Microvalve Bioprinting of MSC-Chondrocyte Co-Cultures

Abstract

Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof-of-concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures to investigate whether the use of MSCs in ACI procedures would stimulate enhanced ECM production by chondrocytes. Microvalve-based bioprinting uses small-scale solenoid valves (microvalves) to deposit cells suspended in media in a consistent and repeatable manner. In this case, MSCs and chondrocytes have been sequentially printed into an insert-based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate ECM production. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This study, therefore, provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.

Keywords: 3D cell culture; autologous chondrocyte implantation; biofabrication; bioprinting; drop-on-demand; micro-tissue; microvalve.

Figure 1

Illustration of the co-culture cell printing configuration using the Transwell^® insert culture platform. Chondrocytes were printed directly onto the surface of the permeable insert membrane followed by MSCs.

Figure 2

Printer configuration showing the microvalve actuator attached to the agitated reservoir. Following ink loading, a small cylindrical magnet is placed within the ink suspension and the lid clamped shut. The magnet is rotated using a motor mounted externally to the ink compartment and pressure maintained via the top-mounted air inlet ports.

Figure 3

Dispensing performance of the microvalve printing platform under a drop-on-demand configuration. (A) Influence of waveform dwell time and backpressure applied to valve on cell density per droplet when printing MSCs at a concentration of 10⁶ cells per mL. (B) Influence of waveform dwell time and backpressure applied to valve on cell density per droplet when printing chondrocytes at a concentration of 10⁶ cells per mL. (C) Dispense volume per droplet number. The gradient of the linear regression trend line corresponds to a droplet volume of 61 nL. (D) Impact of ink cell concentration on cell printing performance. Data represent mean values ± SD. N = 3–6.

Figure 4

Effect of printing process on the viability and function of the MSC and chondrocyte cell lines printed at 10⁶ cells per mL. (A) Cell viability assay showing live (green) and dead (red) cells. Scale bar = 200 μm. (B) Metabolic activity assay. Data represent mean values ± SD. N = 6. Significance as outlined in Section 2.14.

Figure 5

Comparison between the morphology and proliferative activity of printed and manually pipetted cells when deposited at concentrations of 10⁶ cells per mL. (A) Morphology of MSC and chondrocyte cell lines showing cell nuclei visualised using DAPI (blue) and filamentous actin using phalloidin (red) staining. Scale bar = 50 μm. (B) Proliferation rate of MSC and chondrocyte cell lines assessed via cell counting using a trypan blue dye exclusion test. (C) Non-linear regression analysis using an exponential growth model of cell proliferation. Data represent mean values ± SD. N = 6.

Figure 6

Histological characterisation of tissue model. Sections were oriented to display the Transwell^® membrane at the base of each image. (A) Haematoxylin and eosin staining of printed cultures generated from MSCs, chondrocytes and cell co-cultures over a 14-day culture period. (B–D) Immunostaining data comparing the collagen II (COLII, green) and aggrecan (AGCN, red) content of MSC, chondrocyte and MSC-chondrocyte printed cultures. Cell nuclei were visualised using DAPI (blue) staining. Scale bar = 100 μm.

Figure 7

Cellular proliferation and localisation within tissue models. (A–C) Immunofluorescent detection of proliferating cells via EdU (5-Ethynyl-2′-deoxyuridine) incorporation within MSC, chondrocyte and MSC-chondrocyte co-cultures. Cell nuclei were visualised using DAPI (blue) staining. Sections were oriented to display the Transwell^® membrane at the base of each image. (D) Localisation of MSC (green) and chondrocyte (red) cells on the surface of the co-culture over a 14-day culture period. Cells were labelled using CellTracker^® fluorescent dyes prior to printing. Scale bar = 100 μm.