CN102367434A - Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof - Google Patents
Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof Download PDFInfo
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Abstract
The invention relates to the technical fields of tissue engineering and medical wound healing, and provides an amniotic membrane microcarrier capable of simulating a niche microenvironment for the growth of epidermal stem cells in vitro and a skin substitute thereof. A medical waste human amniotic membrane is used as the host material, is subjected to a repeated freeze-thawing & DNA enzyme digestion method to remove amniotic cells, and is further subjected to freeze-cracking to be prepared into the amniotic membrane microcarrier which is 200-500 mu m in size and has a three-dimensional spatial structure. The amniotic membrane microcarrier has the characteristics of common microcarriers, retains the basement membrane structure, has relatively complete Type-IV collagen and laminin, and simultaneously contains NGF, HGF, KGF, bFGF, TGF-beta1, EGF and other growth factors, thereby providing an approximate physiological niche microenvironment for the in-vitro culture and amplification of epidermal stem cells, and quickly amplifying the epidermal stem cells while keeping the characteristics of the stem cells. Besides, the amniotic membrane microcarrier can be directly used as a dermal scaffold for the construction of a skin substitute. Animal full-thickness skin defect and graft experiment shows that the healing quality of the skin substitute is far superior to those of a simple amniotic membrane microcarrier group and a blank control group, a regenerated epidermal layer is obviously thickened and is accompanied with the formation of a mammillary process like structure, and collagenous fibers of a regenerated dermal layer are regularly and orderly arranged.
Description
Technical field
The present invention relates to organizational project and medical science wound repair technical field, be a kind of can be at the amnion microcarrier and the Graftskin thereof of in-vitro simulated epidermal stem cells growth niche microenvironment.
Background technology
The cultivation of epidermal stem cells, amplification are focus and the difficult points in the skin tissue engineering research always.Mainly utilize at present epidermal stem cells that basilar membrane sticked significantly that characteristic is separated, purifying (Barthel R, Aberdam D.Epidermal stem cells.J Eur Acad Dermatol Venereol2005; 19 (4): 405-13.), adopt the two dimensional surface best cultivation to carry out vitro culture and amplification.Go down to posterity when increasing in order to guarantee the single of stem cell; Need at first one of major ingredient IV Collagen Type VI to be tiled in petridish with basilar membrane; But be difficult to avoid cell to cultivate through repeatedly digesting, going down to posterity that the rear clone rate of formation obviously descends, shortcomings such as differentiation, proliferation activity reduction gradually; And because the slow multiplication characteristic of epidermal stem cells inherent, traditional training method be difficult to increase within a short period of time cell of capacity can't in time satisfy the needs of application.
It is the technology of the adherent dependent form cell of a kind of extensive amplification that microcarrier is cultivated; The three-dimensional space that has not possessed when providing cell monolayer to cultivate; Simulated cell growth in vivo environment; Amplifying cells in a large number not only, and help keeping cell phenotype, prevent differentiation, be widely used in field of biological pharmacy at present.The microcarrier of development is how synthetic with biological material artificial at present, lacks basement membrane structure, is unfavorable for adhesion, the propagation of epidermal stem cells.The possessor adopts bionics techniques such as surfaction or modification to handle to the greatest extent, but still is difficult to simulate the interior niche microenvironment of body of epidermal stem cells growth, is unfavorable for keeping its stem cell characteristic.
