CN109771697A - A kind of dermal fibroblast skin graft and its construction method and application - Google Patents
A kind of dermal fibroblast skin graft and its construction method and application Download PDFInfo
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- CN109771697A CN109771697A CN201811635902.7A CN201811635902A CN109771697A CN 109771697 A CN109771697 A CN 109771697A CN 201811635902 A CN201811635902 A CN 201811635902A CN 109771697 A CN109771697 A CN 109771697A
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Abstract
The invention proposes a kind of construction methods of auto derma fibroblast skin graft, the method are as follows: the dermal fibroblast of amplification in vitro is inoculated into and carries out cell curing by the amnion tissue for taking off cell, degreasing, de- nucleic acid processing, construct dermal fibroblast-amnion tissue composite construction, it is enriched with high-purity cell under cell culture condition, obtains the auto derma fibroblast skin graft.The invention also provides the auto derma fibroblast skin grafts constructed by this method.Auto derma fibroblast skin graft of the present invention can be used for the clinical demand of burn trauma, large area skin-grafting and other urgent need large skin defects, has many advantages, such as low rejection, sustainable supply, has wide application prospects.
Description
Technical field
The present invention relates to dermal fibroblast produced in vitro technical fields, and in particular to a kind of dermal fibroblast skin
Piece and its construction method and application.
Background technique
Auto derma Fibroblasts in vitro production technology: the produced in vitro technology of autogenous cell ten has been divided at present
It is ripe.The Fibroblasts in vitro multiplication technique of adult auto derma, has had reached industrialized requirement, has had the production in foreign countries
Injection listing is savored, similar products also occurs in the country.It is now efficient, high at the large-scale production direction of fibroblasts of adult human dermis
Purity, high activity direction develop；Product purpose can directly prepare injection, can also be used as the primary raw materials of other preparations at
Point.
De- epithelial layer amnion constructs solid cell technology: cell curing technology is developed so far, and is gradually divided into two classes, Yi Leishi
It carries out de- cell-refinement born of the same parents using biological tissue's film to handle, another kind of is that solid is made using biogel material fused cell
Gel film.Wherein in biological tissue's film+cell product, in artificial skin, artificial cornea, other artificial tissue films into
Row clinical research.People take off cell amnion as tissue engineering bracket be easy to get, good biocompatibility, degradability
And the advantages that low immunogenicity, have been used to the production of organization engineering skin, tissue engineering comea.But its there is also it is some not
Foot, including intensity is not good enough, there may be rejections etc. after implantation.Currently, the listing tissue of domestic treatment skin burn wound
In engineering skin product, only variant cell " peace body skin ", autogenous cell " PELNAC ".
Summary of the invention
To overcome above-mentioned technical problem of the existing technology, present invention innovation proposes a kind of dermal fibroblast skin
The construction method of piece and the dermal fibroblast skin graft constructed by this method.
The construction method of a kind of dermal fibroblast skin graft proposed by the present invention, by the corium of amplification in vitro at fiber finer
The amnion tissue that born of the same parents are inoculated by degreasing, de- cell, de- nucleic acid processing carries out cell curing, constructs dermal fibroblast-
The composite construction of amnion tissue；Then it is cultivated in amplification culture medium, high-purity cell is enriched under cell culture condition, obtained
The dermal fibroblast skin graft.In the present invention, the external large-scale production of adult somatic cells provides enough, high-purity for skin graft
The production of the living cells of degree, i.e. raw material；Raw cell is immobilized in the consolidated structures of deactivation, i.e. production.
Wherein, the dermal fibroblast is at fibroblasts of adult human dermis (HDFB), it is preferable that adult for FI phenotype
Dermal fibroblast.By form and metabolic rule, seven Bayreuther phenotypes can be divided at fibroblasts of adult human dermis,
Wherein, FI phenotype, FII phenotype, it is most commonly seen at the initial stage of originally culture.FI phenotype cells are spindle shape aligned growth, proliferation
Speed is the major cell types for constituting the most thick and solid position of dermal tissue layer faster, and the form cultivated in vitro is spindle shape, about
It is proliferated within 12 hours one times, and secretes I-type collagen, to promote the proliferation of itself and other cells of skin histology.FII phenotype is thin
Born of the same parents are epithelial, compact growth, and speed is slower.
In the present invention, high-purity is obtained into fibroblasts of adult human dermis.In order to obtain the FI phenotype of high-purity into fiber finer
Born of the same parents, the present invention proposition must be purified at the initial stage of originally culture, that is, the present invention in, originally culture third to five days,
The method directly rejected using micromanipulation removes other phenotype cells by cellular morphology.In the present invention, at the beginning of originally culture
The dermal fibroblast that phase is purified by micromanipulation, the method directly rejected, in originally culture and passage hereafter,
The FI phenotype cells of high-purity can be obtained.By comparison, conventional method is to carry out staining analysis after the cells are obtained, distinguishes table
Type, then with substep digestion method, different cells are separated, which cannot reject heteroproteose cell completely, and time-consuming about needs 7 days left sides
It is right.
