CN105400733A - Primary separation method of animal skin fibroblasts - Google Patents

Primary separation method of animal skin fibroblasts Download PDF

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Publication number
CN105400733A
CN105400733A CN201510862177.7A CN201510862177A CN105400733A CN 105400733 A CN105400733 A CN 105400733A CN 201510862177 A CN201510862177 A CN 201510862177A CN 105400733 A CN105400733 A CN 105400733A
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China
Prior art keywords
skin
cell
separation method
primary separation
animal skin
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CN201510862177.7A
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Chinese (zh)
Inventor
葛啸虎
陈海佳
王一飞
吴子杰
李平
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Priority to CN201510862177.7A priority Critical patent/CN105400733A/en
Publication of CN105400733A publication Critical patent/CN105400733A/en
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Abstract

The invention discloses a primary separation method of animal skin fibroblasts, which utilizes a skin injury healing mechanism, selects a proper healing time period to obtain a large amount of proliferative fibroblasts, shortens enzyme digestion time, can well overcome the technical defects, can increase the number of cells, reduces the influence of enzyme on the cells, and improves the cell separation efficiency and the wall adhesion effect.

Description

A kind of animal skin fibroblast cell primary separation method
Technical field
The present invention relates to a kind of cell primary separation method, particularly relate to a kind of animal skin fibroblast cell primary separation method, belong to stem cell and cultivate field.
Background technology
Aging, the various adventitious agents that current inoblast is mainly used for studying cell are to the damage of cell, cell vicious transformation under in vitro conditions and some inborn error of metabolism, enzyme defect etc.Because skin flbroblast is easy to obtain, be easy to again grow in vitro, skin flbroblast is cultivated and has been obtained using more widely in preclinical medicine and clinic study.
For fibroblastic acquisition, current existing primary separation method mainly contains two kinds: (1) enzyme digestion.With trypsinase or collagenase to containing dual anti-PBS rinsed clean and the skin histology shredded digest, until cellular segregation is out, cross 200 sieves, centrifugal, resuspended, counting cells density inoculation culture.(2) tissue explants adherent method.Respectively with containing dual anti-PBS and the clean skin histology of 75% ethanol rinse, eye scissors shreds into 1mm skin histology 3tissue block, cultivates in 6 orifice plates with culture medium inoculated.
For enzyme digestion, although can obtain cell within a short period of time, enzyme itself has a certain impact to the adherent of cell, repeatedly centrifugally also causes certain physical abuse to cell, and cause Cell viability not high, adherent rate is low.And the relative enzymolysis process step of tissue explants adherent method is comparatively simple, but the cycle is long, and adherent stability is low, and it is low that cell climbs out of rate.
Summary of the invention
The object of the present invention is to provide the animal skin fibroblast cell primary separation method that a kind of Cell viability is higher, adherent rate is higher.
Object of the present invention is achieved by the following technical programs.
A kind of animal skin fibroblast cell primary separation method, it comprises the following steps:
Choose animal skin, preferred skin of abdomen, skin, through the tincture of iodine and/or alcohol disinfecting, does a full-thickness cutaneous wound, preferably does the circular full-thickness cutaneous wound that a diameter is 1.0cm ~ 3.0cm.
In skin injury 3-5 days, take off skin area after damage, subcutis and all blood vessels are removed clean, clean skin 2 ~ 3 times with containing 2% dual anti-PBS.
Skin is gone in centrifuge tube, the centrifuge tube of preferred 50ml, then according to skin area 1mL/cm 2standard, adding quality hundred parts of ratios is the trypsinase of 0.1%-0.25%, 37 DEG C place 1-2 hour.
