CN102499998A - Dermis equivalent constructing method - Google Patents
Dermis equivalent constructing method Download PDFInfo
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- CN102499998A CN102499998A CN2011104348212A CN201110434821A CN102499998A CN 102499998 A CN102499998 A CN 102499998A CN 2011104348212 A CN2011104348212 A CN 2011104348212A CN 201110434821 A CN201110434821 A CN 201110434821A CN 102499998 A CN102499998 A CN 102499998A
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Abstract
The invention discloses a dermis equivalent constructing method. The dermis equivalent constructing method comprises the following steps of: firstly, conducting sterile treatment on a human amniotic membrane, preparing the treated human amniotic membrane and a nitrocellulose membrane into a human amniotic membrane support; taking a milk goat ear skin specimen, removing dirty matters, disinfecting and washing the milk goat ear skin specimen, digesting a resultant dermis layer after the processing with 0.25% of pancreatin, filtering and collecting cells, and culturing the collected cells, thus obtaining dermal fibroblasts; mixing bovine type I collagen solution and DMEM (Dulbecco's Modified Eagle's Medium) solution uniformly, adjusting the pH value of the mixed solution by using NaOH solution, adding the dermal fibroblasts into the mixed solution, and mixing uniformly, thus obtaining collagen cell mixture solution; dropping the obtained collagen cell mixture solution on the human amniotic membrane support, coagulating, and then adding the collagen cell mixture solution in culture solution, culturing in an incubator, and then obtaining a dermis equivalent after the culture. According to the dermis equivalent constructing method of the invention, the human amniotic membrane is used as the support, when being transplanted, the dermis equivalent can be prevented from tearing, the operation operability is improved, after an operation, the human amniotic membrane and organism tissues are compatible, and at the initial stage of the transplanting, certain nutrition ingredients of the organisms can be absorbed, so that the healing of the skin can be promoted.
Description
Technical field
The invention belongs to the bioengineered tissue technical field, being specifically related to a kind of is support with people's amniotic membrane and collagen gel, makes up the method for corium equivalent, promptly a kind of construction method of corium equivalent.
Background technology
Rheinwald in 1975 and Green have invented the epithelium culture technique, make the synthetic possibility that becomes of artificial epidermis.But artificial epidermis can only be used for epidermis injury and epidermis and superficial dermis damage, when damaging when holostrome or near holostrome corium, forms wound surface contracture, scar or open wound easily.Therefore, the research and development dermal substitute becomes clinical demand again.
At present, synthetic corium mainly adopts collagen-GAG, collagen gel, polyglycolic acid, nylon wire etc. as dermis scaffold.For example: the Biobrane skin is a pellosil, and internal layer is imbedded nylon fiber net wherein for part, is full of the pig dermis I type glue of chemical crosslinking in the mesh, to promote itself and the adhesion of wound surface and growing into of fiber blood vessel.Pellosil is the film of half permeability, and the normal skin of its air-and water-permeable performance is strong slightly, helps the medicine local penetration and reduces local hydrops; Can reduce losing of the interior moisture of body on the other hand.Dermagraft-TM then uses degradable polyglycolic acid as dermis scaffold, transplants the after-poppet composition and is degraded gradually, and the Fb of plantation then produces new dermal matrix.Clinical proof, Demagraft can be effective to the treatment of diabetic ulcer.Integra is by Burke and the Yannas a kind of dermal replacement according to material science and engineering principle design, and it is to utilize collagen fiber and chondroitin sulfate to constitute porous support.
When being the artificial dermis of rack making with the collagen gel merely; Along with corium becomes fiber in gel, to increase and collagen and other albumen through secretion self; When forming new dermal matrix; Occur more significantly shrinking, the degree of contraction is relevant with the concentration of collagen, gel component and cell density.Eun Kyung Yang etc. are when doing artificial skin with collagen gel; For reducing the contractility of collagen gel; Screen optimum cell density and best gel strength, simultaneously in order to increase the operability of operation, below collagen, made the toughness that the collagen grid increases artificial skin.Yet, utilize biomembrane to do support, make the corium equivalent at home and abroad research field also belong to blank, the present invention is to be rack making corium equivalent with treated people's amniotic membrane and cattle I Collagen Type VI.