In the present invention, production obtains high yield into fibroblasts of adult human dermis.In the present invention, at originally culture initial stage, mention
The concentration of high FBS promotes the entrance culture vessel adherent growth of cell in primary tissue, shortens primary using high serum environment
The time of culture obtains high yield into fibroblasts of adult human dermis.Originally culture initial stage refers to that cell starts preceding the 8 of vitro propagation
Generation.
Cell in the present invention, after obtaining the inoculation with high viability.In the present invention, in the inoculating process of raw cell
In, in order to improve the survival rate in conjunction with rear cell, increase collagen component in amplification culture medium, i.e. increase cytotrophy,
Gel three-dimensional structure is provided again, makes cell on skin graft, is grown by the fusiform form of script.As cell density improves, collagen
Albumen can be reduced by cell consumption, but will continue to secrete collagen after cell maturation, for itself needs.In the present invention, connect
Contain collagen in kind culture.Preferably, the raw material of inoculation includes dermal fibroblast, collagen, cell amplification training
Support base.
In the present invention, remove the bioactivity of amnion tissue: the present invention carries out de- nucleic acid technique to amnion tissue, with removal
Bioactivity reduces the rejection risk in use.
In one embodiment, the construction method of dermal fibroblast skin graft of the present invention includes:
(1) dermal fibroblast of amplification in vitro is integrated to the amnion group by taking off cell, degreasing, de- nucleic acid processing
Carry out cell curing is knitted, dermal fibroblast-amnion tissue composite construction is constructed；
Preferably, the dermal fibroblast of amplification in vitro is integrated to by de- cell, degreasing, de- albumen, de- nucleic acid
The amnion tissue of processing carries out cell curing, constructs dermal fibroblast-amnion tissue composite construction
(2) it is then cultivated under conditions of amplification culture medium in culture vessel, obtains the dermal fibroblast skin
Piece.
In the step (1), the dermal fibroblast is from the primary tissue at fibroblasts of adult human dermis.It is preferred that
Ground, the dermal fibroblast are the I type phenotype cells (FI phenotype cells) of single phenotype, and such cell proliferation rate is fast,
It is high to secrete I-type collagen content.
In the present invention, at fibroblasts of adult human dermis refer to birth after human individual (in addition to fetus) corium at
Fibrocyte.
Preferably, which can derive from the posterior neck of people, and punch method can be used and obtain
?.Preferably, the volume of the primary tissue is 4-8mm3.It is highly preferred that the volume of the primary tissue is at least 4mm3。
Preferably, the primary tissue of the dermal fibroblast need to be tightly attached to culture and hold after being cut into small tissue blocks
It is cultivated 2 to 3 days in primary culture medium in device.Then, it will be digested, again at the primary tissue of fibroblasts of adult human dermis
Inoculation is hanged and diluted, amplification in vitro is carried out, can get the dermal fibroblast.
Wherein, the digestion, resuspension, dilution inoculation can carry out once；(increase yield) when output demand improves,
Or when being clinically used for multiple times, it can also carry out repeatedly.
Wherein, the condition of the digestion are as follows: under the action of trypsase-EDTA, 37 DEG C, digest 2~3 minutes.
Wherein, the condition of the resuspension are as follows: in containing serum solution, 37 DEG C, mechanical piping and druming.
Wherein, the dilution inoculation refers to, in amplification culture medium, every 1cm2Culture area inoculation 104A cell carries out
Secondary culture.Wherein, in amplification culture medium, every generation needs to cultivate 2-4 days.
Wherein, the condition of the originally culture is to be cultivated under conditions of 37 DEG C, gas concentration lwevel 5%.It is preferred that
Ground, the time of the originally culture are 5-8 days.Preferably, at 3~5 days of originally culture, by cell kenel, other are removed
Phenotype cells can obtain high-purity into fibroblasts of adult human dermis in subsequent primary culture medium and secondary culture.That is, this
The screening technique of dermal fibroblast described in invention are as follows: under microscopy, scrape removal heteroproteose cell with aseptic apparatus.
Wherein, which contains the component of following volumes percentage: 70-86%DMEM high glucose medium,
12-18% fetal calf serum (FBS), 0.5-1% nonessential amino acid, 0.5-1% glutamine and 1-10% blueness strepto-
Element.The present invention is in originally culture initial stage (3~5 days of originally culture), by improving the concentration of FBS, using high serum environment, promotees
Enter culture vessel adherent growth into the cell in primary tissue, shortens the time of originally culture.
Wherein, the DMEM high glucose medium is commercially available commercialization cell culture medium.
Wherein, the nonessential amino acid is lysine.
The amplification culture medium contains the component of following volumes percentage: 84-92%DMEM high glucose medium, 7-15%
Fetal calf serum (FBS), 0.5-1% nonessential amino acid, 0.5-1% glutamine.
