Dog beige adipose-derived stem cell in-vitro separation culture method |
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| CN102485885A (en) * | 2011-06-09 | 2012-06-06 | 臻景生物技术（上海）有限公司 | Separating method and application of fat stem cells |
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| CN101077426A (en) * | 2006-05-25 | 2007-11-28 | 上海国睿生命科技有限公司 | Autologous fat stem cell constructing tissue engineering cartilage |
| CN102666840A (en) * | 2009-10-23 | 2012-09-12 | Rnl生物技术株式会社 | Method for inducing migration of adult stem cells derived from adipose tissue |
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