Examination of ex-vivo viability of human adipose tissue slice culture

PLoS One. 2020 May 26;15(5):e0233152. doi: 10.1371/journal.pone.0233152. eCollection 2020.

Abstract

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 μm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipocytes* / metabolism
  • Adipocytes* / pathology
  • Adipose Tissue* / metabolism
  • Adipose Tissue* / pathology
  • Adolescent
  • Adult
  • Aged
  • Antigens, CD / metabolism
  • Antigens, Differentiation, Myelomonocytic / metabolism
  • Calcium-Binding Proteins / metabolism
  • Cell Survival
  • Female
  • Humans
  • Ki-67 Antigen / metabolism
  • Male
  • Microfilament Proteins / metabolism
  • Middle Aged
  • Models, Biological*
  • Obesity* / metabolism
  • Obesity* / pathology
  • Tissue Culture Techniques*

Substances

  • AIF1 protein, human
  • Antigens, CD
  • Antigens, Differentiation, Myelomonocytic
  • CD68 antigen, human
  • Calcium-Binding Proteins
  • Ki-67 Antigen
  • MKI67 protein, human
  • Microfilament Proteins

Grants and funding

This study was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, www.dfg.de) – Projektnummer 209933838 – SFB 1052, with contributions granted to MG and IB as well as by the Bundesministeriums für Bildung und Forschung (BMBF, German Federal Ministry for Education and Research, www.bmbf.de), with contributions granted to IB and FR (FKZ 031A579). The GSI Helmholtzzentrum für Schwerionenforschung GmbH (Darmstadt, Germany) provided support in the form of salaries for FR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.