Composition for treating ischemic limb disease comprising stem cells derived from adipose tissue
A technology for ischemic diseases and cell therapy, which can be used in extracellular fluid diseases, blood diseases, cardiovascular system diseases, etc., and can solve the problems of unstable storage of cell therapeutic agents.
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Embodiment 1
[0052] Example 1: Isolation of pluripotent stem cells from adipose tissue
[0053] Adipose tissue was obtained by abdominal liposuction, washed with PBS and then microdissected. The dissected tissue was digested at 37° C. for 2 hours in DMEM medium supplemented with collagenase type 1 (1 mg / ml). The digested tissue was washed with PBS and then centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated, and the micelles remaining at the bottom were washed with PBS, and then centrifuged at 1000 rpm for 5 minutes. The resulting micelles were filtered through a 100 μm sieve to remove debris, followed by washing with PBS. The obtained cells were incubated in DMEM medium (10% FBS, 2 mM NAC, 0.2 mM ascorbic acid). After overnight, unattached cells were washed away with PBS, and cultured in keratinocyte-SFM medium (containing 2mM NAC, 0.2mM ascorbic acid, 0.09mM calcium, 5ng / ml rEGF, 50μg / ml BPE, 5μg / ml Insulin and 74ng / ml hydrocortisone) were cultured, and the medium wa...
Embodiment 2
[0054] Example 2: Immunological properties of pluripotent stem cells from adipose tissue
[0055] The adipose tissue-derived pluripotent stem cells obtained in Example 1 were washed with PBS and treated with casein. Treated cells were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, then washed with a mixture of 2% FBS and PBS, and centrifuged at 1000 rpm for 5 minutes. Discard the supernatant, suspend the cells in PBS, and for each sample mix 1 x 10 5Cells are dispersed into cell plates. Antibody (R-phycoerythrin-conjugated mouse anti-human monoclonal antibody) was placed into each well and incubated on ice for 40 minutes. After incubation, the medium was centrifuged at 1000 rpm for 5 minutes. Remove supernatant and wash cells twice with PBS. After washing, the cells were fixed with 1% paraformaldehyde, and then the surface antigens of pluripotent stem cells were analyzed by flow cytometry (Table 1 and figure 2 ).
[0056] Table 1: F...
Embodiment 3
[0060] Example 3: Pluripotent stem cell sphere preparations from human adipose tissue
[0061] The human adipose tissue-derived pluripotent stem cells obtained in Example 1 were stored under conditions of saline solution, saline solution+sucrose, saline solution+sucrose+50% albumin, and PBS+sucrose, and then their sphere-forming ability was examined.
[0062] will be 5×10 4 ~1×10 5 The pluripotent stem cells from human adipose tissue obtained in Example 1 were inoculated into a solution containing 10 μM CORM-2, 5 ml antibiotic antifungal (100×), 1 μg / ml cortisol, 5 μg / ml insulin, 20ng / ml EGF, 40ng / ml FGF, B27 and β-mercaptoethanol in each well of a 6-well plate in serum-free MEBM medium. As a result, cells began to form spherical shapes 3 to 7 days after inoculation, as image 3 As shown, the cells proliferated to form spheres even 7-10 days after inoculation.
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