Research shows; People's amnion is ideal cell cultures amplification vector (Niknejad H; Peirovi H, Jorjani M, et a1.Properties of the amniotic membrane for potential use in tissue engineering.Eur Cell Mater 2008; 29 (15): 88-99.); Have the natural the thickest basilar membrane of human body, its substruction and skin, cornea basilar membrane are similar, contain abundant growth factor in this epimatrix; Like (Koizumi NJ such as NGF, HGF, KGF, bFGF, TGF-β 1, EGF; Inatomi TJ, Sotozono CJ, et al.Growth factor mRNA and protein in preserved human amniotic membrane.Curr Eye Res 2000; 20:173-7.); Can be at the niche of in-vitro simulated stem cell growth microenvironment (Grueterich M; Espana EM, Tseng SC.Ex vivo expansion of limbal epithelial stem cells:amniotic membrane serving as a stem cell niche.Surv Ophthalmol 2003; 48 (6): 631-46.); Therefore amnion is used to the amplification and transplanting (the Shortt AJ of limbal stem cell, mescenchymal stem cell; Secker GA; Lomas RJ, et al.The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells.Biomaterials 2009; 30 (6): 1056-65.Liang HS; Liang P; Xu Y, et al.Denuded human amniotic membrane seeding bone marrow stromal cells as an effective compo site matrix stimulates axonal outgrowth of rat neural cortical cells in vitro.Acta Neurochir (Wien) 2009; 151 (9): 1113-20.).But do not see that so far amnion is prepared to the amnion microcarrier of simulation epidermal stem cells growth niche microenvironment and makes up the report of epidermal stem cells Graftskin with it.
Summary of the invention
The present invention provide a kind of can be at the amnion microcarrier of in-vitro simulated epidermal stem cells growth niche microenvironment and with the epidermal stem cells Graftskin of its structure.
Amnion microcarrier of the present invention is the 3 D stereo particulate amnion with complete basement membrane structure and BA; As a kind of natural novel microcarrier, in conjunction with the rotating and culturing system, the cultured and amplified in vitro that can be epidermal stem cells provides approximate intravital stem cell growth niche microenvironment with it; Epidermal stem cells not only can increase rapidly the short period of time; Keep the epidermal stem cells proliferation activity, keep its stem cell characteristic, and can directly it be made up Graftskin as dermis scaffold.
The present invention with amnion in the discarded placenta of medical treatment as matrix; Adopt multigelation method cracking amnion cell; Auxiliary DNA enzyme is removed nucleic acid; Further have the amnion microcarrier of 3-D solid structure through freezing cracking technique preparation, it has preserved biologically active substance in complete basement membrane structure and the matrix, can physiological niche microenvironment in the approximation be provided for epidermal stem cells.
The construction process of amnion microcarrier of the present invention is following:
1, amnion obtains
Get the healthy fresh human placenta tissue of depleted by routine; Denuded amniotic membrane under aseptic condition, passivity is separated chorion, through containing phosphate buffered saline (PBS) soaking flushing 3 * 15min of 100U/ml penicillium mould, 100 μ g/ml Streptomycin sulphates, 0.25 μ g/ml B fungizone; Amnion is cut into the small pieces of 3 * 2cm size; Put into freeze pipe, add the PBS that contains 10%DMSO, freeze pipe is put-80 ℃ of refrigerators and preserved subsequent use;
2, amnion goes the cell processing
Take out above-mentioned freeze pipe, put 25 ℃ of water-baths and fully thawed the PBS in the sucking-off amnion 10 minutes; Again freeze pipe is positioned in the liquid nitrogen freezingly, takes out after leaving standstill 30min, fully thawed 10 minutes in 25 ℃ of water-baths; Place liquid nitrogen freezing once more, so multigelation is 3 times, then amnion is incubated in enzymolysis among the 1mg/mlDNAase; Hatch the slight concussion of companion in 3 hours under 37 ℃ of conditions, centrifugal 2000rpm * 2min under the room temperature removes supernatant; Fully wash freeze-drying behind the amnion, slabbing amnion by routine with PBS;
3, preparation amnion microcarrier
The sheet amnion is prepared into the amnion microcarrier through freezing cleavage method, and method is following: under the room temperature condition, the sheet amnion of above-mentioned preparation is placed liquid nitrogen rapidly; By its spontaneous microfracture; Become particulate through the freezing cracking sheet amnion that homogenizes, to avoid destroying amnion tissue microstructure, lyophilize under the vacuum seal condition then; The amnion particulate is filtered through 32 orders, 80 order metallic sieves successively, obtain the amnion microcarrier of 200-500 μ m.
