These models are paving the way for a new approach to testing drugs that could be a higher throughput and more efficient screen before in vivo trials of selected drugs.
Fat, also called adipose tissue, is an essential organ for the storage of energy, but is also important as an organ that secretes hormones (called adipokines) that are essential for healthy metabolic regulation. Fat can be extracted as a biopsy and precursor cells extracted that will differentiate into fat cells. Traditionally, adipose cell culture models are 2D, with cells growing on a flat surface side-by-side. These are from either immortalised cell lines, isolated from primary tissues, or differentiated from pluripotent stem cells from humans or animals. They can generally be defined as a collection of cells without a specific multicellular anatomy.
However, 2D adipose cell cultures fall short of the complexity and structure of adipose tissue in vivo. This is an issue that Dr. Alexander Graham and Dr. Rajesh Pandey have been working to improve under the supervision of Dr. Joan Gannon, Dr. Sam Olof and Prof. Roger Cox. They have been developing a drug-responsive 3D adipose model that could be more representative of in vivo tissue and cheaper than testing in vivo.
A key question is whether 3D adipose models develop and organize fat stores that are more like in vivo tissues, for example forming large unilocular droplets (i.e. single droplets) in cells. Further, whether they function more like adipose tissue. For close to three years, Dr. Graham and Dr. Pandey worked collaboratively at OxSyBio (a synthetic tissue spinout company from the University of Oxford) and MRC Harwell Institute respectively to tackle this problem by developing a novel adipose model that can retain its structure for weeks. Using white adipose cells, specifically 3T3-L1 cells an immortalised cell line of mouse origin, the team encapsulated cells in a hydrogel-based matrix containing selected proteins found in the extracellular matrix (ECM) of animal tissues.
This liquid mixture, designed for tissue development, was dispensed as small droplets (150 nL) into lipid-in-oil solution, where the solution formed a spherical geometry and was solidified as a spheroid. Spheroids, after transfer to cell culture medium, were matured over the course of weeks to produce 3D organisations of fat-laden cells, which could be used to model the function of adipose tissue.
The team first accessed Octopus last year through the Bridging for Innovators (B4i) scheme as part of the OxSyBio-MRC Harwell Institute project and acquired very informative images of their structures in order to assist the next stage of product development.
The next step will be to further examine the morphology of lipid droplets, which are used as fat stores, within cells to further validate the model. In order to achieve this, the team have been granted academic access in autumn to use the CLF's Octopus Laser Facility in the Research Complex where microscopy methods that are crucially far more sensitive than conventional light microscopy are available. Working with Octopus link scientists Dr. Alessia Candeo and Prof. Stan Botchway, they will investigate the 3D fat spheroids, assessing fat storage development in response to nutrient feeding and exposure to small molecule compounds such as anti-diabetic drugs.
“We have developed a robust, high throughput and 3D model of white adipose tissue which has been shown to be responsive to anti-diabetic drugs," Dr. Alex Graham said about the research. “It is a powerful platform for potentially identifying new therapeutic interventions for metabolic diseases. This achievement has only been possible through a multi-institutional collaboration between a broad range of specialists at OxSyBio, MRC Harwell Institute and the Central Laser Facility."
Dr. Rajesh Pandey added, “Our 3D white adipose tissue model has potential to reduce animal usage as well as being used to study long-term effect of diet and drugs."
Prof. Stan Botchway from the Octopus Facility at the CLF said about the experimental access, “This is another great example of how the Octopus imaging facility can be deployed in all aspects of fluorescence imaging. In this work, we used the advanced light sheet technique that has several advantages over normal widefield or confocal microscopy. The technique is gentle on live biological samples in terms of light toxicity. Another advantage is that large samples can be imaged in 3D faster than video rate with significant sensitivity at diffraction limited condition. Although the very large rich 3D volume data acquired (terabytes) can be difficult to analyses easily, we are now working with specialist companies, particularly 3DMagination to resolve the data analysis conundrum."
Fig 1: Spheroid of cells at 16 days.
Fig 2: From left to right - Dr. Rajesh Pandey and Dr. Alexander Graham in the MRC lab.
Fig 3: Dr. Rajesh Pandey in the MRC lab.