Engineered Microsystems for Spheroid and Organoid Studies

Abstract

3D in vitro model systems such as spheroids and organoids provide an opportunity to extend the physiological understanding using recapitulated tissues that mimic physiological characteristics of in vivo microenvironments. Unlike 2D systems, 3D in vitro systems can bridge the gap between inadequate 2D cultures and the in vivo environments, providing novel insights on complex physiological mechanisms at various scales of organization, ranging from the cellular, tissue-, to organ-levels. To satisfy the ever-increasing need for highly complex and sophisticated systems, many 3D in vitro models with advanced microengineering techniques have been developed to answer diverse physiological questions. This review summarizes recent advances in engineered microsystems for the development of 3D in vitro model systems. The relationship between the underlying physics behind the microengineering techniques, and their ability to recapitulate distinct 3D cellular structures and functions of diverse types of tissues and organs are highlighted and discussed in detail. A number of 3D in vitro models and their engineering principles are also introduced. Finally, current limitations are summarized, and perspectives for future directions in guiding the development of 3D in vitro model systems using microengineering techniques are provided.

Keywords: 3D in vitro models; mechanical principles; microengineering; organoids; spheroids.

Figure 1.

Schematic representation of 3D cellular structures: spheroid, multicellular spheroid, and organoid. Spheroids are compact aggregates of cells, whereas organoids show realistic microanatomy and typically have a hollow lumen. These 3D cellular structures are important basis for in vitro models of development, disease, drug design. The organization of the 3D cellular structures is composed of three different compartments: a necrotic core in the center, a quiescent zone of non-proliferating cells, and an outermost layer with proliferating cells. Different substance-dependent concentration gradients exist in these layers.

Figure 2.

Mechanical principles for development of in vivo and 3D in vitro model system. The five mechanical principles include gravitational force (F_g), capillary force (F_c), centrifugal force (F_w), adhesive force (F_ad), and hydrodynamic shear stress (τ_s), each of which can uniquely influence the formation of 3D cellular structures.

Figure 3.

Schematic representation of engineered microsystems for 3D in vitro models. Diverse cell sources include, but are not limited to, primary cells, embryonic stem cells, adult stem cells, and induced pluripotent stem cells. Different engineered microsystems with distinct underlying mechanisms are used to generate 3D cellular structures with specific morphological and physiological characteristics.

Figure 4.

Microwell platforms with various microstructures: a) Schematic illustration for the fabrication of the microwell using PEG hydrogels (20 kDa). Reproduced with permission.^[124] Copyright 2017, IOP Publishing. b) Agarose microwell array with improved throughput of hepatocyte spheroid formation. Reproduced with permission.^[125] Copyright 2020, ACS Publications. c) U-shaped polyHEMA microwell for enhanced T47D cell aggregation and sphere formation. The scale bar is 100 μm. Reproduced with permission.^[131] Copyright 2015, Springer Nature Publishing AG. d) Aggregation of bone marrow-derived mesenchymal stem cells in the microwell-mesh and the growth of the cartilage microtissues at day 14 post seeding. The microtissues were too large to pass back through the nylon mesh and were retained in discreet microwells. Reproduced with permission.^[134] Copyright 2015, Elsevier.

Figure 5.

Centrifuge-based 3D cellular structures culture systems: a) rotary culture, where cells are constantly rotated to prevent sediment, and a large number of spheroids are generated in the media. Mouse embryonic stem cells (mESCs) were formed into embryoid bodies (EBs) using rotary culture. Reproduced with permission.^[148] Copyright 2014, Springer US. b) microwell culture of islet cells (ICs, green) and human amniotic epithelial cells (hAECs, red) assisted by centrifugal force. It is essentially the same principle as the microwell platform, but by applying centrifugal force, the denser spheroid is generated faster. Scale bar is 50 μm. Reproduced with permission.^[152] Copyright 2019 Nature Publishing AG. c) Lab-on-a-CD culture, where a disc-shaped chip is rotated to generate various types of multicellular spheroids. Multicellular spheroids of human adipose-derived stem cells (hASC, green) and human lung fibroblasts (MRC-5, red) were generated. Scale bar is 500 μm. Reproduced with permission.^[154] Copyright 2017, IOP Publishing. d) Schematic of centrifugal droplet-generating device and photomicrograph of human malignant melanoma cell line (MEL28) encapsulating microspheres cultured for a time course of 7 days. Scale bar is 200 μm. Reproduced with permission.^[156] Copyright 2020, SAGE Publications.

Figure 6.

Liquid drop-assisted devices with different manipulations of cell suspension: a) Microfluidic hanging drop (μHD) chip operation and docked embryonic stem cells aggregated at bottom center of hanging droplets. The scale bar is 200 μm. Reproduced with permission.^[169] Copyright 2016, MDPI. b) Cross-section of channel structures defined by rims creating uniform-sized interconnected hanging drops (~14 μl). Reproduced with permission.^[170] Copyright 2014 Springer Nature Publishing AG. c) Emulsion-generating microfluidic device for fabrication of mesenchymal stem cells (BMSCs)-laden GelMA microspheres. Scale bars are 100 μm. Reproduced with permission.^[167] Copyright 2016, John Wiley & Sons. d) Electrospray-based device for 3D cell aggregation between MDA-MB-231 (with red fluorescence) and MCF10A cells. Reproduced with permission.^[180] Copyright 2015, The Royal Society.

Figure 7.

Microchannel-based cell culture system: a) Horseshoe-shaped barrier for cell trapping, clustering, spheroid formation and incubation of non-small lung cancer cells (H1650). Reproduced with permission.^[182] Copyright 2011, The Royal Society. b) Pneumatic microstructures (PμSs) for spatial cell trapping fluid flow and formation of 3D tumors (U251 cell). Reproduced with permission.^[183] Copyright 2015, ACS Publications. c) Enhanced vascularization of kidney organoids within a perfusable milli-fluidic system under varying fluidic shear stress. Reproduced with permission.^[186] Copyright 2019, Springer Nature Publishing AG. d) Electrodynamic digital microfluidic (DMF) fluid manipulation for HepG2 liver organoid culture. Reproduced with permission.^[188] Copyright 2014, The Royal Society.