In the present invention, the inoculation refers to: cell is inoculated in culture vessel；The combination refers to cell combination in amnion
On.
In the step (1), the dermal fibroblast after secondary culture is integrated to amnion tissue and carries out cell
Immobilization.Wherein, building raw material includes dermal fibroblast (raw cell), collagen, cell amplification culture medium or life
Manage salt water.
That is, being integrated to amnion group after collagen is added in the dermal fibroblast in amplification culture medium to culture
Knit carry out cell fixation；Alternatively, being integrated to sheep after collagen is added into the physiological saline containing dermal fibroblast
Membrane tissue carries out cell fixation.Preferably, the collagen is I-type collagen.The collagen can increase
To dermal fibroblast-amnion tissue composite construction in viable cell density, improve the inoculation motility rate of cell.
Wherein, the dermal fibroblast may also come from self, non-self, xenogenesis through taming in vitro, homologous
Dermal fibroblast.
Wherein, the solidification/cell fixation refers to above-mentioned inoculation raw material (containing living thin at fibroblasts of adult human dermis
Born of the same parents) it is inoculated into amnion tissue.
In one embodiment, the amnion tissue successively need to take off albumen-by de- cell-degreasing-and take off at nucleic acid
Manage step.
Wherein, the de- cell treatment fluid, main component are Triton X-100 (volume fraction 0.1-0.5%).
Wherein, the ungrease treatment liquid, main component be physiological saline, neutral lipase (solution PH is 7.0 to 7.4, in
Property lipase concentration be 5-10U/mL).
Wherein, the de- albumen treatment fluid, main component are physiological saline, alkaline trypsase.
Wherein, for the de- nucleic acid treatment fluid used in the de- nucleic acid processing for surface activating solution, main component is nucleic acid
Enzyme, SDS (solution PH is 7.0 to 7.4, mass concentration 0.3-0.8%).
Wherein, the treatment process of the de- cell, degreasing, de- albumen, de- nucleic acid, is 37 DEG C, dynamic is handled, amnion quilt
It fixes.
Wherein, each step process finishes, and is cleaned repeatedly with sterile saline.
Wherein, after the completion of amnion is handled through above-mentioned four steps, cryopreservation methods long-term preservation can be used, is such as protected from light guarantor for 4 DEG C
It deposits within 30 days；Wherein, the method for replacing of frozen stock solution is centrifugal process；Freeze the frozen stock solution that formula of liquid is 50% serum, cooling side
Method is program cooling, and store method is liquid nitrogen.
The purity for the cell in dermal fibroblast-amnion tissue composite construction that the method for the present invention constructs is examined
Proved recipe method is antibody act, is incubated for CD73, CD90, CD105, counts to cell fluorescence, is calculated in cell products into fiber finer
Born of the same parents' purity.
In a specific embodiment, the construction method includes the following steps:
Step 1): amnion tissue cleaning takes off cell-degreasing-and takes off the de- nucleic acid sequences processing of albumen-；
Step 2): being inoculated with dermal fibroblast living cells raw material in amnion tissue and carries out cell curing, and building is true
Skin fibroblast-amnion tissue composite construction；
Step 3): in culture vessel, amplification, culture.
In another embodiment, in construction method of the present invention, extracted, screening, amplification, identification and inspection are frozen
It deposits, amnion screening, amnion cleaning and sterilizing, amnion degreasing de- cell, amnion preservation, cell curing, semi-humid culture, obtains purpose
Object, that is, auto derma fibroblast skin graft.
The invention also provides the auto derma fibroblast skin grafts obtained by aforementioned construction method.
The auto derma fibroblast skin graft that construction method of the present invention obtains, can be applied to burn trauma, chronic disease
Deng the clinical treatment of large skin defect.Auto derma fibroblast skin graft of the present invention can be used for burn trauma, large area
The clinical demand of skin-grafting and other urgent need large skin defects, provides the skin injury auxiliary material of low rejection, increases clinical treatment
Method；The auto derma fibroblast skin graft that construction method of the present invention obtains can also be used to prepare skin care item, for preventing, disappearing
Wrinkle is removed and/or reduced, there is wide application prospect.
Detailed description of the invention
Fig. 1 shows the form 100X that auto derma fibroblast in the present invention is cultivated in vitro.
Fig. 2 is the schematic diagram at fibroblasts of adult human dermis of primary FI phenotype in the present invention, and living cells is growing shape
The photomicrograph of state, 40 times of amplifications.
Fig. 3 is the schematic diagram of FI phenotype in the present invention being proliferated in collagen at fibroblasts of adult human dermis, living thin
The photomicrograph of born of the same parents' growth conditions just in the stereochemical structure of collagen, 40 times of amplifications.