Summary of the invention
The objective of the invention is to make the higher defective of shrinkage factor that the corium equivalent exists to existing with collagen gel, providing a kind of is the construction method of the corium equivalent of support with people's amniotic membrane and collagen.Technical scheme of the present invention is to do support with people's amniotic membrane, after certain density dermal fibroblast and gel mixing, is added drop-wise on people's amniotic membrane; People's amniotic membrane can stop the contraction of collagen gel effectively, and end user's amniotic membrane does support, when transplanting, can effectively prevent tearing of corium equivalent; Increase the operability of operation; And postoperative people amniotic membrane and bio-tissue have the good compatibility, can draw some nutritional labelings of organism at transplant early, promote the healing of skin.
In order to address the above problem, the technical scheme that the present invention adopts is:
The present invention provides a kind of construction method of corium equivalent, and said construction method may further comprise the steps:
The aseptic process of a, people's amniotic membrane:
Pretreatment: the Placenta Hominis of taking from the cesarean section delivery puerpera; And the HIV, hepatitis A, hepatitis B and the syphilis serological reaction that detect Placenta Hominis are shown as feminine gender; Placenta Hominis after tested put into be added with three anti-normal saline and carry out soaking disinfection, the time of soaking disinfection is 3～4 hours;
The removal of spongy layer: in aseptic; Blunt separation attached to preliminary treatment after people's amnion on the placenta; People's amnion under peeling off is washed with being added with three anti-physiological saline repeatedly; Wash to blood and other dirt and thoroughly wash off; Wash people's amnion 2～5 times, the spongy layer on the blunt separation people amnion of flushing back with containing three anti-PBS buffer solutions again;
Epithelial removal: the people's amniotic membrane that will remove spongy layer washes 1～3 time with containing three anti-PBS buffer; Then people's amniotic membrane is divided into several fritters; Epithelial surface upwards is tiled in respectively in the glass dish, adds 0.25% pancreatin+0.02% EDTA solution, cold digestion 12～20 h under 4 ℃ of conditions; Cold postdigestive people's amniotic membrane is scraped off except that epithelial cell with cell; Put into conventional DMEM culture fluid then and cultivate 2～5 min, reuse contains three anti-PBS buffer flushings 3～5 times, obtains the people's amniotic membrane after the aseptic process;
The making of b, people's amniotic membrane support: nitrocellulose filter is cut into the square of hollow, is put in sealing in the plastic bag, carry out 5～15 pounds of autoclave sterilizations after the sealing, the sterilization back is for use; Upwards be tiled in the people's amniotic membrane basal surface after the step a aseptic process in the glass dish; Attach on it handling nitrocellulose filter for use well, upset makes its epithelial surface upwards put into another glass dish then; In calorstat, carry out drying, obtain people's amniotic membrane support after the drying;
The preparation of c, dermal fibroblast: get milk goat ear skin sample 0.5 * 0.5cm
2, scrape off the skin surface booty, using mass percentage concentration is 75% alcohol disinfecting 2～4 times; With conventional D-Hank's liquid flushing 3～5 times, wash 5min then at every turn, the dermatological specimens after the sterilization flushing is removed epidermal area; The gained skin corium is cut into rotten shape, adds 3～5ml, mass percentage concentration then and be 0.25% pancreatin, under 37 ℃ of conditions, digest 5～10min; Digestion after-filtration centrifugal collecting cell in the cell inoculation culture dish of collecting, is cultivated with the DMEM culture fluid that contains mass concentration 10% serum; Cultivated 3～4 days, changed liquid once, and obtained dermal fibroblast after the cultivation in per 2 days;
The preparation of d, corium equivalent: with volume be 7ml, concentration be the cattle type i collagen solution of 4.5mg/ml and 5 * DMEM of 1.5～2.5ml (its implication be 5 times of conventional used DMEM solution concentration promptly 5 times concentrate) the solution mix homogeneously; Use concentration to regulate pH value to 6.8～7.5 of mixed solution as the NaOH solution of 1mol/L behind the mix homogeneously; Add the dermal fibroblast mix homogeneously of 1～2ml step c gained then, obtain the collagenocyte mixed liquor behind the mix homogeneously;
E, getting the resulting collagenocyte mixed liquor of 4～5ml steps d, to drip at diameter be on people's amniotic membrane support of obtaining of the step b of 4cm; Obtain being combined with people's amniotic membrane support of collagenocyte mixed liquor after thoroughly solidifying; Add 5～10ml then and contain in the DMEM culture fluid that mass percentage concentration is 20% serum, place CO then
2Cultivated in the incubator 10～15 days, and obtained the corium equivalent after the cultivation.