Remove the tissue and impurity that cannot digest in Digestive system, remove the tissue and impurity that cannot digest in Digestive system preferably by the mode of sieving, then 1000rpm/min low-speed centrifugal 5min, remove supernatant liquor; Pour into appropriate containing 1% dual anti-PBS, then 1000rpm/min low-speed centrifugal 5min again, remove supernatant liquor, to remove residual trypsinase.
Cell after centrifugal, according to (1-10) × 10 5density criterion, add substratum re-suspended cell, excellent substratum is: DMEM+10%FBS+2% is dual anti-.
Resuspended cell suspension is evenly seeded to and has wrapped by the Tissue Culture Dish of 0.2% gelatin, put to 37 DEG C, 5%CO 2cultivation 2-5 days is carried out, preferred 9cm Tissue Culture Dish in incubator.
Described bag is in Tissue Culture Dish, add 0.2% gelatin in advance by the Tissue Culture Dish of 0.2% gelatin, hatches 30min.
Compared with prior art, animal skin fibroblast cell primary separation method of the present invention has the following advantages: the mechanism that (1) the present invention utilizes skin injury to heal, skin is damaged, to stimulate fibroblastic growth, coordinate enzyme digestion again, to increase cell quantity, reduce enzyme to impact cell, obtain the inoblast of more inoblast and the low apoptosis type of high proliferation, improve cellular segregation efficiency and adherent effect.(2) number of days selecting skin injury healing is 3-5 days, and this time, inoblast was in proliferation period, and apoptosis is in low phase.(3) when digesting skin, first remove subcutis, reduce the impact of tissue on isolated cell.(4) shorten the tryptic digestion time, reduce the impact of cell and increase work efficiency.(5) when inoculation, carry out 0.2% gelatin bed board in advance, effectively can improve the adherent speed of skin histology and adherent rate.
Accompanying drawing explanation
Fig. 1 is the fibroblast proliferation curve after the primary separation of Mouse Skin Fibroblasts of embodiment 4;
Fig. 2 is the streaming qualification figure of the control group mice skin flbroblast of embodiment 5, and wherein 2-A is the streaming qualification figure of CK15; 2-B is the streaming qualification figure of Vimentin;
Fig. 3 is the streaming qualification figure of the experimental mice skin flbroblast of embodiment 5, and wherein 3-A is the streaming qualification figure of CK15; 3-B is the streaming qualification figure of Vimentin.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further illustrated.
Embodiment 1
The primary separation method of a kind of Mouse Skin Fibroblasts of the present embodiment, it comprises with step:
1.1 choose mouse part skin, a little hair are cut with elbow scissors, and skin, through the tincture of iodine, alcohol disinfecting, does the circular full-thickness cutaneous wound that a diameter is 2.0cm, and wound exposes and continues to raise.
In 1.2 skin injuries (3-5) sky, put to death mouse, take off skin area after damage, subcutis and all blood vessels are removed clean, clean skin 3 times with containing 2% dual anti-PBS.
1.3 skins go in 50mL centrifuge tube, add 7ml (according to skin area 1mL/cm 2selective agar medium volume) (0.1%-0.25%) trypsinase, place 1-2 hour.
Digestive system is crossed 200 order cell mesh screens by 1.4, removes the tissue and magazine that cannot digest, 1000rpm/min low-speed centrifugal 5min, remove supernatant, pour into appropriate containing 1% dual anti-PBS, remove residual trypsinase, 1000rpm/min low-speed centrifugal 5min, removes supernatant.
1.5 centrifugal after cell, according to (1-10) × 10 5density, add substratum (DMEM+10%FBS+2% is dual anti-) re-suspended cell.
1.6 add 0.2% gelatin in advance in 9cm Tissue Culture Dish, and 1mL/ hole, hatches 30min, reclaim gelatin.Cell suspension is evenly seeded in 9cm Tissue Culture Dish, puts to 37 DEG C, 5%CO 2cultivate in incubator.
Embodiment 2
The present embodiment is the primary separation method of traditional conventional mouse skin flbroblast, comprises the following steps:
2.1 put to death mouse, and a little hair is cut with elbow scissors, skin, through the tincture of iodine, alcohol disinfecting, takes off the skin histology that diameter is 2cm, clean skin 3 times with containing 2% dual anti-PBS.
2.2 skins go in 50mL centrifuge tube, add and add 7mL/cm according to skin area 2(0.1%--0.25%) trypsinase, places 4-6 hour.