Fig. 4 is the schematic diagram of the effect of clinical application auto derma fibroblast treatment face filling；Wherein, a is indicated
Eye circumference under left side before injection；B indicates eye circumference under the right side before injection；C indicates eye circumference under the left side after injection；D indicates injection
Eye circumference under right side afterwards.
Specific embodiment
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail.Implement process of the invention,
Condition, experimental method etc. are among the general principles and common general knowledge in the art, this hair in addition to what is specifically mentioned below
It is bright that there are no special restrictions to content.
Embodiment 1 constructs auto derma fibroblast skin graft
In construction method, operate as follows:
One, dermal fibroblast amplification in vitro:
1) it extracts:
A) under disinfection, from user's posterior neck hair line (fire victim can sample in other positions), skin is used
Punch beats one or more diameter 1.5mm, the pachydermia skin histology of depth 10mm.By pachydermia skin histology loading group immediately
It knits in cleaning solution and is sealed, refrigerates, being kept in dark place, the time must not exceed 4 hours.
B) tissue wash formula of liquid: aseptic injection grade water, penicillin 1Ku/ml, streptomysin 1mg/ml, inculwater (branch
Substance inhibitor) volumetric concentration 0.1%.
C) under the conditions of aseptic experiment, the skin portion of the pachydermia skin histology is cut into (3mm below epidermis), subcutaneously
Fat sites are cut (yellow oily), retain corium area into pars fibrosa.On sterilized petri dishes, tissue is dipped in aseptic cotton carrier repeatedly
Tissue disinfection liquid is cleaned, and is impregnated with the Aseptic sterilisation liquid newly configured, and replace thimerosal.Soaking and washing repeatedly.
D) tissue disinfection formula of liquid: aseptic injection grade water, penicillin 1Ku/ml, streptomysin 1mg/ml, inculwater (branch
Substance inhibitor) volumetric concentration 0.1%.
E) the pachydermia skin histology is cut into small tissue blocks in sterilized petri dishes, enabling tissue block be close to plate must not levitating.
Primitive cell culture liquid is added, is put into 37 DEG C, CO2It is cultivated 3 days in the carbon dioxide incubator of concentration 5%.
F) primitive cell culture formula of liquid :+1% nonessential amino acid+1% of+15% fetal calf serum of DMEM high glucose medium
+ 1% mycillin of glutamine.
2) purifying screening:
G) remove primitive cell culture liquid, sterile saline is added in culture dish, observed under microscopic field primary
Cell.
H) each cell phenotype of observation analysis: where FI type is elongated shuttle shape morphological cellular, is of moderate size, marshalling；
FII is epithelium shape cell, and lesser polygon (similar to epidermal cell), nucleus is larger, and arrangement is close；FIII is biggish
Epithelium shape cell, nucleus are big.
I) in gnotobasis, all cells other than FI type phenotype are carefully scraped.Training is washed with sterile saline
It is multiple to support ware, guarantees that heteroproteose cell completely removes.
J) cell amplification cultivation liquid is added in remaining cell, cultivates 2 in 37 DEG C, the carbon dioxide incubator of CO2 concentration 5%
To 3 days, until cellular morphology is spindle shape, ordered arrangement, density 90%.
3) it expands:
The primary tissue after above-mentioned purified screening is digested using the trypsin-EDTA solutions of non-animal source.
Digestion condition is 37 DEG C, 2 to 3 minutes.The cell obtained after digestion passes through viable count；It is then seeded into primary culture medium
It is cultivated, amplification is then carried out in amplification culture medium and cultivates culture.
Wherein, the primary culture medium contains the component of following volumes percentage: 70-86%DMEM high glucose medium,
12-18% fetal calf serum (FBS), 0.5-1% nonessential amino acid, 0.5-1% glutamine and 1-10% blueness strepto-
Element.
The amplification culture medium contains the component of following volumes percentage: 84-92%DMEM high glucose medium, 7-15%
Fetal calf serum (FBS), 0.5-1% nonessential amino acid, 0.5-1% glutamine.
4) it identifies and examines
It takes in step 3) amplification, the solution obtained after primary tissue is digested is to carry out bacterium, Zhi Yuan containing cell solution
Body, virus examination.Aforementioned inspection result is necessary for negative.
Take step 3) single cell suspension carry out flow cytometer inspection, inspections antibody be CD:73,90,105, positive rate
It need to be 95% or more.
5) it freezes
The cell obtained after amplification is digested unicellular to obtain using pancreatin, is then carried out with the culture medium containing serum
It terminates, the building of subsequent dermal fibroblast skin graft can be carried out.
Remaining cell is freezed after processing, specific steps are as follows: above-mentioned cell is digested to unicellular, process by pancreatin
Sterile saline elutes (centrifugal process elution, centrifugal force 400g, 5 minutes time) twice.Cell after centrifugation, uses frozen stock solution
It is resuspended, is dispensed by the density of 2,000,000/ml.Cooling freezing is carried out by 1 DEG C/min of speed.
