Why pregnancy?
Like many areas of women’s reproductive health, there is a void in pregnancy research, but it is not solely due to historical taboo.
Human pregnancy is an extremely coordinated process, involving the union of gametes, development of the early blastocyst, its successful burying into the prepared lining of the uterus and nurture by the maternal environment – all while convincing her cells that it does not pose a threat. While we have a high-level understanding of these processes, the gritty details have remained a mystery and ultimately limit our ability to understand, prevent and treat pregnancy conditions.
So why the gap? Well, our ability to peer into these critical processes early in pregnancy is restricted by safety and ethical concerns. Sampling from the early placenta, known as chorionic villus sampling, is invasive and risks introducing infection or inducing miscarriage. Alternatively, the entire placenta is birthed at the end of pregnancy and readily available to study, but it is quite different after 9 months of growth and maturation. So, samples from early placental tissue must come from elective pregnancy terminations, impacted by ethical restrictions and the inability to predict whether this pregnancy would have progressed complication-free.
While we have gained important insights from the pregnancies of other animal species, these can differ dramatically from human pregnancies and very few develop complications like preeclampsia.
So, we need an alternative method to study human pregnancy. And this is where the advent of advanced three-dimensional (3D) cell cultures heralds in a new era in human research.
Culturing human cells in a supportive environment that allows them to believe they are within their native tissue can help them to form mini tissues like organoids. Organoids are clusters of cells that grow from one individual cell. They are usually made by suspending cells in a supportive gel that mimics the extracellular matrix they would reside in within a tissue. Cells grown in organoids start to mirror the architecture and function of the organ they’ve come from. Placental organoids have unlocked the ability to peer into the development of the early placenta by watching it unfold in a dish.
The problem
Placental organoids were first described in 2018 and compared to those from organs like the intestine, they are still in their infancy and need some fine tuning.
One limitation of existing organoid cultures is the use of animal-derived gel materials. Materials drawn from mouse-grown tumours are rich in factors to support cell growth and organoid formation, but they’re inherently variable between batches based on individual mice. These materials can be difficult to control for precise cell microenvironments and extend our reliance on animals in research.
Many existing placental organoids have been established in animal-derived matrices, but these don’t necessarily reflect the tissue environment of the uterus where the placenta grows.
A designer home solution
The creation of advanced biomaterials from natural and synthetic sources offers new options for organoid culture.
The capacity to finely tune the rigidity of the material and the addition of specific biomolecules can enhance our ability to design the home of organoid cultures. Pair these materials with 3D printing technologies like bioprinting that can accurately mix and deposit living cells within hydrogels, and you have more reproducible, controllable organoids.
How did we make bioprinted placental organoids?
A/Prof McClements met Prof Gernot Desoye in 2017 at the European Association for the Study of Diabetes in Lisboa (Portugal) where he introduced her to their custom-made ACH-3P cell line established in 2007 as a replacement for the HTR-8/SVneo cell line she was using at the time. This opened up vast opportunities for A/Prof McClements’ reproductive biology research program.
Dr Richards had learnt how to grow endometrial organoids within an undergraduate internship and joined A/Prof McClements’s group as an Honours student. When provided with ACH-3P, she proposed testing their ability to form organoids. At this point, the first placental organoids had only been described the previous year and there was no published research on ACH-3P organoids.
We started by embedding ACH-3P in Matrigel and they readily formed organoids – success. At this time, we had also contributed to procuring a 3D bioprinter, the drop-on-demand RASTRUM cell culture platform from Inventia Life Science, a local start-up. So, we tested ACH-3P organoid formation by screening a range of synthetic polyethylene glycol (PEG) hydrogels that might reflect the environment of the uterus. And again, ACH-3P readily formed organoids within the PEG gel droplets printed by the RASTRUM.
Now to decide which method was most suitable for studying the placenta. After observing some shape and invasion differences within both settings, we took a deep dive in analysis – comparing the organoids grown in Matrigel and bioprinted PEG. We established collaborations with the UTS Microbial Imaging Facility (A/Prof Louise Cole and Dr Amy Bottomley), Proteomics, Metabolomics and Lipidomics Facility (A/Prof Matt Padula and Dr Matthew O’Rourke) and Single Cell Technology Facility (Prof David Gallego-Ortega).
We found that ACH-3P cells spontaneously differentiated when grown in organoid structures, maturing into the major trophoblast subtypes without the need for chemical stimuli or growth factors. We also found that organoids grown in different gels altered how much they differentiated into their subtypes. This highlighted the importance of the environment that organoids are grown in.
To assess how closely our ACH-3P organoids reflected actual placental tissue, we compared their profiles to published data from 6-week and 8-week placental tissue, as well as organoids grown directly from cells extracted from this tissue. Encouragingly, the cell types identified in our ACH-3P organoids were very close to their primary counterparts.
We also found that if we removed young organoids from their matrix and grew them floating in suspension, they rearranged themselves to flip inside-out, better reflecting the structure of placental tissue.
To apply the bioprinted organoid model to drug screening, our Honours student Grace Owen and PhD candidate Ashley Bannister tested organoid growth, metabolism and differentiation under inflammatory conditions and with the addition of current and emerging preeclampsia drugs.
The ability to grow organoids in synthetic materials strengthens the biological relevance of these systems and reduces our use of animals in pregnancy research. Controllable technologies like bioprinting give better reproducibility and the capacity to expand to high throughput drug screening. Further, the use of an immortalised cell line like ACH-3P to study some features of placental development provides a low-risk alternative to primary samples from terminations. In turn, we hope these complex cell cultures will aid our mission of improving the health of women and their babies before, during and after pregnancy.