Microfluidics-based preparation methods for cell-laden hydrogel microspheres are well-suited for large-scale comparative analysis of single or few cells. However, in existing studies, the preparation of cell-laden hydrogel microspheres and the cell culture process are typically separated, requiring the fabricated microspheres to be eluted and transferred from the preparation device to cell culture dishes or plates for cultivation. This transfer process can easily compromise sterility, while conventional cell culture methods consume more reagents and cause microsphere stacking, hindering single-cell observation and analysis. To address these issues, this paper presents an integrated microfluidic chip that sequentially enables droplet generation with cell encapsulation, gel droplet solidification, hydrogel microsphere trapping, and microsphere-based cell culture and analysis, facilitating the cultivation and observation of single or small numbers of cells. Integrating cell-laden microsphere preparation and 3D cell culture within a sealed chip structure reduces contamination risks associated with cell transfer, enables automation of multiple cell analysis workflows, and minimizes reagent and sample consumption. Using polydimethylsiloxane (PDMS) with good gas permeability and processability as the chip material, biocompatible fluorinated oil was selected as the oil phase for microsphere preparation. A mild sodium alginate-calcium ion gelation system was employed, where calcium ions were released under acidic conditions after droplet generation to trigger solidification, yielding uniform hydrogel microspheres. Under optimized conditions, the single-cell encapsulation efficiency for test samples of human myeloid leukemia cells (K562) was 33.8% ± 1.8%, with a size uniformity coefficient of variation (CV) reaching 3.85%. Cells encapsulated within hydrogel microspheres were cultured in 286 on-chip independent cell culture chambers, achieving >95% viability after 24 h.

Membrane composition is a critical factor for electroporation. Although existing research focuses on pore formation driven by local phospholipid headgroup clusters, the large-scale membrane dynamics post-pore formation remain underexplored. In this study, coarse-grained (CG) molecular dynamics (MD) simulations were employed to investigate the effects of phospholipid headgroups (PC, PE and PS), tail characteristics (DLPC, DPPC, POPC and DOPC) and cholesterol (CHOL) content (0 mol%-50 mol%) on membrane electroporation. The results demonstrate that membrane structural parameters, such as area per lipid, hydrophobic layer thickness and interfacial water penetration depth, significantly influence the electroporation threshold electric field and transmembrane substance flux. Phospholipid headgroups can modulate the area per lipid and hydrophobic layer thickness through their size, hydrogen bonding and charge. Regarding phospholipid tails, increasing their length and unsaturation strengthens the hydrophobic layer, and thereby inhibits electroporation. The incorporation of CHOL into the membrane leads to tighter lipid packing, increased layer thickness and restricted water penetration, all of which elevate the threshold electric field. After electroporation, CHOL reduces transmembrane flux by enhancing line tension, although this inhibitory effect is limited at higher concentrations. The simulation results align well with existing experimental data, suggesting our approach can guide protocol design and highlight membrane composition's critical role in electroporation efficiency.