Cell after freezing can be reserved for 2 to 5 years.
When reusing, freeze-stored cell pipe is melted rapidly in the hot water and (is melted to 80%), is transferred to 4 DEG C of sterile physiologicals
In salt water, centrifugal process replaces frozen stock solution.Cell after centrifugation, is counted by living cells count, and living cells can be inoculated with.
Two, epithelial layer amnion is taken off, for constructing solid cell:
1) screening of amnion tissue
According to contributor provide blood samples virus examination as a result, selection non-communicable disease and without amnion it is extremely pregnant
The amnion tissue being pregnent.
2) cleaning and sterilizing of amnion tissue
The fresh amnion given birth to is removed into chorion, removal haemocyte and other impurities is cleaned repeatedly with physiological saline, protects
Stay amnion tissue.This tissue includes epithelium layer, basilar memebrane and the intact amniotic tissue without vascular stroma.
Amnion obtained above is impregnated into rinsing 2 times using the medicinal alcohol of volumetric concentration 75%, reuses tissue wash
Liquid impregnates rinsing 2 times.
3) de- cell-degreasing-of amnion tissue takes off albumen-and takes off nucleic acid sequences processing
A) cell is taken off
Using animal sources lipase, amnion tissue is immersed in lipase solution and is incubated for digestion, digestion condition: 37 DEG C,
Dynamically.Tissue moves on in sterile saline, is scraped off epithelial layer residual cells with cell scraper.
Protein enzyme solution formula: trypsinase concentration 0.25% (mass-volume concentration), 0.005% (quality of EDTA concentration
Volumetric concentration), PH8.4.
B) degreasing
This step is used to remove the membrane tissue of cell membrane and organelle, removes cellular genetic material.
Using non-animal source lipase, amnion tissue is immersed in lipase solution and is incubated for digestion, digestion condition: 37
DEG C, 3 hours, dynamic.
Using surfactant solution, amnion tissue is immersed in lipase solution and is incubated for digestion, incubation conditions: 37
DEG C, 3 hours, dynamic.
Lipase solution formula: by sterile saline, PH=9.0 prepares lipase.
C) albumen is taken off
The de- albumen treatment fluid, main component are physiological saline, alkaline trypsase.
D) nucleic acid is taken off
Amnion tissue is immersed in DNA enzymatic solution kind, 37 DEG C of incubation digestion.All after digestion, the sterile life of amnion
Reason saline rinse 3 times.DNA is removed by this step.
Nuclease solution formula: sterile saline, DNase concentration 1000-3000U/L.
4) preservation of amnion tissue
By the amnion tissue that abovementioned steps are handled, can be immersed in the sterile saline containing 10% mycillin, 4 DEG C
It seals, be kept in dark place.Validity period 30 days.
Three, dermal fibroblast-amnion tissue composite construction is constructed, is cultivated, obtains object i.e. auto derma
Fibroblast skin graft:
1) dermal fibroblast-amnion tissue composite construction is constructed
Aseptically, membrane film is laid in culture vessel.Corium is dissolved in cell amplification at fiber is unicellular
In culture medium, and I-type collagen is added, corium is made into fiber single cell suspension, is integrated on membrane film, it is solid to carry out cell
Change, obtains formation dermal fibroblast-collagen gel dermal fibroblast-amnion tissue on membrane film and answer
Close structure.
2) semi-humid culture
Above-mentioned composite construction tissue is laid in culture vessel, amnion downward, dermal fibroblast-collagen gel
Upward.A small amount of amplification culture medium is accessed, liquid level did not had gel layer to obtain dermal fibroblast skin graft after culture 3 days.
It is inoculated with solution formula: physiological saline or amplification culture medium, HDFB living cells suspension, I-type collagen.
Cultivating obtained dermal fibroblast skin graft can be used for directly spreading at wound.In the skin graft it is main effectively at
It is divided into fibroblast living cells, it is at wound which can continue to secrete I-type collagen under the premise of being close to wound
Remaining cell nutriment is provided, and the external source living cells on skin graft is constantly proliferated, and can be transferred to wound growth, be made wound
Gradually heal.
The dermal fibroblast skin graft that the present invention constructs can promote corium growth, be mainly used for chronic wounds (diabetes
Property festers, long-term bedsore, the long-term bleb of non-infection type etc.)；It can be also used for II degree and following burn treating, can be also used for
Deep burn can be also used for healing phases after debridement early period treatment.
The auto derma fibroblast skin graft of 2 embodiment 1 of embodiment building is examined:
1) active constituent content is examined
Inspection is sampled to batches of product.The method of inspection of Content Test takes the skin graft of 1cm2 area, uses physiology salt
Water rinsing, rinse method: rinsing 2 times in sterile chamber.It is digested with skin graft digestive juice, digestion condition: 37 DEG C, dynamic, point
Secondary digestion is simultaneously collected, and is terminated containing serum solution.Digestive juice is collected and merged, can be centrifuged and be concentrated.