Make up the epidermal stem cells Graftskin with amnion microcarrier of the present invention, method is following:
1) cultivation, amplification epidermal stem cells
Adopt conventional " enzymic digestion+IV Collagen Type VI adheres to fast " method separation of human skin epidermal stem cell (to see for details: Kim DS; Cho HJ; Choi HR; Kwon SB, Park KC.Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents.Cell Mol Life Sci 2004; 61 (21): 2774-81.).
Get the foreskin postoperative skin, with 4 ℃ of digestion of 0.25%Dispase, the separating table cortex was with 37 ℃ of digestion of 0.25% trypsinase 20-30 minute by routine; Metallic sieve filters, centrifugal collecting cell, with cell inoculation in the petridish that is covered with the IV Collagen Type VI in advance; Abandon supernatant behind the adherent 20min, add serum free medium (Keratinocyte serum free medium, K-SFM; Gibco down with), the next day change liquid, to use trysinization to prepare concentration when 70%-80% merges be 5 * 10 when cell reaches
5The epidermal stem cells suspension of individual/ml is subsequent use;
2) make up the epidermal stem cells Graftskin
The amnion microcarrier 100mg injection capacity of getting above-mentioned epidermal stem cells suspension 10ml and above-mentioned preparation is the rotating and culturing system (RCCS) of 10ml; Set speed of rotation 22rpm; The next day change liquid with serum free medium K-SFM half amount, when the cell growth reaches 70%-80% and merges, add new amnion microcarrier; The collision that contacts with each other through between the amnion particulate continues the amplification epidermal stem cells, cultivates 2-3 and forms the Graftskin that contains epidermal stem cells after week.
The present invention also available angle cell plastid or inoblast replaces used epidermal stem cells to make up Graftskin as seed cell.
The advantage of amnion microcarrier of the present invention is: amnion microcarrier size has 3-D solid structure for 200-500 μ m, and it not only possesses the characteristic of general microcarrier; As has a bigger specific surface area; For cell proliferation provides the dimensional culture space, can the rapid amplifying cell, and kept basement membrane structure; Have comparatively complete IV Collagen Type VI, ln; Contain growth factors such as NGF, HGF, KGF, bFGF, TGF-β 1, EGF simultaneously, for the cultured and amplified in vitro of epidermal stem cells provides the niche microenvironment of physiological status in the approximation, can be when keeping the stem cell characteristic rapid amplifying epidermal stem cells; Amnion microcarrier immunogenicity is low in addition, and good biocompatibility can be directly as dermis scaffold load epidermis stem cell transplantation wound repairing.The dual-use function of amnion microcarrier of the present invention improved present Graftskin make up in amplification capacity cell earlier, inoculate the structure pattern that second incubation behind the dermis scaffold forms Graftskin.The present invention utilizes the amnion microcarrier to combine the rotating and culturing system one step to accomplish a large amount of amplifications of epidermal stem cells and the quick structure of Graftskin; Not only shorten vitro culture and made up the time; And avoided digesting repeatedly in the ordinary method damage that pair cell causes; Help keeping cell-proliferation activity, improved Graftskin is building up to clinical Transformation Application from the laboratory efficient simultaneously.
External epidermal stem cells amplification experiment shows; The amnion microcarrier of the present invention epidermal stem cells that can increase rapidly, when cultivating 7,14 days, proliferation activity is respectively 326 ± 28%, 535 ± 47% relatively; Cultural method (232 ± 21% apparently higher than routine; 307 ± 32%, P＜0.05), can effectively prevent cytodifferentiation simultaneously, keep its stem cell characteristic.Nude mice holostrome skin injury surface of a wound transplantation experiments shows; The Graftskin of load epidermal stem cells of the present invention is at simple amnion microcarrier or the blank that are much better than not load epidermal stem cells aspect the nude mice wound healing quality; Not only flat appearance, softness behind the wound healing, shrinkage degree is light, and newborn epidermal area obviously thickens; Form synkaingenesis skin corium arrangement of collagen fibers rule, orderly with the mastoid process spline structure.
