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

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 hydrogels. The device with a 1 cm × 1 cm area creates an array of 3D hydrogel-encapsulated micro-patterns ranging from 80 μm × 80 μm to 250 μm × 250 μm using photolithography, with gaps between the micro-patterns varying from 100 μm to 200 μm (center to center). At a concentration of 2 × 10⁷ cells per mL, the device demonstrated ∼100% patterning efficiency by consistently accommodating ∼13 cells per pattern for the 250 μm × 250 μm micro-pattern array with an excellent cell viability of ∼97.21%. The 80 μm × 80 μm patterns also showed an efficiency of 94.4%, with ∼5 cells per pattern. The probability of encapsulating a single-cell was ∼54.23% for the 80 μm × 80 μm micro-pattern; however, it decreased to 36.1% for the 250 μm × 250 μm 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 the 80 μm × 80 μm 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 the 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.