A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes

Abstract

The lack of relevant in vitro models for adipose tissue makes necessary the development of a more physiological environment providing spatial and chemical cues for the effective maturation of adipocytes. We developed a biofunctionalized hydrogel with components of adipose extracellular matrix: collagen I, collagen VI, and the cell binding domain of fibronectin and we compared it to usual 2D cultures on plastic plates. This scaffold allowed 3D culture of mature adipocytes from the preadipocytes cell lines 3T3-L1 and 3T3-F442A, as well as primary Human White Preadipocytes (HWP), acquiring in vivo-like organization, with spheroid shaped adipocytes forming multicellular aggregates. The size of these aggregates increased with time up to 120 μm in diameter after 4 weeks of maturation, with good viability. Significantly higher lipogenic activity (up to 20-fold at day 28 for HWP cultures) and differentiation rates were also observed compared to 2D. Gene expression analyses highlighted earlier differentiation and complete maturation of 3D HWP compared to 2D, reinforced by the expression of Perilipin protein after 21 days of nutrition. This increase in adipocytes phenotypic and genotypic markers made this scaffold-driven culture as a robust adipose 3D model. Retinoic acid inhibition of lipogenesis in HWP or isoprenalin and caffeine induction of lipolysis performed on mouse 3T3-F442A cells, showed higher doses of molecules than typically used in 2D, underlying the physiologic relevance of this 3D culture system. Biotechnol. Bioeng. 2017;114: 1813-1824. © 2017 Wiley Periodicals, Inc.

Scaffold-free generation of uniform adipose spheroids for metabolism research and drug discovery

“…Two-dimensional culture is not an ideal situation for culture of adipocytes because of limitations on growth and the accumulation of small, multi-locular lipid droplets. Curiously, most previous efforts to generate 3D adipose cultures have relied on the use of exogenous scaffolding material to aggregate cells together 38 – 40 . While successful in creating aggregates, scaffold based strategies introduce additional complexity based on the assumption that the pre-adipocytes and SVF cells are not capable of producing their own supportive matrix.…”

“…However, these strategies fail to create microtissues that mirror both the functional (adiponectin secretion, environmental sensitivity) and histological characteristics (large, unilocular lipid droplet accumulation) of native adipose tissue. Notably most efforts to date have utilized either immature adipose derived stem cells 39 , 41 , 42 , pre-adipocytes 40 , or 3T3-L1 mouse pre-adipocytes 43 possibly contributing to the lack of unilocular lipid droplet morphology typically seen in mature adipocytes in vivo . To date, the closest generation of a 3D adipose microtissue has come from entrapping mature adipocytes within a scaffold.…”

Studying Brown Adipose Tissue in a Human in vitro Context

“…The next step toward recapitulating BAT in vitro is the addition of ECM and supporting cell types. It is possible to generate 3D scaffolds from ECM proteins such as collagens and glycoproteins (71), or to incorporate ECM components into synthetic hydrogels (72,79). However, to recreate the human BAT ECM in vitro, its composition must first be fully characterized in vivo, in both physiological and pathophysiological states.…”

Part 1: A Novel Model for Three-Dimensional Culture of 3T3-L1 Preadipocytes Stimulates Spontaneous Cell Differentiation Independent of Chemical Induction Typically Required in Monolayer

“…The hydrogel can be used for tissue regeneration allowing cells to migrate and populate the structure as seen in in vivo studies and in cell culture where cells are seeded onto the hydrogel and grown. 10,11 Other types of 3D scaffold have also been employed. [12][13][14] Another 3D technique is the spheroid cell model where cells are prevented from attaching to substratum and form a cluster or spheroid of cells.…”