Description of drawings
Fig. 1 is for adopting the situation of microcarrier mAM amplification epidermal stem cells of the present invention, and wherein A and C observe under the phase microscope, and B and D are that Hoechst dyeing back fluoroscope is observed down.
Fig. 2 compares for the relative proliferation activity of cell that adopts microcarrier mAM of the present invention and conventional petridish to cultivate the amplification epidermal stem cells.
Fig. 3 is the wound healing comparison diagram that nude mice holostrome skin injury is transplanted ESC-mAM, mAM and blank group.
Fig. 4 is the nude mice holostrome skin injury post-transplantation pathological section figure in 4 weeks, and A～D is followed successively by the HE dyeing of normal nude mice skin, ESC-mAM transplantation group, blank group and mAM transplantation group.
Embodiment
Combine accompanying drawing and embodiment at present, the present invention is further described.
Embodiment 1. preparations amnion microcarrier mAM of the present invention
1, amnion obtains
Get the medically healthy fresh human placenta tissue of depleted by routine; Denuded amniotic membrane under aseptic condition, passivity is separated chorion, through containing phosphate buffered saline (PBS) soaking flushing 3 * 15min of 100U/ml penicillium mould, 100 μ g/ml Streptomycin sulphates, 0.25 μ g/ml B fungizone; Amnion is cut into the small pieces of 3 * 2cm size; Put into freeze pipe, add the PBS that contains 10%DMSO, freeze pipe is put-80 ℃ of refrigerators and preserved subsequent use;
2, amnion goes the cell processing
Take out above-mentioned freeze pipe, put 25 ℃ of water-baths and fully thawed the PBS in the sucking-off amnion 10 minutes; Again freeze pipe is positioned in the liquid nitrogen freezingly, takes out after leaving standstill 30min, fully thawed 10 minutes in 25 ℃ of water-baths; Place liquid nitrogen freezing once more, so multigelation is 3 times, then amnion is incubated in enzymolysis among the 1mg/mlDNAase; Hatch the slight concussion of companion in 3 hours under 37 ℃ of conditions, centrifugal 2000rpm * 2min under the room temperature removes supernatant; Fully wash freeze-drying behind the amnion, slabbing amnion by routine with PBS;
3, preparation amnion microcarrier mAM
The sheet amnion is prepared into the amnion particulate through freezing cleavage method, and is specific as follows: under the room temperature condition, the sheet amnion of above-mentioned preparation is placed liquid nitrogen rapidly; By its spontaneous microfracture; Become particulate through the freezing cracking sheet amnion that homogenizes, to avoid destroying amnion tissue microstructure, lyophilize under the vacuum seal condition then; The amnion particulate is filtered through 32 orders, 80 order metallic sieves successively, obtain the amnion microcarrier mAM of 200-500 μ m;
Embodiment 2. makes up epidermal stem cells Graftskin ESC-mAM
1) cultivation, amplification epidermal stem cells
Get the foreskin postoperative skin, by routine with 4 ℃ of digestion of 0.25%Dispase, separating table cortex; With 37 ℃ of digestion of 0.25% trypsinase 20-30 minute, metallic sieve filtered, centrifugal collecting cell; Cell inoculation in the petridish that is covered with the IV Collagen Type VI in advance, is abandoned supernatant behind the adherent 20min, add serum free medium K-SFM; The next day change liquid, to use trysinization to prepare concentration when 70%-80% merges be 5 * 10 when cell reaches
5The epidermal stem cells suspension of individual/ml is subsequent use;
2) adopt mAM amplification epidermal stem cells and be built into Graftskin ESC-mAM
The amnion microcarrier mAM 100mg injection capacity of getting above-mentioned epidermal stem cells suspension 10ml and embodiment 1 preparation is the rotating and culturing system of 10ml; Set speed of rotation 22rpm; The next day change liquid with serum free medium K-SFM half amount, when the cell growth reaches 70%-80% and merges, add new amnion microcarrier mAM; The collision that contacts with each other through between the amnion particulate continues the amplification epidermal stem cells, and vitro culture 2-3 forms the Graftskin ESC-mAM that contains epidermal stem cells after week.
