Massively parallel photosensitive hydrogel encapsulated single-cell to a cluster of cells patterning and bone regeneration application

Abstract

Recent advances in cell biology and biomedical research have shifted from traditional two-dimensional (2D) to three-dimensional (3D) cell cultures. To aid in the study of various tissues, we present a high-throughput cell patterning device that encapsulates single-cells to a small cluster of cells within the hydrogels. The device with a 1cm x 1cm area, creates an array of 3D hydrogel-encapsulated micro-patterns ranging from 80µm × 80µm to 250µm × 250µm using photolithography, with gaps between the micro-patterns varying from 100µm to 200µm (center to center). At a concentration of 2 x 10⁷ cells/mL, the device demonstrated ~100% patterning efficiency by consistently accommodating ~13 cells per pattern for 250µm × 250µm micro-pattern array with an excellent cell viability of ~ 97.21%. 80µm × 80µm patterns also showed an efficiency of 94.4%, with ~ 5 cells per pattern. The probability of encapsulating a single-cells was ~ 54.23% for the 80µm × 80µm micro-pattern, however it decreased to 36.1% for 250µm × 250µm micro-pattern with a cell viability of ~95%. Compared to the existing literature, we achieved an improved probability of encapsulating a single-cell with a higher cell viability for 80µm x 80µm micro-pattern, with a 40% reduction in photoinitiator concentration incorporated with gelatin methacryloyl (GelMA) and a 60% decrease in exposure time. This massively parallel cell patterning technique was applied to five cell lines with highest cell viability of 97.2% for NIH-3T3 cells. Furthermore, we engineered the micro-pattern device to enhance patterned osteogenic differentiation by incorporating synthesized nano-hydroxyapatite (nHA) with GelMA by encapsulating MC3T3-E1 preosteoblast cells for bone regeneration applications. The results demonstrated significant increases in osteogenic differentiation with 1%(w/v) nHA treatment after 14 days which was validated through UV absorbance and RT-PCR. Thus, this platform enables comprehensive cellular research through cell-to-cell interaction studies, and patterned biomineralization processes, facilitating tissue architecture simulation and advancing biomedical research.