Title : In vitro evaluation of lyophilized Dedifferentiated Fat cells (DFAT) impregnated artificial dermis
Abstract:
Dedifferentiated fat cells (DFAT) were infiltrated into an artificial dermal collagen sponge under reduced pressure, followed by lyophilization to create lyophilized DFAT-impregnated artificial dermis. In this study, the release of bioactive substances was examined in vitro.
Method: DFAT was prepared by ceiling culture of human subcutaneous fat tissues. Using a simple vacuum infiltration device, 0.25 ml of DFAT suspension (4.0 × 10? cells/ml) was sprayed onto artificial dermis (Pelnac, single-layer collagen-based artificial dermis, GUNZE, Japan) and then vacuum-treated at -70 cmHg for 1 minute to create DFAT-impregnated artificial dermis.
The DFAT-impregnated artificial dermis was divided into the following three groups: 1) fresh group (not preserved), 2) frozen group (frozen and preserved at -80°C for 2 weeks), and 3) lyophilization group (vacuum freeze-dried) (n=8 per group). Each group was immersed in DMEM medium, and the supernatant was collected, and the amounts of human VEGF and bFGF were quantified by ELISA. In addition, the release capacity of physiologically active substances was comprehensively characterized for the 1) fresh group and 3) lyophilaization group. A vacuum freeze-drying machine (CHList, ALPHA1-4/2-4LSCplus, KUBOTA) was used for lyophilization.
Results: DFAT was confirmed within the collagen sponge of the DFAT-impregnated artificial dermis, and although it aggregated after lyophilization, the cell morphology was maintained.
Comprehensive qualitative analysis of physiologically active substances showed that none were lost by the vacuum freeze-drying process, which suggest that properties of DFAT remained unchanged by lyophilization.
Quantitative results for physiologically active substances showed that both VEGF and bFGF were significantly higher in group 3)lyophilization group than in the other groups.
Discussion: DFAT aggregated after lyophilization, but maintained its cellular morphology. Furthermore, returning to a humid environment demonstrated the release of bioactive substances. The amounts released were greater than those in fresh or frozen storage, suggesting that the release occurred all at once due to cell breakdown.
