3D Adipose Tissue Culture Links the Organotypic Microenvironment to Improved Adipogenesis

Abstract

Obesity and type 2 diabetes are strongly associated with adipose tissue dysfunction and impaired adipogenesis. Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold‐free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi‐omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R2 = 0.97). Integration of multi‐omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFβ signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis.

ExAdEx: A simple method to maintain human adipose tissue in long-term cultures reveals differential expansion of adipose progenitor cell subpopulations in fibrotic and pro-inflammatory micro-environments.

“…1c, e and i). This lipid droplet size is similar to the size that can be observed in native tissue and is substantially larger than the 20 µm lipid droplets generated in other 3D models of adipocyte spheroids 4 . Altogether, the data support the claim that the ExAdEx model signi cantly improves on the existing models of human adipose tissue.…”

Gellan Gum Is a Suitable Biomaterial for Manual and Bioprinted Setup of Long-Term Stable, Functional 3D-Adipose Tissue Models

“…Nevertheless, there is optimization potential in the long-term application and the accessibility and storage of the used cells. Although spheroids are shown to be a suitable model for the differentiation and maturation of functional, univacuolar adipocytes [ 62 ], the use of animal cells allows only limited conclusions to be drawn about human cells.…”

Towards better models for studying human adipocytes in vitro

“…They also attach to plastic, can be expanded at least a couple of passages and are compatible with standard cell biology methods. The differentiated 2D-cultured adipocytes accumulate lipid, respond to lipolytic ques, and express all typical adipogenic markers, albeit at lower levels than their mature counterparts [ 9 ]. However, despite numerous attempts to optimize the differentiation protocol, 2D-cultured adipocytes are not mature enough to adapt the typical unilocular morphology seen in vivo , characterized by having only a single large central lipid droplet surrounded by a thin layer of cytoplasm and the nucleus squashed to the side.…”

“…For example, in the past five years alone, nearly 100 papers that describe the development or usage of ‘ adipocyte spheroids ’ have been registered within PubMed, with most of them describing new variants of 3D culturing protocols. The consensus from these studies is that 3D cultured adipocytes show higher adipogenic gene expression, higher adiponectin secretion and, most importantly, much larger lipid droplets than 2D cultures grown in parallel [ 8 , 9 , 11 ]. In addition to the wide range of published protocols [ 12–14 ], recent publications also include a series of useful methodological papers detailing how 3D cultured adipocytes subsequently can be analysed using for example automated screening platforms, mass spectrometry, tissue clearing or transcripomics, thus opening further possibilities to use 3D cultures to generate new discoveries about basic adipocyte biology [ 11 , 15 , 16 ].…”

“…In addition to the wide range of published protocols [ 12–14 ], recent publications also include a series of useful methodological papers detailing how 3D cultured adipocytes subsequently can be analysed using for example automated screening platforms, mass spectrometry, tissue clearing or transcripomics, thus opening further possibilities to use 3D cultures to generate new discoveries about basic adipocyte biology [ 11 , 15 , 16 ]. For example, a recent noteworthy study by the Laushke lab used in-depth multi-omics to identify factors that link 3D culturing to the observed enhanced adipocyte maturation [ 9 ]. The study sequenced human pre-adipocytes that were either differentiated and cultured as scaffold-free 3D spheroids or as traditional 2D cultures and compared them to publicly available sequencing data from freshly isolated mature adipocytes.…”