Construction method according to above-mentioned corium equivalent; Being added with the three anti-normal saline that normal saline adopted described in the step a is that conventional mass concentration is 0.9% NaCl solution, wherein be added with three anti-be 0.1 μ l/mL penicillin, 0.1 μ l/mL streptomycin and 0.1 μ l/mL gentamycin.
According to the construction method of above-mentioned corium equivalent, contain described in the step a in the PBS buffer that three anti-PBS buffer are meant in routine and add 0.1 μ l/mL penicillin, 0.1 μ l/mL streptomycin and 0.1 μ l/mL gentamycin.
Construction method according to above-mentioned corium equivalent is divided into several fritters described in the step a, its specification of counting fritter is 15 * 15cm.
According to the construction method of above-mentioned corium equivalent, 0.25% pancreatin described in the step a+0.02% EDTA solution is the existing product of selling on the market, and its brand is Vellgen, and article No. is N10341.
According to the construction method of above-mentioned corium equivalent, described in the step b nitrocellulose filter is cut into the square of hollow, its foursquare specification is 4 * 4cm; Institute is set forth in when carrying out drying in the calorstat, and the temperature in the calorstat is 30～38 ℃, and be 30～120 min drying time.
According to the construction method of above-mentioned corium equivalent, the preparation of conventional D-Hank's liquid described in the step c is with reference to Pollard et al.1997.
Construction method according to above-mentioned corium equivalent; The dermal fibroblast mix homogeneously that adds 1～2ml step c gained described in the steps d then; The dermal fibroblast that is wherein added is the adjusted product of step c gained dermal fibroblast, and its cell density is 5 * 10
6/ ml.
Construction method according to above-mentioned corium equivalent; Getting the resulting collagenocyte mixed liquor of 4～5ml steps d described in the step e, to drip at diameter be on people's amniotic membrane support of obtaining of the step b of 4cm; This dropping process divides two steps to carry out; At first 45～55% of getting collagenocyte mixed liquor total amount is added drop-wise on people's amniotic membrane support that step b obtains, behind 20～25min, further drips remaining collagenocyte mixed liquor again.
According to the construction method of above-mentioned corium equivalent, CO described in the step e
2That incubator adopts is U.S. WTB CO
2Incubator; Condition in the said incubator is 5%CO2, saturated humidity, changed liquid 1 time in 37 ℃, per 3 days.
Positive beneficial effect of the present invention:
Technical scheme of the present invention is to be support with people's amniotic membrane, after certain density dermal fibroblast and gel are mixed, is added drop-wise on people's amniotic membrane support, and people's amniotic membrane can stop the contraction of collagen gel effectively; And amniotic membrane is done support and is helped the growth of dermal fibroblast in collagen; End user's amniotic membrane is done support, when transplanting, can effectively prevent tearing of corium equivalent, thereby increases the operability of operation; Operation descendant's amniotic membrane and bio-tissue have the good compatibility, can draw some nutritional labelings of organism at transplant early, promote the healing of skin.
Four, description of drawings:
CO among Fig. 1 embodiment 1 step e
2Cultivate the cell growing state of gained corium equivalent after 15 minutes in the incubator;
CO among Fig. 2 embodiment 1 step e
2Cultivate the cell growing state of gained corium equivalent after 6 days in the incubator;
CO among Fig. 3 embodiment 1 step e
2Cultivate the cell growing state of gained corium equivalent after 10 days in the incubator;
CO among Fig. 4 embodiment 1 step e
2Cultivate the cell growing state of gained corium equivalent after 15 days in the incubator.
Five, the specific embodiment:
Following examples have been merely and have further specified the present invention, but do not limit content of the present invention.
