Spherowell, a novel system inducing autonomous spheroid formation of cell monolayers

Abstract

Purpose: Cell spheroids are the most frequently used system for in vitro chondrogenic differentiation and are gaining increasingly importance in clinical applications. However, drawbacks of currently used systems are the lack of early-readout parameters and time as well as reagent intensive procedures. While this can be partially alleviated by process automation, it is often not feasible due to high cost of necessary equipment. Herein we present a completely new concept of spheroid formation (SpheroWell) allowing for mass-production of chondrogenic spheroids from monolayer via growth surface compartmentation with promising applications in cartilage regeneration and in vitro screening applications. Methods: Standard 60 mm cell culture dishes were compartmentalized with a 1 mm or 3 mm raster by laser engraving. Telomerase immortalized adipose derived stromal/stem cells (ASC/TERT1) were seeded in mono-culture and in co-culture with primary human articular chondrocytes (hAC) at a total cell number of 1 x 106 cells/dish on compartmentalized dishes. Co-cultures were conducted at 1:4, 1:1 and 4:1 hAC:ASC ratios. Cells were cultured in differentiation media containing low concentrations of TGFβ-3 and BMP-6 (1 ng/ml). The spheroid formation process was analysed using time-laps imaging and bright field-/confocal microscopy and the resulting spheroid sizes were measured. Chondrogenic differentiation and internal structure were compared to standard pellet culture using qRT-PCR (collagen type 1/2 (Col1/2) and aggrecan (Agg)) and (immuno-) histochemistry (Alcian blue and collagen type II). As a model for tissue regeneration spheroids were embedded into fibrin hydrogel and the cellular outgrowth and matrix deposition were analysed. Finally the system was assessed for its usability as an in vitro testing system using the spheroid formation process as an early readout parameter by assessing the effect of interleukin-1β (IL-1β) on the formation kinetics. Results: After initial seeding, spheroids self-assembled in a multi-phase process. After a short proliferation phase, edge regions (especially in compartment corners) began to retract, followed by roll-up of the monolayer and final condensation into a spheroid (Figure 1). For ASC/TERT1 mono-cultures this process took about 3 weeks, while addition of hAC accelerated the formation time to 1-2 weeks in a cell-ratio dependent manner. However, while ASC and co-cultures up to 1:1 ratio formed pellets of constant size, hAC ratios over 50% lead to variable and smaller spheroids due to faster and incomplete monolayer formation. Apart from cell ratios, spheroid sizes could be controlled by varying the raster size, yielding highly reproducible mean diameters of 340.9 and 134.4 (SD < 10%) for 3 mm and 1 mm raster respectively (with ASC mono-cultures). When comparing spheroids from raster plates with standard pellet culture comparable differentiation capacity was visible despite the 2D-phase of the initial monolayer in SpheroWells. Internal structures revealed differences with spheroids showing strands of matrix homogenously distributed over the cross-section, while pellets generally showed a pronounced aligned cortex region. When embedding spheroids from raster plates into fibrin hydrogels cellular outgrowth into the hydrogel could be observed, with outgrowth speed being controllable via hydrogel density. Matrix deposition could be observed around the outgrowing cells, which was especially pronounced in co-cultures. Finally, first results using the system as an in vitro testing system were obtained. Treatment of raster plate cultures with IL-1β showed a strong reduction in spheroid formation time serving as early readout parameters, while as expected showing abolishment of Col2 expression in endpoint analysis. Conclusions: The new culture system, SpheroWell, is highly efficient in spheroid generation, saving time and culture medium and allowing for additional early read outs via tracking the spheroid formation process. The ability to produce and maintain hundreds of spheroids within one dish, which can be easily integrated into pre-existing production and research workflows makes it attractive for high throughput testing and clinical spheroid application.