Developing vascularized adipose tissue 3D in vitro model: Optimizing mature adipocyte isolation

Abstract

Adipose tissue is vascularized and innervated tissue affecting the whole-body metabolism. The most abundant type of adipose tissue is white adipose tissue. In vitro models of adipose tissue are needed for studying obesity and obesity associated diseases, as obesity is increasing worldwide. There is also need for adipose tissue platforms for drug development and to develop soft tissue grafts. Currently in vitro models containing isolated mature adipocytes are rare. The aims of this thesis are to optimize the isolation of mature adipocytes protocol from liposuction samples as well as to develop three-dimensional (3D) vascularized adipose tissue in vitro model on a microfluidic chip platform.

Six experiments were conducted to optimize the isolation of mature adipocytes following two previous protocols with small modifications to gain viable and functional mature adipocytes. To create vascularized 3D adipose tissue in vitro model isolated mature adipocytes were cocultured in AIM Biotech chips with adipose stromal/stem cells (ASCs) as a supporting cell type to vasculature formation and human umbilical vein endothelial cells (HUVECs) to form the vascular networks. Cell cultures were embedded in fibrin-collagen hydrogels and cultured for two weeks in combination medium consisting of adipogenic medium and endothelial growth medium-2 in 1:1 ratio. The success of the mature adipocyte isolation was quantified from Perilipin-1 staining images and the vasculature formation was observed and quantified from CD31 staining images. Viability of the cells was studied with lactate dehydrogenase assay (LDH), and the functionality of mature adipocytes and ASCs was studied with fatty acid (FA) uptake assay as well as with lipolysis assay measuring lipolysis.

Isolation of mature adipocytes was challenging but enhanced from the first experiments towards the last ones. Finally, isolation of mature adipocytes could be done successfully to gain pure mature adipocytes that could be plated in desired volumes. Adipocyte quantification revealed that the adipocyte amount increased significantly from first experiment to last. Mature adipocytes, ASCs and HUVECs could be all cultured together in a way that viability and functionality was maintained. LDH assay and lipolysis assay showed that viability and functionality of the cells was the highest on day 7 and decreased towards day 14. FA uptake assay did not provide reliable results. Vascular networks were vastest on day 7 and decreased towards day 14. However, best vascular networks formed in controls with ASCs and HUVECs only. Further testing is needed to unveil whether the presence of mature adipocytes, or the free oil they release when they burst, affects the vascular network formation more.

This master’s thesis study enhances the development of vascularized adipose tissue models that include isolated mature adipocytes, for the benefit of tissue engineering and drug development as well as obesity research. Cells could be cultured together, maintaining the viability and functionality of the cells. The isolation of mature adipocytes was successful in the end with the optimized protocol. For future studies, development for more long-term cultures as well as studying the cell-cell interactions more closely, for example with gene expression analysis, would be next steps.