CN100575481C - A kind of culture method of human outer-secretion sweat gland epithelial cell - Google Patents
A kind of culture method of human outer-secretion sweat gland epithelial cell Download PDFInfo
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- CN100575481C CN100575481C CN200610095140A CN200610095140A CN100575481C CN 100575481 C CN100575481 C CN 100575481C CN 200610095140 A CN200610095140 A CN 200610095140A CN 200610095140 A CN200610095140 A CN 200610095140A CN 100575481 C CN100575481 C CN 100575481C
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Abstract
A kind of cultural method of human outer-secretion sweat gland epithelial cell is cultivated, is gone down to posterity and cultivate and the three-dimensional sweat gland sample tubular structure seven step processes of cultivating that make up are formed by human outer-secretion sweat gland picking, enzymic digestion, centrifugal purification, substratum preparation, two dimension.The inventive method compared with prior art has the following advantages: the one, and the preparation method is easy, and is consistent, good reproducibility; The 2nd, adopt collagenase digestion to obtain one cell, it is few to have avoided tissue block method previously to obtain cell concentration, the shortcoming that cell is impure; The 3rd, adopt serum free medium, avoided the restraining effect of serum cell growth; The 4th, in Matrigel, carry out dimensional culture cell is grown, breed sooner, the more important thing is after section statining and to find to have sweat gland sample tubular structure to form, help experiment in vitro and research, for the reconstruction in vitro sweat gland lays the foundation.
Description
One, technical field
The invention belongs to human cell's cultivation, be specifically related to a kind of extracorporeal culturing method of outer-secretion sweat gland epithelial cell.
Two, background technology
Eccrine sweat gland is one of important appendicle of skin, and the effect of secretion, drainage and regulate body temperature is arranged.The meaning of in-vitro separation and cultivation eccrine sweat gland cell is: it can provide the model of research of transepithelial transporting mechanism and the research of eccrine sweat gland relative disease; Secondly, the skin tissue engineering development so far, substantially, solved the problem that the surface of a wound covers, but do not reach the reconstruction fully of real weave construction and function, in at present all organization engineering skin products, all do not have the appendages of skin, separate and cultivate the various cells of eccrine sweat gland and can lay the foundation with the organization engineering skin of studying the band annex for the three-dimensional reconstruction of sweat gland.
The nineties in last century, the beginning had the investigator to carry out the research of human outer-secretion sweat gland cell cultures and pharmacological characteristics aspect abroad, and research previously all adopts tissue mass cell culture to cultivate.Its shortcoming is that the primary cell culture time of tissue block cultural method is long, and is not that each piece all has the cell eruption, so passage number of times and the cell count finally obtained are less, cell category is impure, makes further to continue further investigation and be restricted.At present, Shang Weijian is open relevant for the patent documentation of culture method of human outer-secretion sweat gland epithelial cell; It is open that the Chinese invention patent that a publication number is arranged is CN1082109A applies on February 16th, 1994, it is " a kind of extracorporeal culture of liver cells ", comprise steps such as getting liver, separation, cleaning, precipitation, digestion, cultivation, inoculation, adopt edge strip ginseng liquid synthetic medium to carry out vitro culture, method is easy, the hepatocyte activity of cultivating is good, and biological property is normal, cultivates but be not suitable for human outer-secretion sweat gland epithelial cell.
Three, summary of the invention
The objective of the invention is to defective at the prior art existence, a kind of culture method of human outer-secretion sweat gland epithelial cell that is different from tissue mass cell culture is provided, make it simple, it is purer to cultivate the human outer-secretion sweat gland epithelial cell that obtains, well-grown, cell proliferation is fast, can repeatedly go down to posterity, and also can induce sweat gland sample tubular structure to form.
Take following technical scheme to realize purpose of the present invention.
A kind of cultural method of human outer-secretion sweat gland epithelial cell is cultivated, is gone down to posterity and cultivate and the three-dimensional sweat gland sample tubular structure seven step processes of cultivating that make up are formed by human outer-secretion sweat gland picking, enzymic digestion, centrifugal purification, substratum preparation, two dimension.