Digestive system is crossed 200 order cell mesh screens by 2.3, removes the tissue and magazine that cannot digest, 1000rpm/min low-speed centrifugal 5min, remove supernatant, pour into appropriate containing 1% dual anti-PBS, remove residual trypsinase, 1000rpm/min low-speed centrifugal 5min, removes supernatant.
2.4 centrifugal after cell, according to (1-10) × 10 5density, add substratum (DMEM+10%FBS+2% is dual anti-) re-suspended cell.
2.5 cell suspensions are evenly seeded in 9cm Tissue Culture Dish, put to 37 DEG C, 5%CO 2cultivate in incubator.
Embodiment 3
The present embodiment is the cell quantity contrast after Mouse Skin Fibroblasts is separated
Use the mouse of method process three different batches of embodiment 1 (experimental group) and embodiment 2 (control group) respectively, calculate the cell count of every square centimeter of skin after being separated.Inoculation observation of cell growing state rear every day.
Cell count after two schemes are separated, as following table:
Table 1: the cell quantity contrast table after experimental group is separated with the skin fibroblast of control group
As seen from the table, experiment component from cell count be more than 2 times of control group, prove that the present invention program greatly can improve the cell quantity of separation.
Embodiment 4
The present embodiment is the fibroblast proliferation curve after the primary separation of Mouse Skin Fibroblasts.
CCK-8 method is adopted to detect the fibroblastic proliferation activity cultivated.Be separated according to the separation method of embodiment 1 (experimental group) with embodiment 2 (control group) respectively, when cell confluency degree grows to 90%, respectively collecting cell, inoculating cell in 96 orifice plates, 2000/hole.At 37 DEG C, 5%CO 2cultured continuously 7d in incubator, detects cell at 1d, 3d, 5d, 7d respectively.10 μ L staining agents are added, 37 DEG C, 5%CO in gaging hole to be checked 2cultivate 2h.Survey the light absorption value at 450nm place by microplate reader, each sample does 3 repetitions, the results are shown in Table 2 and Fig. 1.
Table 2: light absorption value contrast table after experimental group is separated with the skin fibroblast small cell of control group
From table 2 and Fig. 1, the propagation of experimental group is obviously greater than control group.
Embodiment 5
The present embodiment is the streaming qualification of Mouse Skin Fibroblasts.
5.1 respectively Example 1 be separated with in embodiment 2 cell growing to more than 90%, digest, stop, resuspended, get 1 × 10 6cells, in average mark to a 2 EP pipe, in contrast, the centrifugal 5min of 1500rpm/min, abandons supernatant to a pipe.
5.2 add 1mL dye solution (PBS containing 10%FBS) re-suspended cell, and the centrifugal 5min of 1500rpm/min, abandons supernatant.Repeat above step 1 time.
5.3 abandon supernatant after, often to add 200 μ L dye solution resuspended for pipe, and sample components does not add 2 μ L vimentin antibodies (Vimentin) and keratin antibody (CK15 antibody).Lucifuge hatches 20min.
5.4 to add 500 μ L dye solution resuspended, and the centrifugal 5min of 1500rpm/min washes residue antibody off.
5.5 abandon supernatant, add 500 μ L sample-loading buffers (10%FBS+1640 substratum), cross 200 mesh sieves, upper machine testing cell-surface antigens.
Table 3: experimental group and cellular control unit streaming qualification contrast table
Vimenit CK-15
Control group (embodiment 2) 92.6％ 0％
Experimental group (embodiment 1) 100％ 0％
Fibroblast marker CK15 expresses negative (≤2%), Vimentin surface marker reaches positive (≤95%).From table 2 and Fig. 2, Fig. 3, control group removes inoblast and has mixed other cells, and in experimental group is 100% inoblast, and this separation method gained cell purity is high.
According to embodiment 3-5, separation method of the present invention is more compared to the one-tenth fiber stem cell of traditional separation method gained, and value-added speed is faster, and purity is higher.
The present invention is not limited to above embodiment, it should be pointed out that for those skilled in the art, and under the premise without departing from the principles of the invention, can also make some accommodations, these accommodations also belong to protection scope of the present invention.