The cell for merging and obtaining is collected to digestion, count regardless of motility rate.Obtain active constituent content result.
Digest formula of liquid: trypsase, collagenase type I, physiological saline.
2) effective component activity assay
Inspection is sampled to batches of product.The method of inspection of Content Test takes the skin graft of 1cm2 area, uses physiology salt
Water rinsing, rinse method: rinsing 2 times in sterile chamber.It is digested with skin graft digestive juice, digestion condition: 37 DEG C, dynamic, point
Secondary digestion is simultaneously collected, and is terminated containing serum solution.Digestive juice is collected and merged, can be centrifuged and be concentrated.
The cell for merging and obtaining is collected to digestion, carries out the counting of AOPI fluorescent dyeing.Obtain the activity of effective component
As a result.
Digest formula of liquid: trypsase, collagenase type I, physiological saline.
Fluorescence dye liquor ingredient: AO dye liquor；PI dye liquor.
3) effective component purity check
Microscopy, growthform, the growth cycle of discernable cell.
Inspection is sampled to batches of product.The method of inspection of Content Test takes the skin graft of 1cm2 area, uses physiology salt
Water rinsing, rinse method: rinsing 2 times in sterile chamber.It is digested with skin graft digestive juice, digestion condition: 37 DEG C, dynamic, point
Secondary digestion is simultaneously collected, and is terminated containing serum solution.Digestive juice is collected and merged, can be centrifuged and be concentrated.
To the cell that digestion obtains, antibody incubation is carried out, antibody includes: CD73, CD90, CD105.Incubation conditions: 4 DEG C, 1
~4 hours.
Antibody detection method: fluorescence counting method.Fluorescence intensity, which calculates, obtains product effective component purity.
Digest formula of liquid: trypsase, collagenase type I, physiological saline.
The adult auto derma Fibroblasts in vitro production of embodiment 3.
In the present embodiment, patient is 33 years old women.Clinical indication is I grades of tail of the eye wrinkle.Concrete operations are as follows:
1) patient examines by blood biochemical, Biochemical urinalysis and virology, and it is different to exclude dysfunction of liver, renal function
Often, virus carries dangerous, enters group later.
2) dermal harvest position is posterior neck.Collection capacity is 4mm3.Skin histology after acquisition is stored in tissue transport liquid
In, it transports 2 hours, transport temperature is 4 DEG C.
3) cleaning 2 times aseptically, is carried out to tissue with tissue-wash solution, with aseptic apparatus removal epidermis and subcutaneously
Fat sites.Dermal tissue position is subjected to sterile cutting, the tissue after cutting is clung in culture dish, and originally culture is added
Base starts to cultivate.
4) after cultivating 2 to 3 days, primary dermal fibroblast is largely grown.Microscopy observation, gnotobasis go down unless
FI phenotype cells retain the cell of high-purity phenotype.Such as Fig. 1.
5) it when cell grows to 90% density, is digested with pancreatin, and cell is suspended again, dilution inoculation, in amplification cultivation
Amplification cultivation is carried out in base.
6) in secondary culture, a part of cell carries out purity check in this process.By antibody CD73, CD90,
Fibroblastic purity in product is examined in the incubation of CD105.By methionine method of inspection, the purity of FI phenotype cells is examined.
7) cell in 28 days, is expanded to 10 after step 5) inoculation7A FI type living cells.
8) the final digestion collecting process of cell digests all cells using pancreatin, and postdigestive cell is with containing
The culture medium of serum is terminated.Culture medium in cell suspension is replaced as physiological saline with centrifugal method.It is given birth to after concentration
Manage the living cells injection of salt water.
9) rest part cell is replaced as frozen stock solution using centrifugal process, cools down by program, is permanently stored in liquid nitrogen,
It is for later use.
Embodiment 4
33 years old female face eye sockets outsides, 1.5 grades of downside wrinkles, auto derma fibroblast injection fillers beauty
(1) dermal tissue harvesting
1) after ear-lobe, 4mm is acquired3The full skin tissue of volume, takes dermal partial, the adhere-wall culture in culture vessel；
2) after originally culture 3 days, cell starts to grow；
3) originally culture 5 days when, micromanipulation remove FI phenotype other than other cells；
4) originally culture 7 days obtain the dermal fibroblast through originally culture；
(2) the fibroblastic production of auto derma
5) dermal fibroblast to above-mentioned through originally culture carries out first time digestion, dilution, amplification, after 14 days, the
Secondary digestion, dilution, amplification, while carrying out the mycoplasma, viral diagnosis, Purity of raw cell；
6) it produces the 21st day, third time digestion, dilution, amplification；
7) it produces the 28th day, collects raw cell；Produce living cells preparation, preparation quality inspection；Clinical use (summary)；Remaining is thin
Born of the same parents freeze；
8) later period produces once again, and clinical application is primary；
(3) clinical application
Wrinkle area is assessed as 0.5 grade.
