Using Microwell Chip for Scalable Generation of Pancreatic Aggregates

Objective: For the production and bio-banking of organoids in large quantities for drug screening and cellular therapies, well-defined procedures for scalable production are required. As well, based on previous studies, co-culture with native pan-creatic mesenchyme promotes self-renewal and expansion of their epithelial progenitors. Therefore, in this study we used a microwell platform as a high throughput three-dimensional (۳D) culture system where human embryonic stem cells derived pancreatic progenitors (hESC-PPCs) can be co-cultured with human fetal pancreatic mesenchyme (hFP-MCs) for large-scale generation of pancreatic aggregates. The advantage of this plat-form compared to conventional suspension culture is its repro-ducibility.Materials and Methods: Non-adherent agarose microwell chip was fabricated by mold-replication technology which con-tained a structured polydimethylsiloxane (PDMS) surface with a standard array of ۴۰۰ µm diameter pyramidal microwells. The PPCs differentiation was achieved from Royan hESC lines. HFP-MCs were isolated from ۱۲-۲۰ week human embryos. For aggregate formation, cell mixture suspension of hESC-PPCs and hFP-MCs was seeded on microwell chips. After aggregates generated by force aggregation and ۲۴ hour incubation, were aspirated from the microwells and placed in static suspension culture.Results: This platform produces approximately ۴۷۰۰ aggre-gates where each aggregate has a diameter of ۷۰–۱۲۰ µm, lead-ing to uniformly sized, shaped and stable aggregates with low variation in diameter. We demonstrated that both cells are pre-sented inside the aggregates after ۴۸ hours of suspension cul-ture and formed aggregates can be harvested easily without any additional forces, which prevent damage to aggregates during harvesting. Aggregates were expressing PP- and MC specific gene and protein markers after ۴۸ hours culturing.Conclusion: Altogether, pancreatic aggregates which are pro-duced by our method formed round-shaped with well-defined borders aggregates and were able to be maintained stable in culture for short-range period in order to apply in in vivo trans-plantations and cell therapies.