Hydrogel-incorporating unit in a well: 3D cell culture for high-throughput analysis

Abstract

The microfluidic 3D cell culture system has been an attractive model because it mimics the tissue and disease model, thereby expanding our ability to control the local cellular microenvironment. However, these systems still have limited value as quantitative assay tools due to the difficulties associated with the manipulation and maintenance of microfluidic cells, and their lack of compatibility with the high-throughput screening (HTS) analysis system. In this study, we suggest a microchannel-free, 3D cell culture system that has a hydrogel-incorporating unit integrated with a multi-well plate (24- to 96-well plate), which can provide better reproducibility in biological experiments. This plate was devised considering the design constraints imposed by various cell biology applications as well as by high-throughput analysis where the physical dimensions of the micro-features in the hydrogel-incorporating units were altered. We also demonstrated that the developed plate is potentially applicable to a variety of quantitative biochemical assays for qRT-PCR, Western blotting, and microplate-reader-based assays, such as ELISA, viability assay, and high content-screening (HCS) as well as the co-culture for biological studies. Human neural progenitor cells (hNPCs) that produce pathogenic Aβ species for modeling Alzheimer's disease (AD) were three-dimensionally cultured, and the efficacy of the inhibitors of Aβ production was assessed by ELISA in order to demonstrate the performance of this plate.

Microfluidic three-dimensional cell culture of stem cells for high-throughput analysis

“…Despite the disadvantages, including the labor and time required, HTS technology using well plates is used extensively for developing various protocols for cell cultures and 2D and 3D screening of cells because the microplate is still a well-established platform for HTS applications so many research groups view it as a user-friendly approach. For this reason, Yu et al[71] developed the well plate-based gel unit array for HTS analysis. This platform has a unique feature in that it has hydrogel-incorporating compartments integrated in a well to culture 3D tissue with uniform thickness while co-culturing with other neighboring cells in a single well.…”

“…Stem cells, including iPSCs, have the potential to serve as a source of cells that can be engineered to suit specific needs in the development of organ-on-chips[5,6]. In recent years, the organ- and organoid-on-a-chip approaches using stem cells have been used extensively to establish the new microengineered models that recapitulate the structure and functional complexity of human organs, such as the liver[74-77], heart[78-86], brain[71,87-94], intestine[95-97], kidney[98-100], and bone[101-103]. Recently, organ-on-a chip technology has been able to integrate multiple organ or tissue models to simulate the human body, and multi-organ systems generated using stem cells have been developed for a human body-on-a chip system[16,75,104,105] It is possible for such a system to provide a predictive model for pharmacokinetics of drugs by mimicking the activities of the human body such as absorbing, distributing, metabolizing, and eliminating drugs.…”

“…The iPSC-derived hepatocytes were aggregated as three-dimensional (3D) organoids and encapsulated in Poly (ethylene glycol) diacrylate hydrogel. These cells were loaded in a C-trap chip subjected to perfusion for a long-term culture (Reproduced from Ref[76] with permission from the Royal Society of Chemistry); B: A miniaturized 96 well-type human iPSC-derived cardiac organoid (hCOs) screening platform, which is called heart dynamometer (Heart-Dyno), facilitates the automated formation of hCOs (Reproduced from Ref[86] with permission from the National Academy of Science); C: Well plate-micropattern hybrid platform for NPC differentiation for modeling Alzheimer’s disease (Reproduced from Ref[71] with permission form the Royal Society of Chemistry). This hybrid-platform is compatible with high-throughput screeninganalysis; D: Organo-plate® comprising 96 microfluidic tissue chips and experimental outline for culturing 3D neuronal-glial networks (reproduced from Ref[94] with permission from the Nature Research).…”

“…Similarly, there have been recent developments in brain-on-a-chip for HTS. The attention for brain models fits in a wider trend towards attention for neural progenitor-cell-derived brain models for diseases, such as Alzheimer’s disease[71,91]. This follows the more generic increase in the popularity of iPSC techniques and progress in controlling the stem cell niche of differentiated tissues.…”

“…Wang et al[93], developed brain-organoids using iPSCs to model neurodevelopment disorders under prenatal nicotine exposure and showed the potential of drug testing. In addition, the 96-well plate-based, HTS-compatible 3D cell culture platform for the brain model was developed for preclinical drug screening applications[71,92]. Especially, Yu et al[71] developed a micropattern array platform combined with conventional well-plate for HTS drug screening to show the proof-of-concept for the Alzheimer’s disease model using neural progenitor cells, as shown in Figure 1C.…”