The result sees accompanying drawing.Visible by Fig. 1, in the time of the 3rd day, the epidermal stem cells among the ESC-mAM attaches to mAM and is three-dimensional growth (Fig. 1-A and B), and cell clone showed increased in the time of the 7th day is clouded in mAM surface (Fig. 1-C and D).Fig. 2 has further compared the relative proliferation activity of cell that adopts microcarrier mAM of the present invention and conventional petridish to cultivate the amplification epidermal stem cells and has compared.Visible by Fig. 2; Beginning in the 3rd day, mAM cultured cells proliferation activity is higher than cultivation (P＜0.05) in the petridish among the ESC-mAM of the present invention, when being cultured to 7,14 days; Proliferation activity is respectively 326 ± 28%, 535 ± 47% relatively; Petridish cultural method (232 ± 21%, 307 ± 32%, P＜0.05) apparently higher than routine.
Experimentation on animals
Get 6-8 week 12 of nude mices (being provided by west, Shanghai pul-Bi Kai laboratory animal ltd), male and female are not limit, by routine with 1% vetanarcol intraperitoneal anesthesia, alcohol disinfecting.Making diameter respectively in both sides, every nude mice back is the circular holostrome skin injury surface of a wound of 1.2cm, and 24 surface of a wound are divided into ESC-mAM group, mAM group and blank group at random.ESC-mAM organizes before transplanting with CM-Dil (Invitrogen; USA) dye marker epidermal stem cells; Marking method sees the CM-Dil working instructions for details, respectively ESC-mAM, mAM equivalent is evenly dripped subsequently to be coated on the surface of a wound, and blank is formed face and dripped and be coated with equivalent PBS; Cover petrolatum gauze again, the petrolatum gauze interrupted suture is fixed in all skin of wound with the 4-0 line.The postoperative routine observation is taken pictures, and compares the wound healing rate through image analysis software, and the wound healing rate is the ratio that the surface-area of non-epithelization accounts for total surface of a wound area, and the capable histological observation of regularly drawing materials.
The result finds, transplants back 2 all (see figure 3)s, and ESC-mAM organizes newborn epidermis and forms; Thinner, the shape that is translucent, the wound healing rate is 97.6 ± 2.1%; And mAM group is formed face with blank and is obviously shunk, and it is thus clear that still the remaining surface of a wound, healing rate is respectively 93.4 ± 4.3%; 91.2 ± 4.9%, be starkly lower than ESC-MAM group (p＜0.01); Transplant 3 weeks of back, ESC-MAM organizes newborn epidermis and thickens, and the surface of a wound heals basically, flat appearance, softness, and shrinkage degree is light, and mAM group and blank are formed the face contraction obviously.Transplant 4 week of back row section HE dyeing (see figure 4); Amnion microcarrier filling dermal matrix under ESC-mAM, the mAM group healing epidermis; Epidermic cell forms the 6-8 layer, and the blank group is the 3-4 layer only, and ESC-mAM association has epidermis mastoid process spline structure to form in addition; Similar normal skin does not see that the mastoid process spline structure forms and mAM group and blank group epidermic cell bases are smooth.
Experimental result shows; Amnion microcarrier of the present invention has complete basement membrane structure and biological active agents; The cultured and amplified in vitro that can be epidermal stem cells provides approximate intravital stem cell growth niche microenvironment; So the epidermal stem cells that not only can increase rapidly the short period of time, keep the epidermal stem cells proliferation activity, and can directly make up Graftskin transplanting reparation holostrome skin injury as dermis scaffold.
