Biomaterial comprising adipose-derived stem cells 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 limited clinical data
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Embodiment 1
[0300] Embodiment 1: the preparation of biological material of the present invention
[0301] hASC
[0302] After informed consent and serological screening, human subcutaneous adipose tissue was collected in the abdominal region by liposuction according to the Coleman technique.
[0303] 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.
[0304] 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...
Embodiment 2
[0324] Example 2: Characterization of Biomaterials
[0325] Materials and methods
[0326] Cytotoxicity
[0327] Cytotoxicity was assessed against L929 cells (mouse fibroblasts cultured in RPMI medium supplemented with 5% FBS). A Transwell insertion procedure was then performed to analyze cytotoxicity.
[0328] The purpose of this method is to assess the toxicity of indirect cell-material contact (diffusion of leachable chemicals in the medium). In this way, hASC and L929 cells were grown at 8000 cells / cm 2 (15200 cells per well) were seeded and incubated in two 24-well plates at 37°C for 72 hours. Then, when the cells are confluent, the medium is removed and a biocompatible material is added to the transwell insert containing the bottom microporous membrane:
[0329] -6.6mg / cm 2 DBM,
[0330] - Three different amounts of HA / β-TCP: for 150cm 2 The container is 1.5cm 3 、2.85cm 3 and 5.91cm 3 ,
[0331] - for 150cm 2 The container is 1.5cm 3 HA pellets, or
[03...
Embodiment 3
[0377] Example 3: Comparison of Biomaterials Formed According to the Invention and Biomaterials Formed According to Different Protocols
[0378] Materials and methods
[0379] tissue preparation
[0380] Adipose stem cells from 2 different donors were used to prepare tissues. NVD-1 was prepared according to the protocol of Example 1 using DBM as the biocompatible material. NVD-0 was made according to the NVD-1 preparation protocol, but with two important changes: (i) 10% FBS was used in the medium (MP and MD) of NVD-0 (instead of 10% FBS in the medium of NVD-1 5% HPL used) and (ii) at passage 4 prepared in NVD-0, cells were grown to confluency in Proliferation Medium (MP) and then cultured in MD until osteogenic differentiation with addition of DBM particles (instead of Cells were cultured directly in MD from passage 4 until osteoprogenitor differentiation, and DBM particles were added to generate NVD-1).
[0381] RTI Surgicals' DBM particles were used to produce NVD-1 a...
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