CN106978387A - The new method of keratinocyte is extracted in a kind of digestion of improvement - Google Patents
The new method of keratinocyte is extracted in a kind of digestion of improvement Download PDFInfo
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- CN106978387A CN106978387A CN201710206875.0A CN201710206875A CN106978387A CN 106978387 A CN106978387 A CN 106978387A CN 201710206875 A CN201710206875 A CN 201710206875A CN 106978387 A CN106978387 A CN 106978387A
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- digestion
- keratinocyte
- tissue
- trypsin solution
- trypsin
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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
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
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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
- C12N2509/10—Mechanical dissociation
Abstract
The invention belongs to histocytology field, and in particular to a kind of external digestion separates the new method of keratinocyte.The invention provides a kind of method of the repetition digestion of dynamic repeatedly.Dynamical fashion carries out alternate agitation clockwise and anticlockwise with suction pipette head to add to be pre-heated to after 37 DEG C of 0.25% trypsin solution in the ware for hold tissue in digestion process in ware, tissue and trypsin solution is fully contacted, the keratinocyte of relatively early digestion is rapidly separated tissue.Stirring digestion suctions out trypsin solution immediately after carrying out 2 minutes, and now trypsin solution contains the keratinocyte of a certain amount of relatively early digestion separation, is added into preprepared digestion and stops solution, that is, completes once to digest.It is repeatedly to be repeated 5 times above-mentioned digestion process.Compare with traditional method, dynamically method repeatedly significantly improves the quantity of separation keratinocyte.
Description
Technical field
The present invention relates to the new method that a kind of external digestion separates keratinocyte, belong to histocytology field.
Background technology
Keratinocyte is the chief component for constituting epidermal cell, is that a kind of can secrete to form keratoprotein
Cell, according to its different differentiation process, in skin texture be distributed as basal cell layer, stratum spinosum epidermidis, granular cell layer,
Cuticula.Keratinocyte is isolated from skin from Briggaman in 1967, so far over 50 years, cultural method is ceaselessly
Development and differentiation, culture technique are constantly improving.The keratinocyte of in vitro culture has the ability that propagation is repaired, Ke Yiyi
Plant and repaired in burn wound.What burn patient often faced in wound repairing is the limited of skin donor site, in minimum confession
It is particularly important that surface product, which turns out more cells to be used for wound repairing,.In culture cell processes, it is necessary first to by cutin
Form cell extraction to separate and then could be cultivated, present wide variety of method is two step enzyme digestions, the first step
Epidermis layer tissue and corium layer tissue are separated with neutral proteinase or Collagenase etc., second again with trypsase by table
Cortical tissue's digestion is separated into individual cells.The wherein cellifugal link of digestion point certainly exists a part of cell not from tissue
Digest and cause to waste, and if conventional trypsinization method crosses digestion tissue for a long time, although be more sufficiently separated thin
Born of the same parents, but due to prolonged digestion damaging cells function, how to obtain maximized thin in minimum skin
Born of the same parents' amount keeps the cell function of greater activity to be the key factor for improving cell culture efficiency simultaneously.
The content of the invention
In order to overcome keratinocyte is thorough in digestion separation process in the prior art to digest and long-time
Thoroughly digest and influence the defect of cytoactive, it is an object of the invention to provide a kind of digestion point of the keratinocyte of improvement
From extracting method, to solve the above problems.
The invention provides a kind of method of the repetition digestion of dynamic repeatedly, including dynamic mode and multiple repetition.
It is further to say that described dynamical fashion is that suction pipette head is used in digestion process in the vessel used in digestion
Alternate agitation clockwise and anticlockwise is carried out, tissue and trypsin solution is fully contacted, makes the keratinocyte of digestion
It is rapidly separated tissue.Stirring digestion suctions out trypsin solution immediately after carrying out 2 minutes, and now trypsin solution contains necessarily
The keratinocyte of the relatively early digestion separation of amount, is added into preprepared digestion and stops (soybean trypsin in solution
Stop solution or the DMEM nutrient solutions containing 10% serum), that is, complete once to digest.
Further say it is repeatedly that above-mentioned dynamic digestion step repeats 5 times.
It is further to say the epidermis layer tissue for needing to be ready to isolate before trypsinization cell, separate epidermis
Need to need through not calcium-magnesium-containing after skin histology is cut into width about 0.3cm, long 1-3cm leather strap, separation epidermis layer tissue before layer
PBS liquid cleans at least 1 time and does not shred the tissue.
It is further say used in digestive ferment be through shifting to an earlier date 0.25% trypsin solution that water-bath preheating is 37 DEG C, and
Kept for 37 DEG C, the tryptose enzyme amount added in the vessel for holding epidermis layer tissue is more than 5 times of tissue volume, it is ensured that completely
Flood tissue.