Claims (5)

1. an animal skin fibroblast cell primary separation method, it comprises the following steps:
Choose animal skin, skin, through the tincture of iodine and/or alcohol disinfecting, does a full-thickness cutaneous wound;
In skin injury 3-5 days, take off skin area after damage, subcutis and all blood vessels are removed clean, clean skin 2 ~ 3 times with containing 2% dual anti-PBS;
Skin is gone in centrifuge tube, then according to skin area 1mL/cm 2standard, adding quality hundred parts of ratios is the trypsinase of 0.1%-0.25%, 37 DEG C place 1-2 hour;
Remove the tissue and impurity, then 1000rpm/min low-speed centrifugal 5min that cannot digest in Digestive system, remove supernatant liquor; Pour into containing 1% dual anti-PBS, then 1000rpm/min low-speed centrifugal 5min again, remove supernatant liquor, to remove residual trypsinase;
Cell after centrifugal, according to (1-10) × 10 5density criterion, add substratum with re-suspended cell, substratum is: DMEM+10%FBS+2% is dual anti-;
Resuspended cell suspension is evenly seeded to and has wrapped by the Tissue Culture Dish of 0.2% gelatin, put to 37 DEG C, 5%CO 2cultivation 2 ~ 5 days is carried out in incubator;
Described bag is in Tissue Culture Dish, add 0.2% gelatin in advance by the Tissue Culture Dish of 0.2% gelatin, hatches 30min.
2. a kind of animal skin fibroblast cell primary separation method according to claim 1, is characterized in that: the preferred skin of abdomen of described animal skin.
3. a kind of animal skin fibroblast cell primary separation method according to claim 1, is characterized in that: the circular full-thickness cutaneous wound of described full-thickness cutaneous wound to be diameter be 1.0cm ~ 3.0cm.
4. a kind of animal skin fibroblast cell primary separation method according to claim 1, is characterized in that: the tissue that cannot digest in described removal Digestive system and impurity adopt the mode of sieving to carry out according to the size of cell.
5. a kind of animal skin fibroblast cell primary separation method according to claim 1, it is characterized in that: described centrifuge tube is 50ml centrifuge tube, culture dish is 9cm Tissue Culture Dish.
CN201510862177.7A 2015-11-30 2015-11-30 Primary separation method of animal skin fibroblasts Pending CN105400733A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560346A (en) * 2020-05-21 2020-08-21 福建省海西细胞生物工程有限公司 Method for efficiently extracting and proliferating autologous fibroblasts by explant adhesion method

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101353642A (en) * 2007-07-26 2009-01-28 上海市针灸经络研究所 Cultivation method of rat distal colon fibroblast
CN102091352A (en) * 2009-12-09 2011-06-15 中国人民解放军总医院第一附属医院 Method for constructing tissue engineering skin model containing sweat gland
CN104548214A (en) * 2015-02-10 2015-04-29 广州赛莱拉干细胞科技股份有限公司 Artificial skin and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101353642A (en) * 2007-07-26 2009-01-28 上海市针灸经络研究所 Cultivation method of rat distal colon fibroblast
CN102091352A (en) * 2009-12-09 2011-06-15 中国人民解放军总医院第一附属医院 Method for constructing tissue engineering skin model containing sweat gland
CN104548214A (en) * 2015-02-10 2015-04-29 广州赛莱拉干细胞科技股份有限公司 Artificial skin and preparation method thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
周琪: "《生物学实验指南丛书 干细胞实验指南》", 31 July 2015, 中央广播电视大学出版社 *
李煜等: "成年小鼠成纤维细胞体外培养", 《内蒙古大学学报(自然科学版)》 *
秦全红等: "成纤维细胞在皮肤创伤愈合中的作用及其调控", 《国外医学创伤与外科基本问题分册》 *
郭恩覃: "《现代整形外科学》", 31 January 2000, 人民军医出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560346A (en) * 2020-05-21 2020-08-21 福建省海西细胞生物工程有限公司 Method for efficiently extracting and proliferating autologous fibroblasts by explant adhesion method

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Application publication date: 20160316