Acoustic assembly of cell spheroids in disposable capillaries

Abstract

Multicellular spheroids represent a promising approach to mimic 3D tissues in vivo for emerging applications in regenerative medicine, therapeutic screening, and drug discovery.Conventional spheroid fabrication methods, such as the hanging drop method, suffer from low throughput, long time, complicated procedure, and high heterogeneity in spheroid size. In this work, we report a simple yet reliable acoustic method to rapidly assemble cell spheroids in capillaries in a replicable and scalable manner. Briefly, by introducing a coupled standing surface acoustic wave, we are able to generate a linear pressure node array with 300 trapping nodes simultaneously. This enables us to continuously fabricate spheroids in a highthroughput manner with minimal variability in spheroid size. In a proof of concept application, we fabricated cell spheroids of mouse embryonic carcinoma (P19) cells, which grew well and retaineddifferentiation potential in vitro. Based on the advantages of the non-invasive, contactless and label-free acoustic cell manipulation, our method employs the coupling strategy to assemble cells in capillaries, and further advances 3D spheroid assembly technology in and easy, cost-efficient, consistent, and high throughput manner. This method could further be adapted into a novel 3D biofabrication approach to replicate compilated tissues and organs for a wide set of biomedical applications.

Engineering Multi‐Cellular Spheroids for Tissue Engineering and Regenerative Medicine

“…[102] Surface acoustic wave (SAW) has been applied to the spheroid formation, in which the cells were loaded into capillary rods or microfluidics chambers and then assembled into spheroid with a uniform size by SAW. [103,104] Mouse embryonic carcinoma cells were assembled to spheroids with high throughput and minimal variability in the spheroid size within capillary rods (average size = 139 µm, generation of 300 spheroids in one capillary) and HepG2 spheroids were formed by SAW in microfluidics within 30 min with the size of spheroids being well-controlled by the seeding density from 30 to 100 µm. [103,104]…”

“…[ 103,104 ] Mouse embryonic carcinoma cells were assembled to spheroids with high throughput and minimal variability in the spheroid size within capillary rods (average size = 139 µm, generation of 300 spheroids in one capillary) and HepG2 spheroids were formed by SAW in microfluidics within 30 min with the size of spheroids being well‐controlled by the seeding density from 30 to 100 µm. [ 103,104 ]…”

“…Controlled size, simplicity [ 71,74] Automated hanging drop Labor-effectiveness, controlled size [75] Superhydrophobic surface Efficiency, controlled size [ 76,77] Insert formed by 3D printing Efficiency, controlled size [ 101] Centrifugal force Pellet culture Controlled size, simplicity [ 14,78] Spinner flask Mass production, reduced apoptosis [ 82,83] Rotary culture Mass production, controlled size [81] Surface chemistry Chemical patterning Mass production (<100 µm), [ 71,93,94,95,96,97] (PNIPAAm/biomolecules) controlled size Microstructure Topological pattern Simplicity, controlled size [ 40,89,90] Micro-well plate Controlled size [ 41,85,86,87,88] Flow-based automation Microfluidics Labor-effectiveness, controlled size, effective medium change [ 98,99,100] Acoustic wave Surface acoustic wave Controlled size [ 103,105] or centrifugal forces) for hanging drop and pellet culture methods, and biomaterials with microstructure and defined surface chemistry for the Aggrewell and the ultra-low attachment plate method, respectively. [14,16,71] These methods have their advantages and disadvantages, and thus, the method is dependent on the intended application.…”

Spatiotemporal quantification of acoustic cell patterning using Voronoï tessellation

“…In this approach, unlabelled cells can be rapidly and dynamically patterned using conventional cell media, culture substrates or biomaterial systems; characteristics that make this technique highly suited to generating complex cell systems. To date, acoustic patterning has been used to generate spheroid cultures,11–14 engineer complex tissue structures15–18 and study processes such as vascularization,19–21 intercellular communication,22 tissue development,23 cell migration,24 natural killer cell activity25,26 and neurite outgrowth 27. This biological versatility arises from the physical mechanisms governing acoustic patterning.…”

“…Given the evident importance of pattern structure and consistency upon the design and outcome of acoustic cell patterning studies, there is a surprising dearth of feature characterization and quality control of the generated cell arrays. The recent, major reports of acoustically patterned cell assemblies have reported only a binary, visual assessment of micrographs to ascertain whether or not a population is patterned 11–14,16–22,24–27,30–34. Only three of these studies provided characterization beyond basic sizing: Christakou et al indirectly evaluated the cluster “compactness” based on the penetration depth of a fluorescent dye,26 Comeau et al used peak-to-peak and peak-to-trough measurements of patterned lines to estimate band spacing and density, respectively,21 while Olofsson et al counted both the number of clusters and the number of single cells patterned in each well of a micro-well plate 13.…”