Embodiment 1:
The construction method of a kind of corium equivalent of the present invention, the detailed step of said construction method is following:
The aseptic process of a, people's amniotic membrane:
Pretreatment: the Placenta Hominis of taking from the cesarean section delivery puerpera; And the HIV, hepatitis A, hepatitis B and the syphilis serological reaction that detect Placenta Hominis are shown as feminine gender; Placenta Hominis after tested put into be added with three anti-normal saline and carry out soaking disinfection (being added with the three anti-normal saline that normal saline adopted is that conventional mass concentration is 0.9% NaCl solution; What wherein be added with three anti-is penicillin, streptomycin and gentamycin; The concentration of penicillin, streptomycin and gentamycin is respectively 0.1 μ l/mL, 0.1 μ l/mL and 0.1 μ l/mL behind the adding normal saline), the time of soaking disinfection is 3～4 hours;
The removal of spongy layer: in aseptic; Blunt separation attached to pretreatment after people's amniotic membrane on the Placenta Hominis; People's amniotic membrane under peeling off is washed (it is the same to be added with three anti-normal saline) repeatedly with being added with three anti-normal saline; Wash to blood and other dirt and thoroughly wash off; Reuse contains three anti-PBS buffer flushing people amniotic membranes 4～5 times, and (contain in the PBS buffer that three anti-PBS buffer are meant in routine and add penicillin, streptomycin and gentamycin, the concentration that adds back penicillin, streptomycin and gentamycin is respectively 0.1 μ l/mL, 0.1 μ l/mL and 0.1 μ l/mL; Its compound method is with reference to Pollard et al.1997), the spongy layer on the blunt separation people amniotic membrane of flushing back;
Epithelial removal: the people's amniotic membrane that will remove spongy layer washes 3 times with containing three anti-PBS buffer (the same), and (specification of number fritter is 15 * 15cm), and epithelial surface upwards is tiled in the glass dish respectively then people's amniotic membrane to be divided into several fritters; (this solution is the existing product of selling on the market to add 0.25% pancreatin+0.02% EDTA solution; Its brand is Vellgen, and article No. is N10341), cold digestion 16h under 4 ℃ of conditions; Cold postdigestive people's amniotic membrane is scraped off except that epithelial cell with cell; Put into conventional DMEM culture fluid then and cultivate 5 min, reuse contains three anti-PBS buffer (the same) flushings 4 times, obtains the people's amniotic membrane after the aseptic process;
The making of b, people's amniotic membrane support: nitrocellulose filter is cut into the square of hollow, and (foursquare specification is 4 * 4cm), is put in sealing in the plastic bag, carries out 10 pounds of autoclave sterilizations after the sealing, and the sterilization back is for use; Upwards be tiled in the people's amniotic membrane basal surface after the step a aseptic process in the glass dish; Attach on it handling nitrocellulose filter for use well; Upset makes its epithelial surface upwards put into another glass dish then, and (temperature in the calorstat is 35 ℃ in calorstat, to carry out drying; Be 90 min drying time), obtain people's amniotic membrane support after the drying;
The preparation of c, dermal fibroblast: get milk goat ear skin sample 0.5 * 0.5cm
2, scrape off the skin surface booty, using mass percentage concentration is 75% alcohol disinfecting 3 times; Use conventional D-Hank's liquid (preparation of conventional D-Hank's liquid is with reference to Pollard et al.1997) flushing 5 times then, wash 5min at every turn, the dermatological specimens after the sterilization flushing is removed epidermal area; The gained skin corium is cut into rotten shape, adds 5ml, mass percentage concentration then and be 0.25% pancreatin, under 37 ℃ of conditions, digest 10min; Digestion after-filtration centrifugal collecting cell in the cell inoculation culture dish of collecting, is cultivated with the DMEM culture fluid that contains mass concentration 10% serum; Cultivated 4 days, changed liquid once, and obtained dermal fibroblast after the cultivation in per 2 days;
The preparation of d, corium equivalent: use volume as 7ml, concentration as the cattle type i collagen solution of 4.5mg/ml and 5 * DMEM of 2.0ml volume (its implication be 5 times of conventional used DMEM solution concentration promptly 5 times concentrate) the solution mix homogeneously; Use concentration to regulate the pH value to 7.2 of mixed solution as the NaOH solution of 1mol/L behind the mix homogeneously; (dermal fibroblast that is wherein added is the adjusted product of step c gained dermal fibroblast, and its cell density is 5 * 10 to add the dermal fibroblast mix homogeneously of 1ml step c gained then
6/ ml), obtain the collagenocyte mixed liquor behind the mix homogeneously;
E, to get that the resulting collagenocyte mixed liquor of 4.8ml steps d drips at diameter be that (this dropping process divides two steps to carry out on people's amniotic membrane support of obtaining of the step b of 4cm; At first 50% of getting collagenocyte mixed liquor total amount is added drop-wise on people's amniotic membrane support that step b obtains; Behind the 25min; Further drip remaining collagenocyte mixed liquor again); Obtain being combined with people's amniotic membrane support of collagenocyte mixed liquor after thoroughly solidifying, add 5.0ml then and contain in the DMEM culture fluid that mass percentage concentration is 20% serum, place CO then
2Cultivate in the incubator 15 days (employing be U.S. WTB CO
2Incubator; Condition in the said incubator is 5%CO2, saturated humidity, changed liquid 1 time in 37 ℃, per 3 days), obtain the corium equivalent after the cultivation.
