Dr Claire Richards1,2, Hao Chen3, Dr Matthew O’Rourke3, A/Prof Matthew Padula1, Prof David Gallego Ortega4,5,6, Prof Philip Hansbro3, Dr Amy Bottomley7, A/Prof Louise Cole7, A/Prof Lana McClements1,2
1School of Life Sciences, Faculty of Science, University of Technology Sydney, Ultimo, Australia, 2Institute for Biomedical Materials and Devices, Faculty of Science, University of Technology Sydney, Ultimo, Australia, 3Centre for Inflammation, Centenary Institute and School of Life Sciences, Faculty of Science, University of Technology Sydney, Ultimo, Australia, 4School of Biomedical Engineering, Faculty of Engineering and Information Technology, University of Technology Sydney, Ultimo, Australia, 5Garvan Institute of Medical Research, The Kinghorn Cancer Centre, Darlinghurst, Australia, 6School of Clinical Medicine, Faculty of Medicine, University of New South Wales, Kensington, Australia, 7g Microbial Imaging Facility at the Australian Institute for Microbial Imaging, Faculty of Science, University of Technology Sydney, Ultimo, Australia
Biography:
Biographies to come
Abstract:
Inappropriate placental development is associated with various pregnancy complications including miscarriage, intrauterine growth restriction and preeclampsia. Trophoblast organoids offer a unique opportunity to investigate mechanisms orchestrating placental growth and development. We aimed to develop a robust, low-cost and reproducible 3D in vitro model of trophoblast organoids using a 3D bioprinting approach suitable for high throughput screening.
ACH-3P first trimester trophoblast cells were either manually embedded in Matrigel or bioprinted in a polyethylene glycol-based hydrogel using a RASTRUM platform (Inventia Life Science) and maintained in Ham’s F12 culture medium (10% FBS, 1% penicillin-streptomycin) for up to 12 days. Organoid growth, metabolism, viability and differentiation were assessed by microscopy and resazurin reduction. Organoids were comprehensively compared at the gene and protein levels by single cell RNA sequencing (scRNAseq) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS).
Organoids demonstrated invasive capabilities and with no significant difference in size between Matrigel and bioprinting conditions. Immunofluorescent labelling revealed spontaneous differentiation into the two main trophoblast lineages, extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs), with no significant difference in proportions between groups (EVTs: 12.33% ± 0.8 Matrigel v 11.23% ± 3.7 bioprinted, p=0.78; STBs: 10.37% ± 2.6 Matrigel v 9.23% ± 2.1 bioprinted, p=0.75). Interestingly, when transcriptomes were clustered by expression of differentiation factor 15 (GDF15), syndecan-1 (SDC1) and matrix metallopeptidase 15 (MMP15), the number of STBs detected was 22% in Matrigel and 3% in bioprinted organoids, indicating altered differentiation or a limitation of STB analysis by scRNAseq. Transcriptomic and proteomic data sets are being further integrated. Additionally, we reversed the inside-out architecture of ACH-3P organoids by suspension, with SDC-1+ STB forming on the periphery of organoids.
Here, we present an alternative, robust and reproducible model to Matrigel-embedded trophoblast organoids using a bioprinting approach that produces trophoblast organoids with crucial features of first trimester placental tissue.
Keywords
placenta, organoid, bioprinting