3D Culture Strategies for the Dynamic Expansion and Preconditioning of Adipose-derived Stromal Cells on Decellularized Adipose Tissue Bioscaffolds
Abstract
Adipose tissue engineering holds promise for the development of therapeutic strategies for subcutaneous adipose tissue regeneration to treat defects resulting from congenital birth defects, invasive surgical procedures and traumatic injuries. Decellularized adipose tissue (DAT) scaffolds represent a potential off-the-shelf tissue substitute for volume augmentation. Seeding the DAT with adipose-derived stromal cells (ASCs) has been shown to enhance adipose tissue regeneration in immunocompetent animals in vivo. Although promising, this strategy is limited by low cell attachment on the DAT. As such, this thesis focused on the development of bioreactor strategies to enhance the capacity of human ASCs to stimulate angiogenesis and adipogenesis within the DAT.
Culturing human ASCs on the DAT scaffolds within a perfusion bioreactor under hypoxia (2% O2) promoted ASC expansion, and altered cell phenotype, upregulating the expression of hypoxia inducible factor 1-alpha (HIF-1a), inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-a) in the ASCs in the peripheral regions of the DAT. Further, bioreactor culture modulated the expression of pro-angiogenic and immunomodulatory paracrine factors, suggesting that the capacity of the ASCs to stimulate regeneration may have been altered by shear stress stimulation. In vivo testing in athymic nude mice demonstrated that angiogenesis and adipogenesis within the DAT were markedly enhanced when the ASCs were cultured for 14 days within the perfusion bioreactor under 2% O2 prior to implantation as compared to bioreactor culture under 20% O2, as well as static-cultured, freshly-seeded and unseeded controls. Analysis of host cell infiltration indicated that there was increased CD31+ endothelial cell recruitment and potentially adipogenic progenitor cell recruitment into the DAT implants, as well as a shift towards a more pro-regenerative macrophage response, in the 2% O2 bioreactor group relative to static cultured controls.
Building from this work, a novel scalable rocking bioreactor platform was explored as an expansion and preconditioning system for ASCs. Preliminary studies indicated that culturing ASCs on DAT coatings within the rocking bioreactor enhanced the expression of the pro-angiogenic and immunomodulatory markers CD146 and iNOS. Collectively, this work supports that dynamic culture systems can be applied to enhance the pro-regenerative potential of ASC-seeded DAT bioscaffolds.