Low-immunogenicity tissue engineering skin construction method
A tissue-engineered skin, immunogenic technology, applied in the field of bioengineering, can solve the problems of immune rejection, low immunogenicity, etc., and achieve the effect of reducing shrinkage, increasing immune tolerance and survival time
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Embodiment 1
[0033] Embodiment 1 Placental Mesenchymal Stem Cells and Epidermal Stem Cells Culture and Differentiation Culture Medium Composition, Culture Method and Results
[0034] Methods: Placental mesenchymal stem cells were isolated and cultured to take fresh placental tissue (generally, placental tissue was taken within 24 hours after delivery, if it could not be separated and cultured in time, it was temporarily placed in a 4°C refrigerator, and experiments proved that the shorter the storage time, the better the state of cultured cells) in On the ultra-clean workbench after ultraviolet disinfection for 30 minutes in advance, use scissors to carefully cut part of the fetal side placenta tissue (carefully avoid large blood vessels when cutting, and the size of the cut is about 2.0cm×1.0cm×0.5cm) respectively Place in two sterile disposable petri dishes, rinse the placental tissue with PBS (adding 1% Anti-Anti) for 3 to 5 times until the cleaning solution is colorless (the purpose is ...
Embodiment 2
[0039] Example 2 Method and Results of Placental Mesenchymal Stem Cells Composite Epidermal Stem Cells Tissue Engineering Skin Construction and Air-liquid Interface Culture Cuticle Formation
[0040] METHODS: 1 day before preparation for inoculation, 3D bovine collagen scaffolds were prepared. Collagen configuration (10ml): 7.5ml 1.5mg / mL type I bovine collagen solution, 1mL 10×DMEM, 0.5mL NaHCO 3 , 1mL 200mM Hepes, 0.1mL 1M NaOH, prepared at low temperature, solidified at 37°C for 30min after preparation. After the collagen becomes solid, slowly add PBS or basal medium and place in the incubator overnight to balance the pH. The next day, discard the equilibrium solution in the culture plate for use. Obtain P2 generation keratinocytes cultured to 70%-80% confluent state, inoculate 8×10 5 The number of cells is placed in the upper chamber of a 24mm nested culture dish, 2ml of medium (Cnt-07, CELLnTEC) is added to the upper chamber, and 3ml of medium (Cnt-07, CELLnTEC) is add...
Embodiment 3
[0042] Example 3 Using the Mouse Skin Transplant Immune Rejection Model to Verify the Function of Low Immunogenicity Artificial Skin
[0043] Methods: The operating area was disinfected with an ultraviolet disinfection vehicle for 30 minutes before the operation, and the surgical instruments were sterilized by autoclaving in a 60°C oven overnight before being used for later use. A C57BL / 6 mouse was taken, and the mouse was anesthetized by intraperitoneal injection of 4% chloral hydrate at a dose of 1 mL / 100 g. After the mouse was unconscious, a circular full-thickness skin defect with a diameter of about 12 mm was cut from the back of the mouse near the left forelimb. They were divided into operation control group, artificial epidermis transplantation group, and fPMSC composite artificial skin group. Artificial skin transplantation was performed, and the dermis was covered on the defect to ensure good skin alignment and avoid residual air bubbles in the contact parts. The pre-p...
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