Generation of immune cell containing adipose organoids for in vitro analysis of immune metabolism

Abstract

Adipose tissue is an organized endocrine organ with important metabolic and immunological functions and immune cell-adipocyte crosstalk is known to drive various disease pathologies. Suitable 3D adipose tissue organoid models often lack resident immune cell populations and therefore require the addition of immune cells isolated from other organs. We have created the first 3D adipose tissue organoid model which could contain and maintain resident immune cell populations of the stromal vascular fraction (SVF) and proved to be effective in studying adipose tissue biology in a convenient manner. Macrophage and mast cell populations were successfully confirmed within our organoid model and were maintained in culture without the addition of growth factors. We demonstrated the suitability of our model for monitoring the lipidome during adipocyte differentiation in vitro and confirmed that this model reflects the physiological lipidome better than standard 2D cultures. In addition, we applied mass spectrometry-based lipidomics to track lipidomic changes in the lipidome upon dietary and immunomodulatory interventions. We conclude that this model represents a valuable tool for immune-metabolic research.

Irradiated Mammary Spheroids Elucidate Mechanisms of Macrophage-Mediated Breast Cancer Recurrence

“…Fibroblasts secrete components that can makeup ECM, and radiation induces fibrosis, which results in excess ECM production 28,29 . Previous studies have incorporated primary fibroblasts into spheroids 30 ; however, we are the first to study the effects of irradiation on spheroid behavior. We confirmed ECM deposition by evaluating collagen IV expression in spheroids ( Figure 2C, Figure S2C ).…”

“…Spheroids were generated from primary cells obtained from the stromal vascular fraction in mouse MFPs using previously published techniques 30 . MFPs were harvested and minced with a blade and digested for 40 minutes in a solution of PBS with 20 ug/mL liberase, and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin).…”

“…The copyright holder for this preprint (which this version posted July 24, 2022. ; https://doi.org/10.1101/2022.07.24.501296 doi: bioRxiv preprint Primary Spheroid Generation. Spheroids were generated from primary cells obtained from the stromal vascular fraction in mouse MFPs using previously published techniques 30 . MFPs were harvested and minced with a blade and digested for 40 minutes in a solution of PBS with 20 ug/mL liberase, and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin).…”

Towards better models for studying human adipocytes in vitro

“…By fattening HUVAS with triglycerides, we hope to have a model that more closely can mimic the initiating disease mechanisms coupled to weight gain and thereby could increase our understanding of how adipocyte function gradually is altered upon obesity. Nevertheless, a major caveat of the HUVAS model remains the absence of tissue-resident macrophages and other immune cells, that to our knowledge to date only have been detected within one 3D adipose tissue culture model [ 29 ]. The successful addition of immune cells to all 3D culture models therefore should be the next major goal for the research field, as it is only when all major cellular components are present that these models start to fully resemble the in vivo adipose tissue and mimic its responses to chronic inflammation, lipid accumulation and insulin resistance.…”

“…These include the difficulty of obtaining in vitro -differentiated adipocytes fully mirroring the in vivo phenotype, with a solely unilocular morphology and lipid droplet sizes in the range of that seen in humans in vivo (60–120 µm). In addition, most published 3D models except one model published by the Weber lab lack significant contributions of immune cells, something that would help to fully recapitulate human adipose tissue phenotype [ 29 ]. As our insights change, the adipose field should also reassess which in vitro characteristics define a fully differentiated adipocyte; is some lipid content and the expression of traditional adipogenic genes such as PPARG, FABP4 and PLIN1 enough to claim full adipogenic differentiation, or are their adipokine secretion, morphology, or total lipid content equal or even more important characteristics?…”