Brief description of the drawings
Fig. 1 is the schematic diagram of operating process of the present invention；
1. the 50ml centrifuge tubes that trypsase stops liquid are filled in figure, 2. facility is 37 DEG C of thermostat water bath, is 3. filled
Concentration is 0.25% trypsase-EDTA container, 4. 1ml suction pipette heads, 5. epidermis layer tissue, 6. culture dish.
The schematic diagram illustrates the main operational steps of the present invention, will be preheated to 37 DEG C of trypsin-EDTA solutions 3.
(the right dotted arrow) culture dish is transferred to, by alternate agitation clockwise and anticlockwise (two solid arrows), is then turned again
Move to (left side dotted arrow) fill trypsase stop liquid 50ml centrifuge tubes 1. carry out termination digestion.
Embodiment
Example further describes and examines the present invention in detail below, and the present invention is not limited only to the example.The present invention's
In the range of or in the juche idea and content for not departing from the present invention, the epidermis that can be used for extracting other animals such as mouse is thin
Born of the same parents, can also make appropriate adjustment, for this area for the concentration of such as trypsase, dosage, time and number of repetition
Technical staff for be it will be apparent that and being included within the scope of the present invention.
Example
Skin sample is peritomized postoperative tissue from Urology Surgery, and tissue specimen is cleaned with containing three anti-DPBS
Bloodstain, trimming hypodermis is carried out with eye scissors, then is cut into width about 0.3cm, and long 1-2cm leather strap is transferred to 15ml centrifuge tubes,
The dispase solution (0.22um membrane filtrations are degerming) that the concentration diluted with DPBS is 0.125% is filled it up with, centrifuge tube is rocked to skin
Piece even suspension in a liquid, lies against 4 DEG C of refrigerator overnights.Next day takes out centrifuge tube, separation and Extraction is carried out in super-clean bench thin
Born of the same parents are tested, and skin graft and solution are poured into culture dish, and leather strap is transferred into another culture dish with small tweezer, add what is resisted containing three
DPBS, is rinsed, and culture dish lid mouthful is positioned over into ultra-clean table top upward, epidermis is isolated from tissue with small tweezer in ware lid
Layer, carries out rinsing 3 times with containing three anti-DPBS again.
By the epidermis layer tissue of separation dry sterile gauze block suck dry moisture, two parts are separated into scissors, are randomly divided into
Experimental group and control group, weigh and record respectively on electronic balance.
Experimental group is the multiple trypsinization method of dynamic, prepares 50ml centrifuge tubes first and adds 15ml containing 10%
The DMEM nutrient solutions of hyclone, separation epidermal cell before in advance by concentration be 0.25% trypsin-EDTA solutions in water
Bathtub is preheated to 37 DEG C, and the experimental group epidermis layer tissue after weighing is put into 60mm culture dishes, adds 3ml and is preheated to 37 in advance
DEG C 0.25% trypsin-EDTA solutions, visible trypsin solution color is persistently stirred after 2min with micropipette tip and is turned
For yellowish-brown, added after solution is drawn in the above-mentioned centrifuge tube for getting nutrient solution ready, micropipettor pressure-vaccum is mixed trypsin solution
And nutrient solution, then take 3ml 0.25% trypsin-EDTA solutions, with after micropipette tip stir about 2min by solution
Be again transferred in centrifuge tube, be repeated 5 times altogether, by liquid in 50ml centrifuge tubes filtered with 200 mesh filter screens to another 50ml from
Heart pipe, adds residual cell in a small amount of DPBS cleaning centrifuge tube and refilters to another centrifuge tube, at room temperature by filtrate carry out from
The heart, 180g, 5min.
Control group is traditional enzyme digestion, and the control group epidermis layer tissue after weighing is put into 60mm culture dishes, is used
Eye scissors shreds into microgranular, size about 1mm；0.25% trypsin-EDTA solutions 3ml is added, is digested under 37 DEG C of environment
Taken out after about 10min, add the DMEM nutrient solutions that 3ml contains 10% hyclone, carry out pressure-vaccum with 5ml pipettes and be mixed, it is seen that
Mixed liquor is sticky, makes cell detachment tissue by pressure-vaccum, and mixed liquor is filtered to 50ml centrifuge tubes and by above-mentioned with 200 mesh filter screens
Mode is centrifuged.