Among the embodiment step e: at CO
2Cultivate in the incubator in the process of corium equivalent, its cell growing state of cultivating gained corium equivalent after 15 minutes, 6 days, 10 days and 15 days sees accompanying drawing 1, accompanying drawing 2, accompanying drawing 3 and accompanying drawing 4 for details.
Embodiment 2:
The construction method of a kind of corium equivalent of the present invention, the detailed step of said construction method is following:
The aseptic process of a, people's amniotic membrane:
Pretreatment: the Placenta Hominis of taking from the cesarean section delivery puerpera; And the HIV, hepatitis A, hepatitis B and the syphilis serological reaction that detect Placenta Hominis are shown as feminine gender; Placenta Hominis after tested put into be added with three anti-normal saline and carry out soaking disinfection (being added with the three anti-normal saline that normal saline adopted is that conventional mass concentration is 0.9% NaCl solution; What wherein be added with three anti-is penicillin, streptomycin and gentamycin; The concentration of penicillin, streptomycin and gentamycin is respectively 0.1 μ l/mL, 0.1 μ l/mL and 0.1 μ l/mL behind the adding normal saline), the time of soaking disinfection is 3～4 hours;
The removal of spongy layer: in aseptic; Blunt separation attached to pretreatment after people's amniotic membrane on the Placenta Hominis; People's amniotic membrane under peeling off is washed (it is the same to be added with three anti-normal saline) repeatedly with being added with three anti-normal saline; Wash to blood and other dirt and thoroughly wash off; Reuse contains three anti-PBS buffer flushing people amniotic membranes 3～4 times, and (contain in the PBS buffer that three anti-PBS buffer are meant in routine and add penicillin, streptomycin and gentamycin, the concentration that adds back penicillin, streptomycin and gentamycin is respectively 0.1 μ l/mL, 0.1 μ l/mL and 0.1 μ l/mL; Its compound method is with reference to Pollard et al.1997), the spongy layer on the blunt separation people amniotic membrane of flushing back;
Epithelial removal: the people's amniotic membrane that will remove spongy layer washes 2 times with containing three anti-PBS buffer (the same), and (specification of number fritter is 15 * 15cm), and epithelial surface upwards is tiled in the glass dish respectively then people's amniotic membrane to be divided into several fritters; (this solution is the existing product of selling on the market to add 0.25% pancreatin+0.02% EDTA solution; Its brand is Vellgen, and article No. is N10341), cold digestion 20h under 4 ℃ of conditions; Cold postdigestive people's amniotic membrane is scraped off except that epithelial cell with cell; Put into conventional DMEM culture fluid then and cultivate 4 min, reuse contains three anti-PBS buffer (the same) flushings 4 times, obtains the people's amniotic membrane after the aseptic process;
The making of b, people's amniotic membrane support: nitrocellulose filter is cut into the square of hollow, and (foursquare specification is 4 * 4cm), is put in sealing in the plastic bag, carries out 15 pounds of autoclave sterilizations after the sealing, and the sterilization back is for use; Upwards be tiled in the people's amniotic membrane basal surface after the step a aseptic process in the glass dish; Attach on it handling nitrocellulose filter for use well; Upset makes its epithelial surface upwards put into another glass dish then, and (temperature in the calorstat is 30 ℃ in calorstat, to carry out drying; Be 120 min drying time), obtain people's amniotic membrane support after the drying;
The preparation of c, dermal fibroblast: get milk goat ear skin sample 0.5 * 0.5cm
2, scrape off the skin surface booty, using mass percentage concentration is 75% alcohol disinfecting 4 times; Use conventional D-Hank's liquid (preparation of conventional D-Hank's liquid is with reference to Pollard et al.1997) flushing 4 times then, wash 5min at every turn, the dermatological specimens after the sterilization flushing is removed epidermal area; The gained skin corium is cut into rotten shape, adds 4ml, mass percentage concentration then and be 0.25% pancreatin, under 37 ℃ of conditions, digest 8min; Digestion after-filtration centrifugal collecting cell in the cell inoculation culture dish of collecting, is cultivated with the DMEM culture fluid that contains mass concentration 10% serum; Cultivated 4 days, changed liquid once, and obtained dermal fibroblast after the cultivation in per 2 days;
