Platelet Lysate as a Serum Substitute for 2D Static and 3D Perfusion Culture of Stromal Vascular Fraction Cells from Human Adipose Tissue

Abstract

Fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 are key supplements for the culture of stromal vascular fraction (SVF) cells from adipose tissue, both for typical monolayer (2D) expansion and for streamlined generation of osteogenic-vasculogenic grafts in 3D perfusion culture. The present study investigates whether factors present in human platelet lysate (PL) could substitute for FBS and FGF-2 in 2D and 3D culture models of SVF cells from human lipoaspirates. SVF cells were grown in medium supplemented with 10% FBS þ FGF-2 or with 5% PL. In 2D cultures, PL initially supported SVF cell proliferation, but resulted in growth arrest shortly after the first passage. Freshly isolated SVF cells cultured with both media under perfusion for 5 days within 3D ceramic scaffolds induced bone formation after subcutaneous implantation in nude mice. However, blood vessels of donor origin were generated only using FBS þ FGF-2-cultured cells. This was unexpected, because the proportion of CD34 þ =CD31 þ endothelial lineage cells was significantly higher with PL than that of FBS þ FGF-2 (33% vs. 3%, respectively). These results support the use of PL as a substitute of FBS þ FGF-2 for short-term culture of human SVF cells, and indicate that more specific serum-free formulations are required to maintain a functionally vasculogenic fraction of SVF cells expanded under 3D perfusion.

Intraoperative engineering of osteogenic grafts combining freshly harvested, human adipose-derived cells and physiological doses of bone morphogenetic protein-2

“…The fractions of different SVF subpopulations were highly variable across different donors, as assessed by the large standard deviations in the percentage of positive cells for different typical surface markers (Fig. 1A) and in accordance with previous reports (Müller et al, 2009;Müller et al, 2010). Once embedded in a fibrin gel around ceramic granules, SVF cells were viable and homogenously distributed throughout the construct, as evidenced by MTT metabolic staining (Fig.…”

“…78/07). Minced tissue from excised fat samples or lipoaspirates were processed as previously described Müller et al, 2010) and the cell pellets resuspended in complete medium (CM), consisting of α-MEM supplemented with 10 % of foetal bovine serum (FBS), 1 % HEPES, 1 % sodium pyruvate and 1 % Penicillin-Streptomycin Glutamate (100x) solution (all from Gibco, www.invitrogen.com).…”

“…One or four millions SVF cells were suspended in the fibrinogen phase (30 µL) of a polymerising fibrin gel (Tisseel ® , Baxter, www.baxter.com), as described previously (Bensaid et al, 2003;Müller et al, 2010), with or without addition of 250 ng of rhBMP-2 (own-produced or from R&D Systems). Briefly, following mix with the…”

Stem cells from adipose tissue

“…Human ASCs in vitro [19] Human ASCs in athymic nude rats [39] Chondrogenic differentiation Human ASCs in vitro [56,57,91,92] Human ASCs in SCID mice [54] Osteogenic differentiation Human ASCs in vitro [93][94][95][96] Human ASCs in nude mice [97] Human ASCs in SCID mice [19,98] Human ASCs in athymic mice [45] Human ASCs in rats [75] Rat ASCs in SD rats [40] Myogenic differantiation Human ASCs in vitro [60,[99][100][101] Human ASCs in mdx mice [102] Human ASCs in nude mice [34] Cardiomyogenic differentiation Human ASCs in vitro [103,104] Rat ASCs in rats [105] Neuronal differentiation Human ASCs in vitro [43,106] Canine ASCs in dogs with spinal cord injuries [107] Rat ASCs in rats [70] Osteogenic constructs with intrinsic vascularization Human ASCs in nude mice [66,69] Epithelial differentiation Human ASCS in vitro [59,74] Kidney differentiation Human ASCs in C57BL/6 mice [108] Pancreatic differentiation Human ASCs in vitro [72,73] Hepatocyte differentiation Human ASCs in Pfp/Rag2 knockout mice [77] Human ASCs in Ccl4 mice [76] Periodontal tissue regeneration Rat ASCs in rats [109] The proposed uses for ASCs in tissue engineering and regenerative medicine are collected in Tab. 1.…”

Comparative study assessing effects of sonic hedgehog and VEGF in a human co-culture model for bone vascularisation strategies

“…In this context several potential autologous cell sources for pro-angiogenic cell types such as cells from adipose tissuederived stromal fraction (SVF) (Scherberich et al, 2007;Muller et al, 2009), endothelial progenitor cells from different sources such as the bone marrow (Quirici et al, 2001), the cord blood (Murohara et al, 2000; Shh and VEGF on bone vascularisation al., 2004) or adult peripheral blood (Kalka et al, 2000;Peichev et al, 2000) are currently discussed. The mechanisms by which pro-angiogenic cells contribute to the neovascularisation process include paracrine mechanisms based on the production of angiogenic growth factors leading to the angiogenic activation and proliferation of endothelial cells resident in the tissue (Gulati et al, 2003;Rehman et al, 2003).…”