Inducing liquid for differentiation of bone marrow mesenchymal stem cell (BMSC) into epithelial cell and preparation and application thereof
A technology for bone marrow mesenchymal and epithelial cells, applied in biochemical equipment and methods, microorganisms, tissue culture, etc., can solve problems such as overgrowth, uncertain induction results, safety, etc., and achieve economical sources, simple and practical preparation methods. Effect
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Embodiment 1
[0044] Embodiment 1 prepares the epithelial cell induction liquid of the present invention
[0045] ●Collection of epithelial cell culture medium
[0046] (1) One adult healthy Beagle dog, weighing 11kg (provided by the Animal Experiment Center of Sichuan University). After general anesthesia, the mouth was opened, iodine was repeatedly wiped and disinfected, rinsed three times with a solution containing 500 U / ml gentamycin, the oral mucosa was peeled off, and the submucosal tissue was removed with scissors on a clean workbench, and the tissue was cut into about 4mm×4mm size, put into a Petri dish filled with 0.25% Dispase II, and digest overnight at 4°C. Ophthalmic tweezers were used to separate the epidermis to obtain the oral mucosal epithelial tissue including the basal layer. The separated epithelial tissue was placed in 0.25% trypsin, digested at 37°C for 10min, and FBS neutralized the trypsin. Centrifuge at 1200r / min×5min, discard the supernatant, add Definded Kerati...
Embodiment 2
[0051] Example 2 In vitro induction of bone marrow mesenchymal stem cells
[0052] A. BMSC cultivation:
[0053] Under sterile conditions, 5ml of adult healthy Beagle dog bone marrow (heparin anticoagulant) was obtained by puncturing through the posterior superior spinous bone of the ilium, mixed with 5ml of low-sugar DMEM medium (L-DMEM), and centrifuged at 1200r / min for 5min at room temperature. Fat layer, serum layer, medium were discarded. Take the lower layer of blood cells and an equal volume of L-DMEM to make a suspension of about 5ml, gently blow and mix with a pipette, and spread the suspension slowly in a centrifuge tube prepared in advance with 5ml of canine lymphocyte separation medium. After centrifugation at 2000r / min at room temperature for 20 minutes, carefully collect the buffy coat layer with nucleated cells on the interface, add L-DMEM to wash, centrifuge, and collect the cells. Resuspend with L-DMEM, count the cells, adjust the cell density, and divide th...
Embodiment 3
[0057] Example 3 Combination of oral mucosal epithelial cells and materials to construct tissue-engineered esophagus to repair canine esophageal defect
[0058] A: Oral mucosal epithelial cell culture
[0059] One adult healthy Beagle dog weighing 11kg (provided by Animal Experiment Center of Sichuan University). After general anesthesia, the mouth was opened, iodine was repeatedly wiped and disinfected, rinsed three times with a solution containing 500 U / ml gentamycin, the oral mucosa was peeled off, and the submucosal tissue was removed with scissors on a clean workbench, and the tissue was cut into about 4mm×4mm size, put into a Petri dish filled with 0.25% Dispase II, and digest overnight at 4°C. Ophthalmic tweezers were used to separate the epidermis to obtain the oral mucosal epithelial tissue including the basal layer. The separated epithelial tissue was placed in 0.25% trypsin, digested at 37°C for 10min, and FBS neutralized the trypsin. Centrifuge at 1200r / min×5min...
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