The preparation of d, corium equivalent: use volume as 7ml, concentration as the cattle type i collagen solution of 4.5mg/ml and 5 * DMEM of 1.8ml volume (its implication be 5 times of conventional used DMEM solution concentration promptly 5 times concentrate) the solution mix homogeneously; Use concentration to regulate the pH value to 7.0 of mixed solution as the NaOH solution of 1mol/L behind the mix homogeneously; (dermal fibroblast that is wherein added is the adjusted product of step c gained dermal fibroblast, and its cell density is 5 * 10 to add the dermal fibroblast mix homogeneously of 1.2ml step c gained then
6/ ml), obtain the collagenocyte mixed liquor behind the mix homogeneously;
E, to get that the resulting collagenocyte mixed liquor of 5.0ml steps d drips at diameter be that (this dropping process divides two steps to carry out on people's amniotic membrane support of obtaining of the step b of 4cm; At first 50% of getting collagenocyte mixed liquor total amount is added drop-wise on people's amniotic membrane support that step b obtains; Behind the 22min; Further drip remaining collagenocyte mixed liquor again); Obtain being combined with people's amniotic membrane support of collagenocyte mixed liquor after thoroughly solidifying, add 8.0ml then and contain in the DMEM culture fluid that mass percentage concentration is 20% serum, place CO then
2Cultivate in the incubator 15 days (employing be U.S. WTB CO
2Incubator; Condition in the said incubator is 5%CO2, saturated humidity, changed liquid 1 time in 37 ℃, per 3 days), obtain the corium equivalent after the cultivation.
Embodiment 3:
The construction method of a kind of corium equivalent of the present invention, the detailed step of said construction method is following:
The aseptic process of a, people's amniotic membrane:
Pretreatment: the Placenta Hominis of taking from the cesarean section delivery puerpera; And the HIV, hepatitis A, hepatitis B and the syphilis serological reaction that detect Placenta Hominis are shown as feminine gender; Placenta Hominis after tested put into be added with three anti-normal saline and carry out soaking disinfection (being added with the three anti-normal saline that normal saline adopted is that conventional mass concentration is 0.9% NaCl solution; What wherein be added with three anti-is penicillin, streptomycin and gentamycin; The concentration of penicillin, streptomycin and gentamycin is respectively 0.1 μ l/mL, 0.1 μ l/mL and 0.1 μ l/mL behind the adding normal saline), the time of soaking disinfection is 3～4 hours;
The removal of spongy layer: in aseptic; Blunt separation attached to pretreatment after people's amniotic membrane on the Placenta Hominis; People's amniotic membrane under peeling off is washed (it is the same to be added with three anti-normal saline) repeatedly with being added with three anti-normal saline; Wash to blood and other dirt and thoroughly wash off; Reuse contains three anti-PBS buffer flushing people amniotic membranes 3～5 times, and (contain in the PBS buffer that three anti-PBS buffer are meant in routine and add penicillin, streptomycin and gentamycin, the concentration that adds back penicillin, streptomycin and gentamycin is respectively 0.1 μ l/mL, 0.1 μ l/mL and 0.1 μ l/mL; Its compound method is with reference to Pollard et al.1997), the spongy layer on the blunt separation people amniotic membrane of flushing back;
Epithelial removal: the people's amniotic membrane that will remove spongy layer washes 3 times with containing three anti-PBS buffer (the same), and (specification of number fritter is 15 * 15cm), and epithelial surface upwards is tiled in the glass dish respectively then people's amniotic membrane to be divided into several fritters; (this solution is the existing product of selling on the market to add 0.25% pancreatin+0.02% EDTA solution; Its brand is Vellgen, and article No. is N10341), cold digestion 14h under 4 ℃ of conditions; Cold postdigestive people's amniotic membrane is scraped off except that epithelial cell with cell; Put into conventional DMEM culture fluid then and cultivate 5 min, reuse contains three anti-PBS buffer (the same) flushings 3 times, obtains the people's amniotic membrane after the aseptic process;
The making of b, people's amniotic membrane support: nitrocellulose filter is cut into the square of hollow, and (foursquare specification is 4 * 4cm), is put in sealing in the plastic bag, carries out 8 pounds of autoclave sterilizations after the sealing, and the sterilization back is for use; Upwards be tiled in the people's amniotic membrane basal surface after the step a aseptic process in the glass dish; Attach on it handling nitrocellulose filter for use well; Upset makes its epithelial surface upwards put into another glass dish then, and (temperature in the calorstat is 38 ℃ in calorstat, to carry out drying; Be 40 min drying time), obtain people's amniotic membrane support after the drying;
The preparation of c, dermal fibroblast: get milk goat ear skin sample 0.5 * 0.5cm