Auto derma fibroblast in the present invention is prepared into injection, is injected directly into eye socket wrinkle area, as a result
As shown in Figure 4, wherein a indicates eye circumference under the left side before injection；B indicates eye circumference under the right side before injection；After c indicates injection
Eye circumference under left side；D indicates eye circumference under the right side after injection, and comparison is as it can be seen that dominant wrinkle disappears.
Protection content of the invention is not limited to above embodiments.Under the spirit and scope without departing substantially from present inventive concept,
Various changes and advantages that will be apparent to those skilled in the art are all included in the present invention, and are with appended claims
Protection scope.
Claims (10)
1. a kind of construction method of dermal fibroblast skin graft, which is characterized in that the method are as follows:
Step (1): the dermal fibroblast of amplification in vitro is integrated to the amnion by taking off cell, degreasing, de- nucleic acid processing
Tissue carries out cell curing, constructs dermal fibroblast-amnion tissue composite construction；The dermal fibroblast is FI
Phenotype is at fibroblasts of adult human dermis；
Step (2): cultivating in amplification culture medium, obtains the dermal fibroblast skin graft.
2. construction method as described in claim 1, which is characterized in that the primary tissue of dermal fibroblast is in originally culture
It is cultivated in base；Then the amplification in vitro is carried out, the method for the amplification in vitro is digestion-resuspension-dilution inoculation；Its
In, the condition of the digestion are as follows: under the action of trypsase-EDTA, 37 DEG C, digest 2~3 minutes；The condition of the resuspension
Are as follows: in containing serum solution, 37 DEG C, mechanical piping and druming；The dilution inoculation refers to, in amplification culture medium, every 1cm2Culture face
Product inoculation 104A cell carries out secondary culture.
3. construction method as described in claim 1, which is characterized in that the primary tissue of the dermal fibroblast is primary
It is cultivated in culture medium, the primary culture medium contains the component of following volumes percentage: 70-86%DMEM high sugar culture
Base, 12-18% fetal calf serum, 0.5-1% nonessential amino acid, 0.5-1% glutamine and 1-10% mycillin.
4. construction method as described in claim 1, which is characterized in that the dermal fibroblast be integrated to amnion tissue into
The method of row cell curing is inoculated and cultured；The building raw material of the inoculated and cultured include dermal fibroblast, collagen,
Cell amplification culture medium or physiological saline.
5. construction method as described in claim 1, which is characterized in that the amnion carry out de- cell, degreasing, de- albumen,
De- nucleic acid processing.
6. construction method as claimed in claim 1 or 5, which is characterized in that wherein, the de- cell treatment fluid includes
TritonX-100, volume fraction 0.1-0.5%；And/or the ungrease treatment liquid includes physiological saline, neutral lipase, pH
It is 7.0 to 7.4, neutral fat enzyme concentration is 5-10U/mL；And/or the de- albumen treatment fluid includes physiological saline, alkaline pancreas
Protease；And/or the de- nucleic acid treatment fluid includes nuclease, SDS, pH value of solution is 7.0 to 7.4, mass concentration 0.3-
0.8%.
7. construction method as described in claim 1, which is characterized in that the dermal fibroblast is logical at originally culture initial stage
Cross micromanipulation, the method directly rejected is purified.
8. a kind of dermal fibroblast skin graft that construction method according to claim 1 is prepared.
9. dermal fibroblast skin graft as claimed in claim 7 is lacked in preparation treatment burn trauma, chronic disease or skin
The application in product that damage, slow wound are burnt, or the application in product of the preparation for skin-grafting.
