High-throughput culture and embedment of spheroid array using droplet contact-based spheroid transfer

Abstract

Spheroids are one of the most representative models of 3D cell culture, which can be easily formed using conventional hanging drop method. However, medium change and spheroid transferring process are the bottlenecks that reduce the throughput of the entire process in the hanging drop culture. In addition, the embedment of spheroid into hydrogel still depends on the individual pipetting process. To overcome these issues, we present poly(dimethylsiloxane) (PDMS)based simple devices which can exploit droplet contact-based spheroid transfer using a drop array chip (DAC) having an array of well structures and peripheral rims. When the upper spheroid-containing drops were in contact with the lower liquid drops, the air-liquid interface disappeared at the merged surface and the spheroids settled down due to gravitational force. This method was applied to repetitive medium change and live/dead staining of spheroids cultured with the hanging drop method. To simultaneously embed the spheroids into the corresponding collagen hydrogel drops, a PDMS-based pillar array chip (PAC) was contacted in advance with the spheroid-containing DAC. The contacted PAC then contained the spheroids trapped in small drops of liquid reduced in volume to around 0.5 ll. Consequently, the spheroids were embedded into the collagen drops at once by contacting the spheroid-containing PAC with the collagen-loaded DAC. The embedded spheroids using the DAC-PAC contacting method showed a reliable invasion behavior compared to the embedded spheroids using conventional manual pipetting.

Recent Advances in Three-Dimensional Multicellular Spheroid Culture and Future Development

“…They found that the diameter of the spheroids on the oxygen-permeable chip increased significantly, the hypoxia and survival thresholds of the HepG2 spheroids cultured on it reached 400 and 600 mm, respectively, the cell growth was significantly enhanced, and anaerobic glycolysis was significantly reduced [ 80 ]. To overcome the shortcomings of the hanging drop method that the culture medium is not easy to replace and the spheroids are not easy to transfer, Kim et al used a method in which a droplet array chip (DAC) containing the spheroids was contacted with a DAC to transfer the spheroids and replace the medium [ 81 ].…”

Spheroids and organoids as humanized 3D scaffold‐free engineered tissues for SARS‐CoV‐2 viral infection and drug screening

“…Spheroids can be produced by different 3D cell culture techniques 35,40 . Different studies have already been published showing high‐throughput production of spheroids from different cell types by using the hanging‐drop technique 61‐65 . Other techniques already describe high‐throughput spheroid production consisting of using nonadhesive agarose microwell systems, 66 cell culture plates with a higher number of wells, as described by Grinner et al, 67 which used a 1536‐well plate format to engineer tumor spheroids for drug screening tests.…”

“…35,40 Different studies have already been published showing high-throughput production of spheroids from different cell types by using the hanging-drop technique. [61][62][63][64][65] Other techniques already describe high-throughput spheroid production consisting of using nonadhesive agarose microwell systems, 66 cell culture plates with a higher number of wells, as described by Grinner et al, 67 which used a 1536-well plate format to engineer tumor spheroids for drug screening tests. Other studies were already performed using well plates for high-throughput applications, as performed by Liao and collaborators 68 ; however, these authors used a 96well plate to cast agarose microwells.…”

Methods for in vitro modeling of glioma invasion: Choosing tools to meet the need

“…Otherwise this assay closely resembles the later assembly steps found in the sandwich invasion assay. Cells suspended in an ECM mixture are placed on the inside cover of a cell culture plate then inverted to hang down as the ECM solidifies (Figure 3c; H. Kim, Cho, & Park, 2018;Kuo et al, 2017;Marcello, Richards, Mason, & See, 2017). Several commercially available, specialized plates have been developed to optimize hanging drop sphere formation (H. Kim et al, 2018;Kuo et al, 2017;Marcello et al, 2017).…”