2, scrape off the skin surface booty, using mass percentage concentration is 75% alcohol disinfecting 4 times; Use conventional D-Hank's liquid (preparation of conventional D-Hank's liquid is with reference to Pollard et al.1997) flushing 3 times then, wash 5min at every turn, the dermatological specimens after the sterilization flushing is removed epidermal area; The gained skin corium is cut into rotten shape, adds 4.5ml, mass percentage concentration then and be 0.25% pancreatin, under 37 ℃ of conditions, digest 7min; Digestion after-filtration centrifugal collecting cell in the cell inoculation culture dish of collecting, is cultivated with the DMEM culture fluid that contains mass concentration 10% serum; Cultivated 4 days, changed liquid once, and obtained dermal fibroblast after the cultivation in per 2 days;
The preparation of d, corium equivalent: use volume as 7ml, concentration as the cattle type i collagen solution of 4.5mg/ml and 5 * DMEM of 2.2ml volume (its implication be 5 times of conventional used DMEM solution concentration promptly 5 times concentrate) the solution mix homogeneously; Use concentration to regulate the pH value to 7.2 of mixed solution as the NaOH solution of 1mol/L behind the mix homogeneously; (dermal fibroblast that is wherein added is the adjusted product of step c gained dermal fibroblast, and its cell density is 5 * 10 to add the dermal fibroblast mix homogeneously of 1.6ml step c gained then
6/ ml), obtain the collagenocyte mixed liquor behind the mix homogeneously;
E, to get that the resulting collagenocyte mixed liquor of 5.0ml steps d drips at diameter be that (this dropping process divides two steps to carry out on people's amniotic membrane support of obtaining of the step b of 4cm; At first 50% of getting collagenocyte mixed liquor total amount is added drop-wise on people's amniotic membrane support that step b obtains; Behind the 25min; Further drip remaining collagenocyte mixed liquor again); Obtain being combined with people's amniotic membrane support of collagenocyte mixed liquor after thoroughly solidifying, add 7.0ml then and contain in the DMEM culture fluid that mass percentage concentration is 20% serum, place CO then
2Cultivate in the incubator 15 days (employing be U.S. WTB CO
2Incubator; Condition in the said incubator is 5%CO2, saturated humidity, changed liquid 1 time in 37 ℃, per 3 days), obtain the corium equivalent after the cultivation.
Claims (10)
1. the construction method of a corium equivalent is characterized in that, said construction method may further comprise the steps:
The aseptic process of a, people's amniotic membrane:
Pretreatment: the Placenta Hominis of taking from the cesarean section delivery puerpera; And the HIV, hepatitis A, hepatitis B and the syphilis serological reaction that detect Placenta Hominis are shown as feminine gender; Placenta Hominis after tested put into be added with three anti-normal saline and carry out soaking disinfection, the time of soaking disinfection is 3～4 hours;
The removal of spongy layer: in aseptic; Blunt separation attached to preliminary treatment after people's amnion on the placenta; People's amnion under peeling off is washed with being added with three anti-physiological saline repeatedly; Wash to blood and other dirt and thoroughly wash off; Wash people's amnion 2～5 times, the spongy layer on the blunt separation people amnion of flushing back with containing three anti-PBS buffer solutions again;
Epithelial removal: the people's amniotic membrane that will remove spongy layer washes 1～3 time with containing three anti-PBS buffer; Then people's amniotic membrane is divided into several fritters; Epithelial surface upwards is tiled in respectively in the glass dish, adds 0.25% pancreatin+0.02% EDTA solution, cold digestion 12～20 h under 4 ℃ of conditions; Cold postdigestive people's amniotic membrane is scraped off except that epithelial cell with cell; Put into conventional DMEM culture fluid then and cultivate 2～5 min, reuse contains three anti-PBS buffer flushings 3～5 times, obtains the people's amniotic membrane after the aseptic process;
The making of b, people's amniotic membrane support: nitrocellulose filter is cut into the square of hollow, is put in sealing in the plastic bag, carry out 5～15 pounds of autoclave sterilizations after the sealing, the sterilization back is for use; Upwards be tiled in the people's amniotic membrane basal surface after the step a aseptic process in the glass dish; Attach on it handling nitrocellulose filter for use well, upset makes its epithelial surface upwards put into another glass dish then; In calorstat, carry out drying, obtain people's amniotic membrane support after the drying;
The preparation of c, dermal fibroblast: get milk goat ear skin sample 0.5 * 0.5cm