Claims (4)
1. simulate the amnion microcarrier of epidermal stem cells growth niche microenvironment, construction process is following:
1) amnion obtains
Get discarded healthy fresh placenta tissue by routine; Denuded amniotic membrane under aseptic condition, passivity is separated chorion, through containing the PBS soaking flushing 3 * 15min of 100U/ml penicillium mould, 100 μ g/ml Streptomycin sulphates, 0.25 μ g/ml B fungizone; Amnion is cut into the small pieces of 3 * 2cm size; Put into freeze pipe, add the PBS that contains 10%DMSO, freeze pipe is put-80 ℃ of refrigerators and preserved subsequent use;
2) amnion goes the cell processing
Take out above-mentioned freeze pipe, put 25 ℃ of water-baths and fully thaw the PBS in the sucking-off amnion; Again freeze pipe is positioned in the liquid nitrogen freezingly, so thaws and freezing multigelation 3 times, then amnion is incubated in enzymolysis among the 1mg/mlDNAase; Hatch the slight concussion of companion in 3 hours under 37 ℃ of conditions; Centrifugal removal supernatant under the room temperature fully washes freeze-drying behind the amnion, slabbing amnion by routine with PBS;
3) preparation amnion microcarrier
Above-mentioned sheet amnion is placed liquid nitrogen rapidly; Through the freezing cracking that homogenizes, the sheet amnion is cracked into the amnion particulate, then lyophilize under the vacuum seal condition; The amnion particulate is filtered through 32 orders, 80 order metallic sieves successively, obtain the amnion microcarrier of 200-500 μ m.
2. the epidermal stem cells Graftskin that makes up of the said microcarrier of claim 1, construction process is following:
1) cultivation, amplification epidermal stem cells
Get the discarded skin of postoperative, by routine with 4 ℃ of digestion of 0.25%Dispase, separating table cortex; With 37 ℃ of digestion of 0.25% trypsinase 20-30 minute, metallic sieve filtered, centrifugal collecting cell; Cell inoculation in the petridish that is covered with the IV Collagen Type VI in advance, is abandoned supernatant behind the adherent 20min, add serum free medium K-SFM; The next day change liquid, to prepare the epidermal stem cells suspension with trysinization when 70%-80% merges subsequent use when cell reaches;
2) make up the epidermal stem cells Graftskin
Get the described amnion microcarrier of above-mentioned epidermal stem cells suspension and claim 1 and inject the cultivation of rotating and culturing system by a certain percentage; The next day change liquid with serum free medium K-SFM half amount; When the cell growth reaches the 70%-80% fusion; Add new amnion microcarrier and continue the amplification epidermal stem cells, cultivate 2-3 and form the Graftskin that contains epidermal stem cells after week.
3. the application of amnion microcarrier in making up Graftskin of the said simulation epidermal stem cells growth of claim 1 niche microenvironment.
