Autonomous spheroid formation by culture plate compartmentation

Abstract

Scaffold-free 3D cell cultures (e.g. pellet cultures) are widely used in medical science, including cartilage regeneration. Their drawbacks are high time/reagent consumption and lack of early readout parameters. While optimisation was achieved by automation or simplified spheroid generation, most culture systems remain expensive or require tedious procedures. The aim of this study was to establish a system for resource efficient spheroid generation with additional early readout parameters. This was achieved by a new approach for spheroid generation via self-assembly from monolayer via compartmentation of cell culture surfaces utilising laser engraving (grid plates). The compartmentation triggered contraction and rolling up of the cell monolayer, finishing in condensation into a spheroid in human adipose-derived stem cell (ASC/TERT1) and human articular chondrocytes (hACs)-ASC/TERT1 co-cultures, when cultivated on grid plates under chondrogenic conditions. Plates with 1 and 3 mm grid size yielded stable diameters (about 140 µm and 300 µm, respectively). ASC/TERT1 spheroids fully formed within 3 weeks while co-cultures took 1–2 weeks, forming significantly faster with increasing hAC ratio (p < 0.05 and 0.01 for 1:1 and 1:4 ASC/TERT1:hAC ratio, respectively). Co-cultures showed slightly lower spheroid diameters, due to earlier spheroid formation and incomplete monolayer formation. However, this was associated with a more homogeneous matrix distribution in the co-culture. Both showed differentiation capacity comparable to standard pellet culture in (immune-)histochemistry and RT-qPCR. To assess usability for cartilage repair, spheroids were embedded into a hydrogel (fibrin), yielding cellular outgrowth and matrix deposition, which was especially pronounced in co-cultures. The herein presented novel cell culture system is not only a promising tool for autonomous spheroid generation with the potential of experimental and clinical application in tissue engineering, but also for the generation of ‘building blocks’ for subsequential biofabrication strategies such as bioprinting.

Gelatin methacryloyl as environment for chondrocytes and cell delivery to superficial cartilage defects

“…Alginate, a natural biomaterial frequently used in cartilage research (Häuselmann et al., 1996 ; Lee et al., 2003 ), is not suitable for intra‐articular application and cannot be degraded by cells to be replaced by new matrix. Fibrin, often used as a tissue glue and for cell encapsulation (Fürsatz et al., 2021 ; Perka et al., 2000 ; Salam et al., 2018 ), can be easily applied but bears low mechanical stability and degrades rapidly.…”

Progress of 3D Organoid Technology for Preclinical Investigations: Towards Human In Vitro Models

“…Not like the direct force of forming cell pellets, the rolling-up method takes a longer duration from the cell seeding as a monolayer to the final formation of spheroids, usually taking at least two weeks. The circularity of rollingup spheroids is not as good as the ones derived from directly compacted cell aggregates [57].…”

“…In addition to the above methods, another productive way of generating spheroid/organoids with similar diameters is by rolling up monolayer cell cultures via compartmentation of cell culture surfaces utilising laser engraving (grid plates) [57]. Cells are cultured as monolayers inside the dish, subdivided by compartments; the plate then triggers the retraction at the edge of each compartment and therefore rolls the monolayer up as a spheroid [58].…”

Unlocking Potential: Low Bovine Serum Albumin Enhances the Chondrogenicity of Human Adipose-Derived Stromal Cells in Pellet Cultures

“…Cells were expanded in Endothelial Cell Growth Basal Medium (EGM-2, Lonza, Vienna, Austria CC-3156 and CC-4176), based on previous studies [32,33]. hASCs in passage 2 were differentiated towards the chondrogenic lineage using different BSA concentrations.…”