2, scrape off the skin surface booty, using mass percentage concentration is 75% alcohol disinfecting 2～4 times; With conventional D-Hank's liquid flushing 3～5 times, wash 5min then at every turn, the dermatological specimens after the sterilization flushing is removed epidermal area; The gained skin corium is cut into rotten shape, adds 3～5ml, mass percentage concentration then and be 0.25% pancreatin, under 37 ℃ of conditions, digest 5～10min; Digestion after-filtration centrifugal collecting cell in the cell inoculation culture dish of collecting, is cultivated with the DMEM culture fluid that contains mass concentration 10% serum; Cultivated 3～4 days, changed liquid once, and obtained dermal fibroblast after the cultivation in per 2 days;
The preparation of d, corium equivalent: with volume is that 7ml, concentration are the cattle type i collagen solution of 4.5mg/ml and 5 * DMEM solution mix homogeneously of 1.5～2.5ml; Use concentration to regulate pH value to 6.8～7.5 of mixed solution as the NaOH solution of 1mol/L behind the mix homogeneously; Add the dermal fibroblast mix homogeneously of 1～2ml step c gained then, obtain the collagenocyte mixed liquor behind the mix homogeneously;
E, getting the resulting collagenocyte mixed liquor of 4～5ml steps d, to drip at diameter be on people's amniotic membrane support of obtaining of the step b of 4cm; Obtain being combined with people's amniotic membrane support of collagenocyte mixed liquor after thoroughly solidifying; Add 5～10ml then and contain in the DMEM culture fluid that mass percentage concentration is 20% serum, place CO then
2Cultivated in the incubator 10～15 days, and obtained the corium equivalent after the cultivation.
2. the construction method of corium equivalent according to claim 1; It is characterized in that: being added with the three anti-normal saline that normal saline adopted described in the step a is that conventional mass concentration is 0.9% NaCl solution, wherein be added with three anti-be 0.1 μ l/mL penicillin, 0.1 μ l/mL streptomycin and 0.1 μ l/mL gentamycin.
3. the construction method of corium equivalent according to claim 1 is characterized in that: contain described in the step a in the PBS buffer that three anti-PBS buffer are meant in routine and add 0.1 μ l/mL penicillin, 0.1 μ l/mL streptomycin and 0.1 μ l/mL gentamycin.
4. the construction method of corium equivalent according to claim 1 is characterized in that: be divided into several fritters described in the step a, its specification of counting fritter is 15 * 15cm.
5. the construction method of corium equivalent according to claim 1 is characterized in that: 0.25% pancreatin described in the step a+0.02% EDTA solution is the existing product of selling on the market, and its brand is Vellgen, and article No. is N10341.
6. the construction method of corium equivalent according to claim 1 is characterized in that: described in the step b nitrocellulose filter is cut into the square of hollow, its foursquare specification is 4 * 4cm; Institute is set forth in when carrying out drying in the calorstat, and the temperature in the calorstat is 30～38 ℃, and be 30～120 min drying time.
7. the construction method of corium equivalent according to claim 1 is characterized in that: the preparation of conventional D-Hank's liquid described in the step c is with reference to Pollard et al.1997.
8. the construction method of corium equivalent according to claim 1; It is characterized in that: the dermal fibroblast mix homogeneously that adds 1～2ml step c gained described in the steps d then; The dermal fibroblast that is wherein added is the adjusted product of step c gained dermal fibroblast, and its cell density is 5 * 10
6/ ml.
9. the construction method of corium equivalent according to claim 1; It is characterized in that: getting the resulting collagenocyte mixed liquor of 4～5ml steps d described in the step e, to drip at diameter be on people's amniotic membrane support of obtaining of the step b of 4cm; This dropping process divides two steps to carry out; At first 45～55% of getting collagenocyte mixed liquor total amount is added drop-wise on people's amniotic membrane support that step b obtains, behind 20～25min, further drips remaining collagenocyte mixed liquor again.
10. the construction method of corium equivalent according to claim 1 is characterized in that: CO described in the step e
2That incubator adopts is U.S. WTB CO
2Incubator; Condition in the said incubator is 5%CO2, saturated humidity, changed liquid 1 time in 37 ℃, per 3 days.
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| CN110484491B (en) * | 2019-08-20 | 2022-03-18 | 北京京蒙高科干细胞技术有限公司 | Method for obtaining amniotic membrane and amniotic fluid derived endothelial progenitor cells and purification culture method thereof |
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