PhD Defence Marieke Meteling | Guiding cell behaviour via intracellular crowding

Guiding cell behaviour via intracellular crowding

Marieke Meteling is a PhD student in the department Developmental BioEngineering. (Co)Promotors are dr. J.C.H. Leijten and prof.dr. H.B.J. Karperien from the Faculty of Sciences & Technlogy (TNW), University of Twente.

To successfully engineer functional living tissues, it is essential to have an in depth understanding of how cell behaviour can be guided. This knowledge is equally relevant for engineering tissues on a small scale (e.g. to create physiologically relevant disease models), as it is for engineering tissues on a larger scale (e.g. to engineer donor replacement organs). While it is well established that cell behaviour is mediated in a reciprocal manner with its microenvironment, it remains a challenge to re-create tissue-specific cell behaviour in the lab. In the last decades, it has become clear that the cell behaviour in three-dimensional (3D) cell culture systems more closely resembles native-like cell behaviour compared to the historically conventional 2D cell culture system (i.e. monolayer cell culture on plastic). While many advances in the field have led to ever more complex 3D cell culture systems, which has greatly advanced our control over cell functionality, we still do not fully comprehend how 3D cell culture orchestrates this more native-like cell behaviour.

This thesis highlights how the interplay of key intracellular processes in conjunction with intracellular macromolecular crowding orchestrates 3D-induced cell behaviour. For studying the 3D cell culture induced cell behaviour, we used chondrogenesis (i.e. cartilage formation) as a model system as 3D cell culture, unlike 2D cell culture, is well known to drive chondrogenesis. Following the transition from 2D to 3D cell culture, we demonstrated that microtissue condensation occurs via cell volume reduction. This resulted in increased cell mass density, which is indicative of increased intracellular macromolecular crowding. Notably, this phenomenon was neither cell type specific, nor exclusive to microtissue culture. Further investigation revealed that primary cells increase in volume during prolonged 2D expansion culture, which led to a decrease in cell mass density. Notably, prolonged 2D expansion culture of primary cells is associated with a loss in cell phenotype. Delving further into the mechanism, we revealed that cell culture dimensionality directs cell behaviour in a mechano-metabolic manner. In addition, studies on primary chondrocytes demonstrated that medium osmolarity-induced alternations in intracellular crowding could at least in part emulate the osteoarthritic and healthy-like phenotype, in healthy and osteoarthritic chondrocytes, respectively. This highlighted that changes in intracellular crowding likely also play a role in pathology. Lastly, to gain a better understanding of the reciprocal nature between the cell and its microenvironment, a novel read-out platform was developed. This technology enables the analysis of deposited nascent matrix proteins within 3D microenvironments in a non-destructive manner at the single cell level in high-throughput. This was achieved by combining single cell microgels with immunolabelling and flow cytometry. This newly developed method, termed Extracellular Protein Identification Cytometry (EPIC), allowed us to study population heterogeneity in matrix deposition. Due to its inherent compatibility with fluorescence activated cell sorting (FACS), subpopulations of interest could be isolated for downstream analysis such as confocal imaging. Moreover, it was demonstrated that EPIC is also compatible with multi-cell read-outs, which enabled us to study the influence of cell-cell contact on matrix deposition at single-cell level in a high-throughput manner.

Overall, this thesis aims to contribute to our understanding of how cell behaviour is orchestrated by microenvironmental cues, such as cell culture dimensionality or nascent matrix deposition. A articular focus was placed on intracellular crowding as an important parameter of the cell’s state. The findings of this thesis may present a new design strategy to engineer living tissue constructs in a more predictable manner, and thus help optimize current cell culture systems and disease models. In addition, the newly developed single cell analysis technique, EPIC, opens up the possibility to study the influence of different environmental and cellular parameters (e.g. viscoelasticity, drug treatments, intracellular crowding levels) on matrix deposition at the single cell level in high-throughput. This may yield additional insights on how to optimize cell culture systems, and help identify new therapeutic treatment strategies.