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

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.

In vitro Lipolysis and Leptin Production of Elephant Seal Blubber Using Precision-Cut Adipose Tissue Slices

“…tissue, the best results regarding metabolic activity of the slices, based on conversion of 14 C glucose into lipids (Faulhaber et al, 1972;Ramsay and Rosebrough, 2005), expression of lipid droplet surface protein by immunofluorescence (Schopow et al, 2020) as well as morphological investigations (Anayama et al, 2015), were observed with slices thickness of 400 to 1,000 µm. The thickness of the slices obtained here falls within this range.…”

“…They also have to be thick enough to preserve the natural architecture of the tissue and minimize the proportion of damaged cells (on the cutting edges) ( de Kanter et al, 2005 ; Fisher and Vickers, 2013 ). Within the few publications preparing thin slices of adipose tissue, the best results regarding metabolic activity of the slices, based on conversion of 14 C glucose into lipids ( Faulhaber et al, 1972 ; Ramsay and Rosebrough, 2005 ), expression of lipid droplet surface protein by immunofluorescence ( Schopow et al, 2020 ) as well as morphological investigations ( Anayama et al, 2015 ), were observed with slices thickness of 400 to 1,000 μm. The thickness of the slices obtained here falls within this range.…”

“…Most of the publications on adipose tissue slices report a good viability up to 24 h of culture ( Mersmann and Shparber, 1993 ; Ramsay and Rosebrough, 2005 ; Cai et al, 2011 ). Only two of them cultured thin adipose tissue slices for longer periods, i.e., five ( Anayama et al, 2015 ) and 14 days ( Schopow et al, 2020 ).…”

“…On the other hand, the method appears much less developed for adipose tissue, which might be due in part to the technical difficulties linked to its texture. Very recently, Schopow et al (2020) successfully established a slice culture system from human adipose tissue to examine cellular mechanisms involved in obesity, highlighting the interest of this promising tool to study the complex processes underlying adipose function and dysfunction. Such a model however, is lacking for wild animals such as marine mammals.…”

Clinical Application of the Novel Cell-Based Biosensor for the Ultra-Rapid Detection of the SARS-CoV-2 S1 Spike Protein Antigen: A Practical Approach

“…This condition renders the wide use of cell-based biosensor principles practically impossible, especially in resource-limited environments. Overcoming the hurdle of limited cell viability by using specific cell types (e.g., fish gills) or integrating microfluidic/organ-on-chip circuits in the consumable modules of biosensing platforms is only one of the potential areas for improving the performance of advanced CBAs [45][46][47]. Equally important is the ability to achieve highly reproducible test results within the linear range of responses; this is not always guaranteed when using cell suspension cultures, in particular when cell densities are not kept constant or are not clearly defined.…”