One) human outer-secretion sweat gland picking
At first obtain leather strap after healthy people's the skin surgical blanking, and place D-Hank ' s liquid to wash 3-5 time repeatedly immediately, again with eye scissors shred be placed in the phosphate buffered saline buffer resuspended, then tissue suspension is placed under at least 20 times of dissecting microscopes and observe, with the direct picking human outer-secretion sweat gland of ophthalmology tweezer.
Two) enzymic digestion
It is in 0.4% the IV Collagen Type VI enzyme that the human outer-secretion sweat gland of picking is inserted volume fraction, 37 ℃ of digestion 10-14 hour, and shakes frequently, makes tissue digestion evenly, fully.
Three) centrifugal purification
D-Hank ' s liquid 40-50ml is joined in the good human outer-secretion sweat gland tissue of enzymic digestion, centrifugal 3-5 time repeatedly, each 2000-3000rpm * 5min, the human outer-secretion sweat gland epithelial cell of acquisition purifying is expected blue living cell counting number with 0.2% then.
Four) substratum preparation
In serum-free epidermic cell substratum, add the Urogastron (EGF) of 5 μ g/L and the Niu Chuiti extract of 50mg/L, obtain the substratum of sweat gland epithelial cell, preserve standby.
Five) two dimension is cultivated
The human outer-secretion sweat gland epithelial cell of purifying is resuspended with the substratum of sweat gland epithelial cell, make the resuspended liquid of human outer-secretion sweat gland epithelial cell, join again in the culturing bottle that scribbles collagen and inoculate, inoculum density is 1-100 * 10
3Individual cell/cm
2, cover tight plug, place 37 ℃, 5%CO
2Incubator cultivate, the substratum of changing sweat gland epithelial cell in per 3 days once, every day, sampling placed microscopically observation of cell growing state, reached 70%～80% to the cytogamy degree.
Six) cultivation of going down to posterity
Degrees of fusion reached 70%～80% cell and add pancreatin 0.5ml for every bottle, in 37 ℃ of incubators, digest 5～10min, when cell rounding, when the intercellular substance increases, stop digestion with pancreatin inhibitor, add D-Hank ' s liquid 10ml, it is resuspended that centrifugal 2000-3000rpm * 5min, the cell of acquisition add the substratum of sweat gland epithelial cell again, be inoculated in bottle and be primed with in the culturing bottle of collagen, inoculum density is 1-20 * 10
4Individual/cm
2, place 37 ℃, 5%CO
2Continue amplification cultivation in the incubator; The substratum of per 3 days replacing sweat gland epithelial cells is once taken a sample and is placed microscopically observation of cell growing state every day.
Seven) the three-dimensional sweat gland sample tubular structure that makes up is cultivated
Under condition of ice bath, with the human outer-secretion sweat gland epithelial cell of purifying with 1-50 * 10
3The density of individual/ml, be mixed in the Matrigel gel, join in 12 orifice plates with every hole 2ml, dimensional culture is carried out in the substratum 2ml/ hole that adds sweat gland epithelial cell again, and the substratum of per 3 days replacing sweat gland epithelial cells once, every day sampling places microscopically observation of cell growing state, and the cell mass sampling of incubation growth after 9 days is fixing, section, HE dyeing are observed tubular structure and formed situation.
In culture method of human outer-secretion sweat gland epithelial cell of the present invention, used raw and auxiliary material holostrome skin, phosphate buffered saline buffer, IV Collagen Type VI enzyme, serum-free epidermic cell substratum, Urogastron, Niu Chuiti extract, collagen and Matrigel gel etc. all should meet aseptic requirement.
The inventive method compared with prior art has the following advantages: the one, and the preparation method is easy, and is consistent, good reproducibility; The 2nd, adopt collagenase digestion to obtain one outer-secretion sweat gland epithelial cell, it is few to have avoided tissue block method previously to obtain cell concentration, the shortcoming that cell is impure; The 3rd, adopt serum free medium, avoided the restraining effect of serum cell growth; The 4th, in the Matrigel gel, carry out dimensional culture cell is grown, breed sooner, the more important thing is after section statining and to find to have sweat gland sample tubular structure to form, help experiment in vitro and research, for the reconstruction in vitro sweat gland lays the foundation.
Four, embodiment
Be concrete cultivation example of culture method of human outer-secretion sweat gland epithelial cell of the present invention below, but need to prove that the inventive method never is confined to the cultivation example lifted.