10. application as claimed in claim 9, which is characterized in that the dermal fibroblast skin graft is for preventing, eliminating
And/or reduce wrinkle.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110269868A (en) * | 2019-06-12 | 2019-09-24 | 江苏艾尔康生物医药科技有限公司 | A kind of construction method containing auto derma fibroblast hyaluronic acid derivatives agent |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006068972A2 (en) * | 2004-12-21 | 2006-06-29 | Ethicon, Inc. | Tissue engineering devices for the repair and regeneration of tissue |
| CN101361989A (en) * | 2008-09-03 | 2009-02-11 | 陕西瑞盛生物科技有限公司 | Double membrane tissue patching material and preparation method thereof |
| CN101361990A (en) * | 2008-09-03 | 2009-02-11 | 陕西瑞盛生物科技有限公司 | Double layer artificial skin and preparation method thereof |
| CN101757690A (en) * | 2010-02-05 | 2010-06-30 | 陕西瑞盛生物科技有限公司 | Method for preparing tissue engineering cornea |
| CN101954124A (en) * | 2010-08-31 | 2011-01-26 | 中国人民解放军第二军医大学 | Tissue engineered skin with basilar membrane and construction method thereof |
| CN103114073A (en) * | 2013-01-23 | 2013-05-22 | 宁波大学 | Method for removing cells from human amnion |
| CN103893831A (en) * | 2012-12-28 | 2014-07-02 | 广州暨南大学医药生物技术研究开发中心 | Organ-type artificial skin as well as preparation method and application thereof |
| WO2015195506A1 (en) * | 2014-06-15 | 2015-12-23 | Amnio Technology Llc | Acellular amnion derived therapeutic compositions |
| CN105688287A (en) * | 2016-01-26 | 2016-06-22 | 深圳爱生再生医学科技有限公司 | Amniotic membrane patch for treating skin wound and preparation method thereof |
| CN105797212A (en) * | 2016-04-22 | 2016-07-27 | 黑龙江天晴干细胞股份有限公司 | Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal |
| CN106668955A (en) * | 2017-01-09 | 2017-05-17 | 广州润虹医药科技有限公司 | Double-layer tissue engineering skin and preparation method thereof |
| WO2017120371A1 (en) * | 2016-01-08 | 2017-07-13 | Cryolife, Inc. | Human placental tissue graft products, methods, and apparatuses |
| CN107019816A (en) * | 2017-03-30 | 2017-08-08 | 贵州省人民医院 | A kind of compound method for going epidermis dermis to build organization engineering skin of amniotic epithelial cells |
| CN107254431A (en) * | 2017-05-25 | 2017-10-17 | 武汉珺睿再生医学科技研究有限公司 | A kind of novel tissue engineering skin preparation method |
-
2018
- 2018-12-29 CN CN201811635902.7A patent/CN109771697B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006068972A2 (en) * | 2004-12-21 | 2006-06-29 | Ethicon, Inc. | Tissue engineering devices for the repair and regeneration of tissue |
| CN101361989A (en) * | 2008-09-03 | 2009-02-11 | 陕西瑞盛生物科技有限公司 | Double membrane tissue patching material and preparation method thereof |
| CN101361990A (en) * | 2008-09-03 | 2009-02-11 | 陕西瑞盛生物科技有限公司 | Double layer artificial skin and preparation method thereof |
| CN101757690A (en) * | 2010-02-05 | 2010-06-30 | 陕西瑞盛生物科技有限公司 | Method for preparing tissue engineering cornea |
| CN101954124A (en) * | 2010-08-31 | 2011-01-26 | 中国人民解放军第二军医大学 | Tissue engineered skin with basilar membrane and construction method thereof |
| CN103893831A (en) * | 2012-12-28 | 2014-07-02 | 广州暨南大学医药生物技术研究开发中心 | Organ-type artificial skin as well as preparation method and application thereof |
| CN103114073A (en) * | 2013-01-23 | 2013-05-22 | 宁波大学 | Method for removing cells from human amnion |
| WO2015195506A1 (en) * | 2014-06-15 | 2015-12-23 | Amnio Technology Llc | Acellular amnion derived therapeutic compositions |
| WO2017120371A1 (en) * | 2016-01-08 | 2017-07-13 | Cryolife, Inc. | Human placental tissue graft products, methods, and apparatuses |
| CN105688287A (en) * | 2016-01-26 | 2016-06-22 | 深圳爱生再生医学科技有限公司 | Amniotic membrane patch for treating skin wound and preparation method thereof |
| CN105797212A (en) * | 2016-04-22 | 2016-07-27 | 黑龙江天晴干细胞股份有限公司 | Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal |
| CN106668955A (en) * | 2017-01-09 | 2017-05-17 | 广州润虹医药科技有限公司 | Double-layer tissue engineering skin and preparation method thereof |
| CN107019816A (en) * | 2017-03-30 | 2017-08-08 | 贵州省人民医院 | A kind of compound method for going epidermis dermis to build organization engineering skin of amniotic epithelial cells |
| CN107254431A (en) * | 2017-05-25 | 2017-10-17 | 武汉珺睿再生医学科技研究有限公司 | A kind of novel tissue engineering skin preparation method |
Non-Patent Citations (3)
| Title |
|---|
| ANAHI SANLUIS-VERDES ET AL: "Production of an acellular matrix from amniotic membrane for the synthesis of a human skin equivalent", 《CELL TISSUE BANK》 * |
| MAHNAZ MAHMOUDI RAD ET AL: "Application of Allogeneic Fibroblast Cultured on Acellular Amniotic Membrane for Full-thickness Wound Healing in Rats", 《WOUNDS》 * |
| SARUMATHI GOBINATHAN ETAL: "Decellularization and genipin crosslinking of amniotic membrane suitable for tissue engineering applications", 《JOURNAL OF BIOAMTERIALS SCIENCE,POLYMER EDITION》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110269868A (en) * | 2019-06-12 | 2019-09-24 | 江苏艾尔康生物医药科技有限公司 | A kind of construction method containing auto derma fibroblast hyaluronic acid derivatives agent |
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