Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits

Abstract

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.

Preparation strategy and illumination of three-dimensional cell cultures in light sheet–based fluorescence microscopy

“…[20][21][22] A spatially resolved analysis of drug response in 3-D models is of pivotal interest for anticancer compound research as gradients of hypoxia and nutrient distribution, which are observed in tumors and reproduced in tumor cell spheroids, influence the sensitivity towards anticancer drugs. 23 Since phototoxicity can interfere with anticancer drug effects, minimization of the light dose by SPIM again proves to be essential.…”

Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening

“…The use of micropatterns that provide a controllable microenvironment for spheroid growth has been investigated in the past, but in 55 mm Petri dish format only. 25 Preliminary experiments investigated the effects of micropattern geometry, diameter, pitch, and cell-seeding density on HT-29 spheroid growth dynamics and size. Based on these initial results, a specific micropatterned 96-well plate was designed in which the dimensions and arrangement of the micropatterns addressed issues of (1) spheroid size heterogeneity, (2) the undesired detachment of spheroids during wash procedures or long-term growth, (3) spheroids touching and growing into one another as they expand, and (4) overcrowding that prevents optimal growth conditions for the production of large spheroids.…”

A microfluidic array with cellular valving for single cell co-culture

“…24 Native PDMS surfaces provide biologically inert backgrounds which resist cell adhesion. [26][27][28] Hydrophilic patterns for cell adhesion were provided within the microfluidic system by plasma stencilling using a Tesla generator. 22 As with the aqueous flows, the plasma was routed along the linear path of least resistance, oxidizing the surface to produce a hydrophilic state which supported cell adhesion, while the neighbouring hydrophobic PDMS surfaces confined cell adhesion to the trap regions.…”