4. the application of the said Graftskin of claim 2 in preparation wound repair graft materials.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114073A (en) * | 2013-01-23 | 2013-05-22 | 宁波大学 | Method for removing cells from human amnion |
| CN105018417A (en) * | 2015-07-22 | 2015-11-04 | 中国人民解放军第二军医大学 | Amnion innate stem cell carried frozen active amnion particle and conditioned medium and application thereof |
| CN106520677A (en) * | 2016-11-01 | 2017-03-22 | 浙江译美生物科技有限公司 | Recovery liquid and method for cryopreserved epidermal stem cells |
| CN107583109A (en) * | 2017-09-22 | 2018-01-16 | 吉林大学 | A kind of corium deep layer filler and its preparation method and application |
| CN108624581A (en) * | 2018-05-15 | 2018-10-09 | 中国科学院苏州生物医学工程技术研究所 | A kind of microballoon and brainpower insufflation system of mescenchymal stem cell materials for binding biological |
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| CN115105636A (en) * | 2022-07-16 | 2022-09-27 | 沈阳汇智细胞产业技术创新研究院有限公司 | Preparation method of acellular human amniotic membrane |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563366A (en) * | 2004-04-09 | 2005-01-12 | 西北农林科技大学 | Preparation method and application of epidermal stem cell constructed tissue engineering corneal epithelial implant |
| CN101352586A (en) * | 2008-08-26 | 2009-01-28 | 程树军 | Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation |
-
2011
- 2011-06-03 CN CN 201110148123 patent/CN102367434B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563366A (en) * | 2004-04-09 | 2005-01-12 | 西北农林科技大学 | Preparation method and application of epidermal stem cell constructed tissue engineering corneal epithelial implant |
| CN101352586A (en) * | 2008-08-26 | 2009-01-28 | 程树军 | Method for preparing full-thickness skin for toxicity test by stem cell raft type cultivation |
Non-Patent Citations (2)
| Title |
|---|
| 刘德伍等: "表皮干细胞复合羊膜构建组织工程皮肤修复糖尿病难愈创面", 《透析与人工器官》, vol. 21, no. 3, 30 September 2010 (2010-09-30) * |
| 钟清玲等: "羊膜负载表皮干细胞促进糖尿病大鼠创面的愈合", 《中国组织工程研究与临床康复》, vol. 14, no. 32, 6 August 2010 (2010-08-06), pages 6010 - 6014 * |
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| CN103114073A (en) * | 2013-01-23 | 2013-05-22 | 宁波大学 | Method for removing cells from human amnion |
| CN103114073B (en) * | 2013-01-23 | 2014-11-05 | 宁波大学 | Method for removing cells from human amnion |
| CN105018417A (en) * | 2015-07-22 | 2015-11-04 | 中国人民解放军第二军医大学 | Amnion innate stem cell carried frozen active amnion particle and conditioned medium and application thereof |
| CN105018417B (en) * | 2015-07-22 | 2018-11-23 | 中国人民解放军第二军医大学 | The load intrinsic stem cell of amnion freezes active amnia particle and its conditioned medium and application |
| CN106520677A (en) * | 2016-11-01 | 2017-03-22 | 浙江译美生物科技有限公司 | Recovery liquid and method for cryopreserved epidermal stem cells |
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| CN107583109B (en) * | 2017-09-22 | 2020-09-25 | 吉林大学 | Dermal deep filler and preparation method and application thereof |
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| CN109701076A (en) * | 2019-01-29 | 2019-05-03 | 北京颢美细胞基因生物技术有限公司 | Regenerating tissues matrix particles, preparation method and application for micro-shaping |
| CN109513044A (en) * | 2019-01-29 | 2019-03-26 | 北京颢美细胞基因生物技术有限公司 | For the regenerating tissues matrix particles implant of micro-shaping, preparation method and application |
| CN109646719B (en) * | 2019-01-29 | 2022-02-11 | 北京颢美细胞基因生物技术有限公司 | Articular cartilage repair composition containing regenerated tissue matrix microparticles |
| CN109701076B (en) * | 2019-01-29 | 2022-02-15 | 北京颢美细胞基因生物技术有限公司 | Regenerated tissue matrix particles for micro-plastic, preparation method and application |
| CN109513044B (en) * | 2019-01-29 | 2022-02-18 | 北京颢美细胞基因生物技术有限公司 | Regenerated tissue matrix particle implant for micro-plastic, preparation method and application |
| CN115105636A (en) * | 2022-07-16 | 2022-09-27 | 沈阳汇智细胞产业技术创新研究院有限公司 | Preparation method of acellular human amniotic membrane |
| CN115105636B (en) * | 2022-07-16 | 2024-03-19 | 沈阳汇智细胞产业技术创新研究院有限公司 | Preparation method of acellular human amniotic membrane |
| CN117757719A (en) * | 2023-12-25 | 2024-03-26 | 中国人民解放军总医院第一医学中心 | Micro-tissue for accelerating skin injury repair and application thereof |
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