Making cartilage one drop at a time: harnessing droplet microfluidics for cartilage tissue engineering

Abstract

Osteoarthritis and other musculoskeletal disorders affect a large proportion of the aged population worldwide. Cartilage tissue engineering approaches have emerged to replace invasive surgical techniques aiming to restore hyaline articular cartilage. Human bone marrow stromal stem cells (HBMSCs) offer the potential as a cell source for differentiation into cartilage tissue. However, their application is hindered by a crucial limitation: subpopulation heterogeneity. The current study provides proof of concept for the implementation of droplet microfluidics for functional assessment of cell aggregates. The goal is to achieve high-throughput cell aggregation simulating early chondrogenic condensation with a view to enriching HBMSC chondroprogenitor subpopulations on the basis of functional, condensation-dependent markers such as SOX9. The in vitro characterisation of multicellular spheroids exposed differences in their chondrogenic differentiation outcome linked to cell number. SOX9 expression in large spheroids did not change as a function of time. In contrast, SOX9 expression declined in small spheroids in late chondrogenesis and did not vary significantly during the first week. This indicated that SOX9 could be a potential early marker to predict chondrogenesis in HBMSCs and was further validated by the lack of significant differences in SOX9 mRNA levels during the first week of chondrogenic differentiation. SOX9 heterogeneity was observed on an intra-spheroid level, which highlighted a potential advantage in harnessing even smaller aggregates for enriching stem cells for cartilage tissue engineering. The suitability of SOX9-Cy5 nanoflares was assessed for their application for sorting of cell aggregates. The Cy5-positive cell subpopulations corresponded with high SOX9 mRNA levels only in those instances where there was a clear difference in terms of order of magnitude of Cy5 fluorescence signal between positive and negative gated cells by flow cytometry. Thus, the data suggest that nanoflares were not sufficiently sensitive to dissect HBMSC subpopulations on the basis of biologically relevant differences in SOX9 expression. The optimisation process of a microfluidic device concluded that soft lithography designs incorporating silanisation and plasma bonding generated highly monodisperse droplet diameters that were promising for cell encapsulation. A functional stability assay demonstrated the higher effectiveness of the Bio-Rad QX200TM droplet generation oil commercial variation compared to the in-house Soton2 surfactant. The droplet microenvironment was suitable for the formation of minimal HBMSC aggregates (comprising only a few cells). HBMSCs aggregates displayed maintained colony formation functionality when re-plated from aggregates incubated in short-term chondrogenic differentiation cultures for up to 3 days. In addition, HBMSC aggregates self-assembled in microdroplets expressed higher levels of SOX9 than cells cultured in monolayer as early as 24 h of chondrogenic induction. The work presented in this thesis confirms the potential of droplet microfluidics for culturing multicellular spheroids as vehicles for chondrogenesis. The application of this platform for cell aggregation, short-term culture and sorting on the basis of early stage functional markers opens new prospects for harnessing HBMSCs for cartilage tissue engineering.

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Identifiers

ORCID for Jonathan Dawson: orcid.org/0000-0002-6712-0598

ORCID for Rahul Tare: orcid.org/0000-0001-8274-8837

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