Get holostrome skin 3 * 3cm size of human body armpit anaplasty excision, immediately skin histology is placed D-Hank ' s liquid to wash repeatedly five times, after being cut into lingel, shred with eye scissors again, place the 50ml phosphate buffered saline buffer resuspended again, the tissue suspension that obtains places under 20 times of dissecting microscopes and observes, with the direct picking human outer-secretion sweat gland of ophthalmology tweezer; It is among 0.1% the IV Collagen Type VI enzyme 30ml that the human outer-secretion sweat gland of picking is inserted volume fraction, 37 ℃ of digestion 12 hours, and shakes frequently, makes tissue digestion evenly, fully; In the good human outer-secretion sweat gland tissue of enzymic digestion, add D-Hank ' s liquid to 50ml, centrifugal repeatedly 5 times, each 2500rpm * 5min, the human outer-secretion sweat gland epithelial cell cell of acquisition purifying is expected blue living cell counting number with 0.2% then.
The human outer-secretion sweat gland epithelial cell of purifying is resuspended with the substratum of sweat gland epithelial cell, make the resuspended liquid of human outer-secretion sweat gland epithelial cell, this suspension is joined in the culturing bottle that scribbles mouse tail collagen inoculate again, inoculum density is 5 * 10
4Individual cell/cm
2, cover tight plug, place 37 ℃, 5%CO
2Incubator cultivate, changed the epithelioglandular substratum of sweat gland once in per 3 days, sampling every day places microscopically to observe, can be observed cell attachment after 24 hours, observe cell about 3-4 days and be a plurality of clone's sample wheat sample growths, 3 all left and right sides cytogamy are the growth of paving stone sample, substantially at the bottom of being paved with bottle, sweat gland glandular epithelium after the fusion is flat polygon, and there is circular kernel the centre, and cell grows to degrees of fusion and reaches at 70%～80% o'clock and can be used for experiment or go down to posterity.
Degrees of fusion reached 70%～80% cell and add pancreatin 0.5ml for every bottle, in 37 ℃ of incubators, digest 5～10min, when cell rounding, when the intercellular substance increases, stop digestion with pancreatin inhibitor, add D-Hank ' s liquid 10ml, centrifugal 2500rpm * 5min, it is resuspended that the cell of acquisition adds the epithelioglandular substratum of sweat gland, be inoculated in bottle and be primed with in the culturing bottle of collagen, inoculum density is 1 * 10
4Individual/cm
2, place 37 ℃, 5%CO
2Continue amplification cultivation in the incubator; The substratum of per 3 days replacing sweat gland epithelial cells once, every day, sampling placed microscopically observation of cell growing state, as seen passage cell is adherent in 24 hours, I is similar to former generation for cellular form, major part is flat polygon, become big since II for cell volume, it is irregular that form becomes gradually, and the cell that has stretches out long pseudopodium and adjacent cells joins.The human outer-secretion sweat gland glandular epithelium that present method is cultivated can go down to posterity 3～5 times.
It is three-dimensional that to make up that sweat gland sample tubular structure cultivates be under condition of ice bath, with the human outer-secretion sweat gland epithelial cell of purifying with 5 * 10
3The density of individual/ml is mixed in the Matrigel gel, joins in 12 orifice plates with every hole 2ml, and dimensional culture is carried out in the substratum 2ml/ hole that adds sweat gland epithelial cell again, and the substratum of per 3 days replacing sweat gland epithelial cells once; Every day, sampling placed microscopically observation of cell growing state, and as seen, the cell mass diameter increased about one times in average per two days in preceding 9 days in the Matrigel gel, and there were significant differences between per two days, but proliferation slows after 9 days, no significant difference between the 11st day and the 13rd day.Get the 9th day cell mass HE dyeing, among the visible Matrigel cell is arranged, cell is lumps and distributes, and is arranged in the tube chamber spline structure between the cell mutually, and nucleus indigo plant is dyed, and tenuigenin is red to be dyed.Cell has polarity, surrounds the tube chamber spline structure, is the outer-secretion sweat gland epithelial cell of monolayer alignment on every side, and central authorities are circular tube chamber.
