Microfluidic Biofabrication of 3D Multicellular Spheroids by Modulation of Non-geometrical Parameters

Abstract

Three-dimensional (3D) cell spheroids are being increasingly applied in many research fields due to their enhanced biological functions as compared to conventional twodimensional (2D) cultures. 3D cell spheroids can replicate tissue functions, which enables their use both as in vitro models and as building blocks in tissue biofabrication approaches. In this study, we developed a perfusable microfluidic platform suitable for robust and reproducible 3D cell spheroid formation and tissue maturation. The geometry of the device was optimized through computational fluid dynamic (CFD) simulations to improve cell trapping. Experimental data were used in turn to generate a model able to predict the number of trapped cells as a function of cell concentration, flow rate, and seeding time. We demonstrated that tuning non-geometrical parameters it is possible to control the size and shape of 3D cell spheroids generated using articular chondrocytes (ACs) as cellular model. After seeding, cells were cultured under perfusion at different flow rates (20, 100, and 500 µl/min), which induced the formation of conical and spherical spheroids. Wall shear stress values on cell spheroids, computed by CFD simulations, increased accordingly to the flow rate while remaining under the chondroprotective threshold in all configurations. The effect of flow rate on cell number, metabolic activity, and tissue-specific matrix deposition was evaluated and correlated with fluid velocity and shear stress distribution. The obtained results demonstrated that our device represents a helpful tool to generate stable 3D cell spheroids which can find application both to develop advanced in vitro models for the study of physiopathological tissue maturation mechanisms and to obtain building blocks for the biofabrication of macrotissues.

Recent advances in spheroid-based microfluidic models to mimic the tumour microenvironment

“…The significance of fluid dynamics on the culture of 3D spheroids and tissue maturation by developing a perfusable microfluidic platform with computational fluid dynamics-based simulation was demonstrated by Lopa et al 65 A perfusion system combined with digital systems allowed the generation of a model to predict cell trapping efficacy, flow rate, and seeding time, demonstrating the possibility of controlling the size and shape of spheroids through fluid flow. The effect of shear stress on spheroid formation was also evaluated; lower shear stress to the cells resulted in larger and less rounded spheroids, whereas higher shear stress resulted in smaller and spherical spheroids.…”

“…When MCF7 cells were co-cul- 62 (C) A microfluidic system able to induce different hydrogel stiffness in which spheroids proliferate, demonstrating higher proliferation in spheroids grown in stiff hydrogels. 65 (D) Metabolic gradient induced in a microfluidic device, comparing cellular uptake of metabolic gradients in tumour spheroids and monolayer-culture cells. 67 tured with other cells (macrophages and fibroblasts) to form a spheroid, the cells exhibited higher proliferation regardless of gel stiffness.…”

“…(B) Microfluidic platform developed to control the concentration of oxygen in the tumour microenvironment, inducing different oxygen concentrations to tumour spheroids and promote different cell behaviour 62. (C) A microfluidic system able to induce different hydrogel stiffness in which spheroids proliferate, demonstrating higher proliferation in spheroids grown in stiff hydrogels 65. (D) Metabolic gradient induced in a microfluidic device, comparing cellular uptake of metabolic gradients in tumour spheroids and monolayer-culture cells 67.…”

Spheroid Engineering in Microfluidic Devices

“…Conventional methods have limited control over the size and geometry of 3D cell spheroids . However, robust, reproducible, and high-throughput 3D cell spheroid formation can be achieved using microfluidic technologies . Spheroids generated using advanced 3D culture techniques have been emerging as tissue precursors used to develop a variety of on-a-chip tissue and disease models, particularly drug delivery systems, by simulation of the complex multicellular architecture, barriers to mass transport, extracellular matrix synthesis, various protein and gene expressions, and in vivo physiological conditions …”

One-Step Generation and Purification of Cell-Encapsulated Hydrogel Microsphere With an Easily Assembled Microfluidic Device

“…Droplet microfluidics is a versatile technology that allows for the manipulation of small volumes of liquids on a microfluidic chip (Zhu et al, 2019;Mohamed et al, 2020;Balasubramanian et al, 2021). Previous studies have applied droplet microfluidics in the fields of molecular and cellular biology and analytical chemistry for applications such as clonal development (Dolega et al, 2015), drug screening (Courtney et al, 2017), singlemolecule/single-cell analysis (Najah et al, 2012;Shembekar et al, 2018;Shao et al, 2020), tissue engineering (Liu et al, 2018), organoid modeling (Vadivelu et al, 2017;Zhang et al, 2021), and spheroid fabrication (Lopa et al, 2020;Mohamed et al, 2020). Droplet microfluidic technology is especially suitable for 3D cell cultures (Eqbal et al, 2021).…”