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The correction of soft-tissue defects by adipose tissue transplantation often produces poor and unpredictable results. The implantation of isolated and cultured preadipocytes offers a solution to this problem since these cells differentiate into adipocytes when implanted in vivo. A field of major interest is to maximize the yield of preadipocytes isolated from adipose tissue showing only low contamination with other cell types. Aspiration and excision are two concurrent clinical ways of harvesting adipose tissue for the isolation of preadipocytes. This tissue is usually discarded after surgery. In this study, the yield of preadipocytes obtained from liposuction material was compared to that of excised adipose tissue. Furthermore, we determined the loss of precursor cells if isolation of preadipocytes was delayed for 24 h. Preadipocytes were isolated from the stromal cell fraction of human subcutaneous adipose tissue samples. Harvesting of adipose tissue by suction was performed according to the Coleman procedure (manually applied negative pressure using a 10-ml syringe with a blunt tip cannula). Isolation was either carried out within 60 min after extraction or after storage for 24 h in culture medium at 4°C. Isolated preadipocytes were cultured for 24 h, trypsinized and counted in a Neubauer chamber. Our results show clearly that the yield of preadipocytes isolated from liposuction material (within 60 min after extraction and after 24 h of storage) is higher than the cell yield from excised adipose tissue. Overnight storage for 24 h leads to a significant loss of preadipocytes in excised tissue but not in liposuction material. The high yield of cells isolated from liposuction material proves that extraction by suction does not damage the stromal cell fraction in the adipose tissue. If cell isolation is not performed immediately after the operation, liposuction material is clearly the better alternative for storage.

Extended soft tissue defects after extensive deep burns or tumor resections are still an unresolved problem in plastic and reconstructive surgery. There is a clinical need for an adequate solution to this problem but currently, no adequate implant material is available for the correction of these defects. Since the autologous transplantation of mature adipose tissue gives poor results, this study explores the advantages of using human preadipocytes in collagen sponges for tissue reconstruction purposes. Human preadipocytes of young adults were isolated, cultured, seeded onto collagen sponges with uniform pore size, and implanted into immunodeficient mice. After 24 hours of incubation in vitro and after explantation at 3, 8, and 12 weeks, sponges were examined for macroscopic appearance, weight, thickness, histology, immuno-histochemistry, and ultrastructure. We find good penetration of cells into the scaffold, layers of adipose tissue, and new vessels on all grafts while controls appear unchanged. These results are promising for improving the reconstruction of soft tissue defects.

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Adipose tissue engineering with preadipocyte-loaded scaffolds requires adequate tracking of preadipocytes to allow evaluation and quantification of cell proliferation, expansion and differentiation in three-dimensional systems. To differentiate between graft and host cells, labeling of preadipocytes before implantation and tracking of these cells until harvest would be useful. Immunohistochemistry enables the differentiation between cells of different species but is time-consuming, expensive, elaborate, and not applicable for autologous transplantation. So far, there is no published method to use externally applied dyes for tracking of human preadipocytes in adipose tissue engineering. We tested the cell dyes PKH26, CM-DiI, and CFSE to analyze their applicability for labeling human preadipocytes. CM-DiI had toxicity levels of 45–70%, while 3–4% proliferating cells were stained on day 35. CFSE revealed clear cytoplasmic coloring in proliferating cells with 5–6% stained cells after 35 days and toxicity ranging from 55 to 90% dead cells. PKH26 demonstrates lowest levels of toxicity and best labeling results after 4 weeks in proliferating preadipocytes in monolayer. Although none of the dyes showed long-lasting labeling during proliferation, all three dyes demonstrated permanent staining in differentiated cells. The results reveal the problems of preadipocyte tracking with fluorescent dyes but justify the dye application for limited time periods.