Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof
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A technology of epidermal stem cells and microcarriers, which is applied in the fields of tissue engineering and medical wound repair, can solve the problems of no amnion, etc., and achieve the effect of maintaining stem cell characteristics, good biocompatibility, and maintaining proliferation activity
Inactive Publication Date: 2013-06-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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But so far, there is no report that amnion is prepared as amnion microcarrier simulating the niche microenvironment of epidermal stem cell growth and used to construct epidermal stem cell skin substitute
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Embodiment 1
[0029] Embodiment 1. prepare amniotic membrane microcarrier mAM of the present invention
[0030] 1. Acquisition of amniotic membrane
[0031] Take the healthy fresh placental tissue that was discarded in medicine routinely, peel off the amniotic membrane under aseptic conditions, bluntly separate the chorion, and phosphate buffered with 100U / ml penicillin, 100μg / ml streptomycin, 0.25μg / ml amphotericin Soak and rinse in saline (PBS) for 3×15min, cut the amniotic membrane into small pieces of 3×2cm, put them into a cryovial, add PBS containing 10% DMSO, and store the cryovial in a -80°C refrigerator for later use;
[0032] 2. Decellularization of amniotic membrane
[0033] Take out the above cryovial, thaw it fully in 25°C water bath for 10 minutes, suck out the PBS in the amniotic membrane, then place the cryovial in liquid nitrogen to freeze, let it stand for 30 minutes, take it out, fully thaw it in 25°C water bath for 10 minutes, and put it in liquid nitrogen again. Freez...
Embodiment 2
[0036] Example 2. Construction of epidermal stem cell skin substitute ESC-mAM
[0037] 1) Culture and expansion of epidermal stem cells
[0038] After circumcision, the skin was digested with 0.25% Dispase at 4°C as usual, the epidermis was separated, digested with 0.25% trypsin at 37°C for 20-30 minutes, filtered with a metal mesh, centrifuged to collect cells, and the cells were inoculated on pre-coated IV In a type collagen culture dish, discard the supernatant after 20 minutes of adherence to the wall, add serum-free medium K-SFM, change the medium every other day, and digest with trypsin when the cells reach 70%-80% confluence to prepare a concentration of 5×10 5 Each / ml epidermal stem cell suspension is ready for use;
[0039] 2) Using mAM to expand epidermal stem cells and construct a skin substitute ESC-mAM
[0040] Get the above-mentioned epidermal stem cell suspension 10ml and the amniotic membrane microcarrier mAM 100mg that embodiment 1 prepares to inject into th...
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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 [0001] The invention relates to the technical field of tissue engineering and medical wound repair, and is an amnion microcarrier capable of simulating the growth niche microenvironment of epidermal stem cells in vitro and a skin substitute thereof. Background technique [0002] The cultivation and expansion of epidermal stem cells has always been a hot and difficult point in the research of skin tissue engineering. At present, epidermal stem cells are mainly used to separate and purify the remarkable adhesion properties of basement membrane (Barthel R, Aberdam D. Epidermal stem cells. J Eur Acad Dermatol Venereol2005; 19(4): 405-13.), using two-dimensional plane culture in vitro culture and expansion. In order to ensure the singleness of stem cells during subculture and expansion, it is necessary to first spread collagen type IV, one of the main components of the basement membrane, on a culture dish. Due to the inherent slow proliferation characteristics o...
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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074A61L27/38A61L27/60
Inventor 夏照帆肖仕初纪世召黄国锋朱世辉王光毅罗鹏飞
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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