Claims (1)
1, a kind of culture method of human outer-secretion sweat gland epithelial cell is characterized in that:
1) it is cultivated, is gone down to posterity and cultivate and the three-dimensional sweat gland sample tubular structure seven step processes of cultivating that make up are formed by human outer-secretion sweat gland picking, enzymic digestion, centrifugal purification, substratum preparation, two dimension;
2) the human outer-secretion sweat gland picking is at first to obtain leather strap after healthy people's the skin surgical blanking, and place D-Hank ' s liquid to wash 3-5 time repeatedly immediately, again with eye scissors shred be placed in the phosphate buffered saline buffer resuspended, then tissue suspension is placed under at least 20 times of dissecting microscopes and observe, with the direct picking human outer-secretion sweat gland of ophthalmology tweezer;
3) enzymic digestion is that the human outer-secretion sweat gland of picking is inserted in the IV Collagen Type VI enzyme that volume fraction is 0.01-0.8%, 37 ℃ of digestion 4-18 hour, and shakes frequently, makes tissue digestion evenly, fully;
4) centrifugal purification is that D-Hank ' s liquid 40-50ml is joined in the good human outer-secretion sweat gland tissue of enzymic digestion, centrifugal 3-5 time repeatedly, and each 2000-3000rpm * 5min, the human outer-secretion sweat gland epithelial cell of acquisition purifying is expected blue living cell counting number with 0.2%;
5) the substratum preparation is to add the Urogastron of 5 μ g/L and the Niu Chuiti extract of 50mg/L in serum-free epidermic cell substratum, obtains the substratum of sweat gland epithelial cell, preserves standby;
6) the two dimension cultivation is that the human outer-secretion sweat gland epithelial cell of purifying is resuspended with the substratum of sweat gland epithelial cell, makes the resuspended liquid of human outer-secretion sweat gland epithelial cell, joins in the culturing bottle that scribbles collagen again and inoculates, and inoculum density is 1-100 * 10
3Individual cell/cm
2, cover tight plug, place 37 ℃, 5%CO
2Incubator cultivate, the substratum of changing sweat gland epithelial cell in per 3 days once, every day, sampling placed microscopically observation of cell growing state, reached 70%～80% to the cytogamy degree;
7) go down to posterity that to cultivate be degrees of fusion to be reached 70%～80% cell add pancreatin 0.5ml for every bottle, in 37 ℃ of incubators, digest 5～10min, when cell rounding, when the intercellular substance increases, stop digestion with pancreatin inhibitor, add D-Hank ' s liquid 10ml, it is resuspended that centrifugal 2000-3000rpm * 5min, the cell of acquisition add the substratum of sweat gland epithelial cell again, be inoculated in bottle and be primed with in the culturing bottle of collagen, inoculum density is 1-20 * 10
4Individual/cm
2, place 37 ℃, 5%CO
2Continue amplification cultivation in the incubator, the substratum of per 3 days replacing sweat gland epithelial cells is once taken a sample and is placed microscopically observation of cell growing state every day;
It is 8) three-dimensional that to make up that sweat gland sample tubular structure cultivates be under condition of ice bath, with the human outer-secretion sweat gland epithelial cell of purifying with 1-50 * 10
3The density of individual/ml, be mixed in the Matrigel gel, join in 12 orifice plates with every hole 2ml, dimensional culture is carried out in the substratum 2ml/ hole that adds sweat gland epithelial cell again, and the substratum of per 3 days replacing sweat gland epithelial cells once, every day sampling places microscopically observation of cell growing state, and the cell mass sampling of incubation growth after 9 days is fixing, section, HE dyeing are observed tubular structure and formed situation.
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| 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 |
| CN105936888B (en) * | 2015-09-14 | 2017-09-15 | 南京医科大学附属南京儿童医院 | The Isolation and identification method of human tonsil's epithelial cell |
| WO2019031500A1 (en) * | 2017-08-09 | 2019-02-14 | 株式会社マンダム | Immortalized sweat gland myoepithelial cell |
| CN112255398A (en) * | 2020-10-19 | 2021-01-22 | 福建医科大学 | Method for fluorescence labeling of cell mass cells by non-paraffin section method |
| CN112410288A (en) * | 2020-11-12 | 2021-02-26 | 安徽惠恩生物科技股份有限公司 | Method for extracting human exocrine gland stem cells with hair follicle regeneration function |
| CN114107174B (en) * | 2021-11-24 | 2023-10-13 | 中国人民解放军总医院 | In-vitro separation method of primary sweat gland cells |
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