Two groups of cell suspension 10ul described above are respectively taken to be separately added into two 0.6mlEP pipes, it is each to add 10ul trypan blues
Mix, counted after 1min on cell counting count board after solution, non-viable non-apoptotic cell is blueness, living cells is colorless and transparent, uses blood cell
Tally distinguishes living cell counting and non-viable non-apoptotic cell under inverted phase contrast microscope.Unit of account weight epidermis tissue extraction is lived
Cell quantity [living cells quantity ÷ epidermis layer tissues are weighed], calculating cell survival rate [living cells quantity ÷ (living cells quantity+
Non-viable non-apoptotic cell number) × 100%].This experiment is repeated 8 times.As a result represented with mean ± standard deviation, the pairing t in Statistics Application
The method of inspection is compared.
Dynamic be repeated several times digestion method and traditional digestion method digestive efficiency compare (n=8,)
The cell that this experiment is extracted by Trypan Blue and blood counting chamber to two methods is counted, by upper table knot
Fruit understands that the number of viable cells that new method is extracted is significantly more than conventional method, about the 3 of conventional method times, although living cells is accounted for
The ratio of total cell number is slightly relatively low compared with conventional method, and this is probably due to what is digested is enough thorough, by epidermis layer tissue most top layer
Adjust the hypercellularity digestion separation died.The horn cell number for the work extracted in a word is significantly improved.
Claims (3)
1. a kind of method by the multiple enzymic digestion of dynamic extracts keratinocyte, it is characterised in that dynamical fashion and multiple
Reuse 37 DEG C of trypsin solutions and carry out digestion separation epidermal cell.
2. dynamical fashion as claimed in claim 1 refers to stand after being preheated to 37 DEG C of trypsin solutions addition epidermis layer tissues
Persistently give and stir, tissue and trypsin solution is fully contacted by alternate agitation clockwise, counterclockwise, after 2 minutes,
Trypsin solution is suctioned out immediately to add in the trypsase termination liquid being made ready beforehand for.It is characterized in that digestion process is dynamic
And nonstatic.
3. as claimed in claim 1 be repeated several times refers to repeat the mode in claim 25 times.It is characterized in that
Carry out avoiding disposable damage of the digestion to cell for a long time by several times, it also avoid short time digestion and cause digestion not thorough enough
Bottom.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710206875.0A CN106978387A (en) | 2017-03-31 | 2017-03-31 | The new method of keratinocyte is extracted in a kind of digestion of improvement |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710206875.0A CN106978387A (en) | 2017-03-31 | 2017-03-31 | The new method of keratinocyte is extracted in a kind of digestion of improvement |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004011598A2 (en) * | 2002-05-24 | 2004-02-05 | The Research Foundation Of State University Of New York | Long lived keratinocytes |
| CN103003415A (en) * | 2010-05-21 | 2013-03-27 | 罗德里戈·福西翁·索托帕雷哈 | Process for the production of patches or dressings of autologous skin through cultivation of autologous keratinocytes and fibroblasts with autologous serum for the generation of skin |
-
2017
- 2017-03-31 CN CN201710206875.0A patent/CN106978387A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004011598A2 (en) * | 2002-05-24 | 2004-02-05 | The Research Foundation Of State University Of New York | Long lived keratinocytes |
| US20040214323A1 (en) * | 2002-05-24 | 2004-10-28 | Marcia Simon | Long lived keratinocytes |
| CN103003415A (en) * | 2010-05-21 | 2013-03-27 | 罗德里戈·福西翁·索托帕雷哈 | Process for the production of patches or dressings of autologous skin through cultivation of autologous keratinocytes and fibroblasts with autologous serum for the generation of skin |
Non-Patent Citations (5)
| Title |
|---|
| ANDERS PATRIK GUNNARSSON ET AL.: ""Isolating subpopulations of human epidermal basal cells based on polyclonal serum against trypsin-resistant CSPG4 epitopes"", 《EXPERIMENTAL CELL RESEARCH》 * |
| XIN WANG ET AL.: ""Efficient isolation and high yield of epidermal cells from foreskin biopsies by dynamic trypsinization"", 《BURNS》 * |
| 李伟 等: ""人角质形成细胞库的建立"", 《第三军医大学学报》 * |
| 欧阳安力 等: ""胰酶对皮肤角质细胞分离和传代的影响"", 《生物工程学报》 * |
| 王鑫: ""表皮细胞联合MEEK微型皮片移植技术修复深度创面的实验研究"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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| TA01 | Transfer of patent application right |
Effective date of registration: 20191118 Address after: 100048 Beijing city Haidian District Fuchengmen Road No. 51 Applicant after: Fourth Medical Center, General Hospital of the Chinese People's Liberation Army Address before: 100048 No. 51 Fu Cheng Road, Beijing, Haidian District Applicant before: Shen Chuanan Applicant before: Wang Xin Applicant before: Li Dawei |
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Application publication date: 20170725 |
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