Biomaterial comprising adipose-derived stem cells and gelatin and method for producing the same
An adipose stem cell and biomaterial technology, applied in the field of multi-dimensional biomaterial production, can solve problems such as confusion in literature
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Embodiment 1
[0212] Example 1: Production of Biomaterials of the Invention
[0213] 1.1. Isolation of hASCs
[0214] After informed consent and serological screening, human subcutaneous adipose tissue was collected in the abdominal region by liposuction according to the Coleman technique.
[0215] Human adipose stem cells (hASCs) were immediately isolated from freshly harvested adipose tissue. Lipoaspirate can be stored at +4°C for 24 hours, or at -80°C for longer than 24 hours.
[0216] First, for quality control purposes, a portion of the lipoaspirate is isolated and the remaining volume of the lipoaspirate is measured. Then, the adipose aspirate was digested by collagenase solution (NB 1, Serva Electrophoresis GmbH, Heidelberg, Germany) prepared in HBSS (final concentration about 8 U / mL). The volume of enzyme solution used for digestion is twice the volume of adipose tissue. Digestion was performed at 37°C ± 1°C for 50 minutes to 70 minutes. The first intermittent shaking was per...
Embodiment 2
[0232] Example 2: Characterization of Biomaterials
[0233] 2.1. Materials and methods
[0234] 2.1.1 Structure / Histology
[0235] The formation of 3D structures obtained from ASC and Cutispher G and S particles was tested. Cutispher particles were added to confluent ASCs at passage 4 from 6 different donors. Different volumes were tested: for each 150cm 2 The container is 1cm 3 、1.5cm 3 、2cm 3 particle. Cells were maintained in differentiation medium (DMEM, 4.5 g / L glucose, Ultra glutamine + 1% penicillin / streptomycin + 0.5% amphotericin AB + dexamethasone (1 μM), ascorbic acid (0.25 mM) and sodium phosphate (2.93mM), and replace the medium every 3 to 4 days.
[0236] To compare cultures in MP and MD, 3D structures in MD were biopsied 5 days, 14 days and 8 weeks after particle addition.
[0237] To evaluate cell density, 3D structures were biopsied at 4, 8 and 12 weeks after addition of Cultispher particles.
[0238] They were fixed in formaldehyde and prepared fo...
Embodiment 3
[0294] Example 3: Study of Angiogenesis and Osteogenic Properties in Vivo
[0295] 3.1. Materials and methods
[0296] 3.1.1 In vivo experiments using nude rats
[0297] On day 0, ten replicates of the biomaterial of the invention (as described in Example 1 during 7.5 weeks of maturation with 1.5 cm 3 ASCs cultured together with Cutispher G or S) were sutured on the cauterized lumbar muscles of nude rats. On day 29 post-implantation, biomaterial was collected for imaging and histological analysis.
[0298] 3.1.2 In vivo experiments using Wistar rats
[0299] On day 0, ten replicates of the biomaterial of the invention (as described in Example 1 during 7.5 weeks of maturation with 1.5 cm 3 ASCs cultured together with Cutispher G or S) were sutured on the cauterized lumbar muscles of Wistar rats. On day 29 after implantation, biomaterial was collected for analysis by imaging and histology.
[0300] During the experiment, the general clinical status of the animals was che...
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