High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures

Abstract

High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology.

Keywords: high content imaging; miniaturized cell-based assay; predictive toxicology; three-dimensional (3D) cell culture.

Figure 1

Commonly used 3D cell culture techniques for HCI. (A) Hanging droplet plate containing 3D spheroids. To generate 3D spheroids, cell suspension is dispensed through the access holes of the hanging droplet plate such that the droplets are attached to the hydrophilic surface. Individual cells are aggregated within hours of incubation due to gravity, forming a single spheroid. (Adapted from [57] with permission of The Royal Society of Chemistry.) (B) Liquid overlay on top of cells in a 96-well plate. The bottom of the 96-wells is coated with non-adhesive polymer in a serum-free medium, which is followed by cell seeding. The 96-well plate is centrifuged to induce cell aggregation and hydrogel in a serum-supplemented medium is overlaid on top of the aggregated cells. (C) Hydrogel matrix mixed with cells in a 96-well plate. Cell suspension is mixed with hydrogel and dispensed in the 96-well plate. Cells naturally form 3D structures within the hydrogel matrix while growing.

Figure 2

Miniaturized 3D cell culture systems for HCI. (A) Microwell platform (Adapted from Ref [90] with permission of The Royal Society of Chemistry). Overview of hydrogel microwell arrays fabrication process: (Step 1) A polydimethylsiloxane (PDMS) stamp containing an array of micropillars is cast on a silicon master. (Steps 2 and 3) Poly(ethylene glycol) (PEG) gel is cross-linked to contain complementary microwell array topography using the PDMS stamp as a template. (Step 4) Individual cells are trapped on the hydrogel surface after swelling and washing of the surface. (B) Cellular microarrays on a functionalized glass slide. A mixture of cells and hydrogel precursor is printed on a glass slide coated with poly(styrene-co-maleic anhydride) (PS-MA). Various polymer coating is done on top of the PS-MA coating to attach different hydrogels to the glass slide. Cells are encapsulated in a hydrogel matrix, forming 3D structures after gelation (which occurs via various mechanisms). (C) Cellular microarrays on a micropillar/microwell chip platform (Adapted by permission from Macmillan Publishers Ltd: Nature Communications, Ref [84]). Cells mixed with hydrogel are printed on top of the micropillar chip. After gelation, the micropillar chip containing cells encapsulated in hydrogel is sandwiched with a complementary microwell chip containing growth media or other reagents. (D) Microfluidic device. (i) Top view of a bilayer microfluidic chip fabricated with PDMS on top of a glass slide. Several inlet and outlet channels provide parallel access to cell suspension, growth medium and other reagents. (ii) Overview of the cell culture process in the microfluidic device: (Step 1) Bi-layer chip is fabricated with PDMS containing several channels on top of a glass slide. (Step 2) A mixture of cells and hydrogel precursor is fed from the cell inlet channel. (Step 3) A growth medium is supplied from the medium inlet channel for cell culture.