Speaker
Description
"Introduction
Organoids have the potential to revolutionise drug discovery and development, providing an in vitro platform to determine drug efficacy and toxicity while reducing the need for animal testing. However, the wider adoption of organoids is held back by reliance on animal derived matrices for the production and culture of organoids. Animal derived materials are not only unethical but also lead to high batch-to-batch variability and xenogenic contamination and are therefore unsuitable for preclinical processes. Currently the field lacks a suitable, widely adopted, synthetic 3D cell culture environment for organoid culture and wider adoption into the drug development pipeline.
The aim of this project is to develop a new, fully synthetic (non-animal derived) 3D matrix which is optimised for the growth of colorectal organoids and can be applied in the future for industrial-scale organoid production and high throughput applications for several preclinical studies.
Methodology
PeptiGels® were produced by Manchester BIOGEL Ltd. The stiffness was measured using oscillatory rheology. The suitability for organoid-relevant cell culture was determined by qualitatively analysing the structural integrity of 50µL domes of different PeptiGels® over a course of 21 days under normal cell culture conditions. Colorectal organoids provided by Cellesce Ltd were cultured for up to 21 days, viability was assessed by LIVE/DEAD assay. DIC light microscopy was used to obtain images of organoids during their culture. Using ImageJ analysis, the cross-sectional area of organoids was generated.
Results
We assessed PeptiGels® Gamma 2 included within the hydrogels were 4 different functional biomimetic peptide motifs -GFOGER (Collagen), -RGD (fibronectin), -IKVAV (Laminin) and -YIGSR (Laminin). All combinations of the hydrogels had stiffnesses matching physiological tissue stiffness (between 0.5 and 3 kPa). Creating hydrogels using the ‘dome method’ (a gold-standard method for organoid culture), the acellular hydrogels were stable for up to 21 days in culture. Next, human primary colorectal organoids from Cellesce Ltd were cultured within the hydrogels. The ISO68 organoid line formed viable organoids of an equivalent size and shape to organoids grown in Matrigel, the organoids increased in cross-sectional area over the course of 14 days. Finally, the inclusion of functional motifs -IKVAV (Laminin) and -GFOGER (Collagen) into Gamma2 PeptiGels® showed enhanced organoid growth when compared to Gamma 2, Matrigel, Gamma 2-YIGSR and Gamma2-RGD.
Conclusion
This initial work highlights the potential for PeptiGel® Gamma2 for use in colorectal organoid manufacture and culture. Future work will focus on expanding the potential of PeptiGels® for use with other organoid types."
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