Miniaturized droplet microarray platform enables maintenance of human induced pluripotent stem cell pluripotency

Abstract

The capacity of human induced pluripotent stem cells (hiPSCs) for indefinite self-renewal warrants their application in disease modeling, drug discovery, toxicity assays and efficacy screening. However, their poor proliferation ability, inability to adhere to surfaces without Matrigel coating and tendency to spontaneously differentiate in vitro hinder the application of hiPSCs in these fields. Here we study the ability to culture hiPSCs inside 200 ​nL droplets on the droplet microarray (DMA) platform. We demonstrate that (1) hiPSCs can attach to the Matrigel (MG)-free surface of DMA and show good viability after 24 h culture; (2) hiPSC do not spontaneously differentiate when cultured on the MG-free surface of DMAs; (3) culturing of hiPSCs in 200 ​nL as compared to 2 ​mL culture leads to higher expression of the Nanog pluripotency marker. Overall, the results demonstrate the possibility to culture undifferentiated hiPSCs in 200 ​nL droplets on DMA, thereby opening the possibility for high-throughput screenings of hiPSCs with various factors without compromising the results through the involvement of animal-derived materials, such as Matrigel.

Droplet Microarray Based Screening Identifies Proteins for Maintaining Pluripotency of hiPSCs

“…The viability was measured by a live–dead staining as described previously. [ 44 ] The viability of cells after noncontact printing was higher than that of manual seeding. This might be due to the higher volume of each spot after printing (200 nL) than the volume of manual seeding (≈80 nL) could provide more nutrient for cell growing and survival.…”

“Cells‐to‐cDNA on Chip”: Phenotypic Assessment and Gene Expression Analysis from Live Cells in Nanoliter Volumes Using Droplet Microarrays

“…In our laboratory, we have developed a "Droplet Microarray" (DMA) platform, formed by superhydrophobic-hydrophilic patterning. [36][37][38][39][40][41][42][43] Using this platform, various cell-based experiments are possible in hundreds to thousands of micro-or nanoliter cell culture reservoirs, separated by superhydrophobic borders on a single microscope glass slide. DMAs can be prepared on various types of substrates, e.g., nanostructured glass, [37] porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) polymer (HEMA-EDMA), [38,39] poly(thioether) dendrimers [44] etc.-having a broad range of surface topography and roughness.…”

“…[36][37][38][39][40][41][42][43] Using this platform, various cell-based experiments are possible in hundreds to thousands of micro-or nanoliter cell culture reservoirs, separated by superhydrophobic borders on a single microscope glass slide. DMAs can be prepared on various types of substrates, e.g., nanostructured glass, [37] porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) polymer (HEMA-EDMA), [38,39] poly(thioether) dendrimers [44] etc.-having a broad range of surface topography and roughness. We have demonstrated that HeLa-CCL2 cells cultured on different biomaterial-coatings used for fabrication of DMA, and in droplets of as low as 9 nL volumes exhibit no drastic transcriptomic alternations in comparison to cells grown in microtiter plates.…”

“…We have demonstrated that HeLa-CCL2 cells cultured on different biomaterial-coatings used for fabrication of DMA, and in droplets of as low as 9 nL volumes exhibit no drastic transcriptomic alternations in comparison to cells grown in microtiter plates. [45] The DMA platform has enabled culturing and screening of variety of cells in thousands of separated nanoliter droplets, making it a versatile, miniaturized, wall-free, high throughput platform for stem cells, [37] bacterial cells, [41] 2D and 3D mammalian cell culture, [36,46] single cells, [42] embryoid bodies [43] and even zebrafish (Danio rerio) embryos. [47] In this study, we for the first time established and evaluated "Cells-to-cDNA on Chip" methodology on DMA platform, which includes culturing cells, microscopy-based evaluation, mRNA isolation, and conversion to cDNA-all on an array of 200 nL droplets, followed by gene expression analysis from as low as a single cell per sample.…”

“…We have demonstrated that HeLa‐CCL2 cells cultured on different biomaterial‐coatings used for fabrication of DMA, and in droplets of as low as 9 nL volumes exhibit no drastic transcriptomic alternations in comparison to cells grown in microtiter plates. [ 45 ] The DMA platform has enabled culturing and screening of variety of cells in thousands of separated nanoliter droplets, making it a versatile, miniaturized, wall‐free, high throughput platform for stem cells, [ 37 ] bacterial cells, [ 41 ] 2D and 3D mammalian cell culture, [ 36,46 ] single cells, [ 42 ] embryoid bodies [ 43 ] and even zebrafish ( Danio rerio ) embryos. [ 47 ]…”