Enhancing metabolic activity and differentiation potential in adipose mesenchymal stem cells via high-resolution surface-acoustic-wave contactless patterning

Abstract

Acoustofluidics has shown great potential for label-free bioparticle patterning with excellent biocompatibility. Acoustofluidic patterning enables the induction of cell-cell interactions, which play fundamental roles in organogenesis and tissue development. One of the current challenges in tissue engineering is not only the control of the spatial arrangement of cells but also the preservation of cell patterns over time. In this work, we developed a standing surface acoustic wave-based platform and demonstrated its capability for the well-controlled and rapid cell patterning of adipose-derived mesenchymal stem cells in a high-density homogenous collagen hydrogel. This biocompatible hydrogel is easily UV crosslinked and can be retrieved within 3 min. Acoustic waves successfully guided the cells toward pressure nodal lines, creating a contactless alignment of cells in <5 s in culture media and <1 min in the hydrogel. The acoustically patterned cells in the hydrogel did not show a decrease in cell viability (>90%) 48 h after acoustic induction. Moreover, 45.53% and 30.85% increases in metabolic activity were observed in growth and differentiation media, respectively, on Day 7. On Day 14, a 32.03% change in metabolic activity was observed using growth media, and no significant difference was observed using differentiation media. The alkaline phosphatase activity showed an increase of 80.89% and 24.90% on Days 7 and 14, respectively, for the acoustically patterned cells in the hydrogel. These results confirm the preservation of cellular viability and improved cellular functionality using the proposed high-resolution acoustic patterning technique and introduce unique opportunities for the application of stem cell regenerative patches for the emerging field of tissue engineering.

Keywords: Engineering; Materials science.

Fig. 1. SSAW-patterning platform and workflow of the patterning mechanism and retrievability.

a Schematic of the SSAW platform. b Cell-hydrogel solution is seeded into the platform. c SSAW waves are applied to the IDTs via a function generator to create cell patterns. d UV light is applied to the patterned cells in the hydrogel to crosslink the scaffold. e Brief incubation of the UV-crosslinked cell-hydrogel scaffold is conducted to complete the gelation of the cell-patch. f Cell-patch is retrieved from the platform for potential implantation. g Cell analysis is performed on Days 1, 7, and 14

Fig. 2. Optimization experimental parameters for cell patterning.

a Cells prior SSAW activation and after SSAW activation with kymograph image showing nodal line distribution and distance. b Velocity vs voltage plot. c Velocity distribution of cells in culture media. Scale Bar is 500 μm

Fig. 3. Cell patterning sample optimization

a Retrievability of the cell-hydrogel construct after 3 min of UV exposure. b Velocity distribution of different cell suspension conditions. c Cell patterning for various cell densities (I) 2.5 × 106, (II) 1.3 × 106, and (III) 0.6 × 106 cells/mL showing defined patterned lines. Scale bar is 250 μm

Fig. 4. Cell viability and metabolic activity for patterned and non-patterned cells.

a Cell viability for patterned ASCs and MC3T3-E1 cells with and without PhotoCol^® for up to 48 h after SSAW induction (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). b Live/Dead assay showing high cell viability after (I) at time zero and (II) after 48 h of SSAW induction. c Acoustically patterned ASCs showing aligned nuclei (Hoechst 33342) and actin (Phalloidin-iFluor 594) fibers after 1 week in growth media. d ASC cell–cell structure alignment of non-patterned (I) and patterned (II) cells in PhotoCol^® on Day 14. e Metabolic activity of acoustically patterned and non-patterned ASCs cultured in growth media and differentiation media. Scale bar is 250 μm

Fig. 5. Osteogenic differentiation potential of acoustically patterned cells.

a ALP activity for non-patterned and patterned cells encapsulated in PhotoCol^®-LAP hydrogel on Days 1, 7 and 14 (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). b Confocal images of non-patterned (control) cells (I) and acoustically patterned cells (II) with an equal density of 2.5 × 106 cells/mL after 14 days of culture in differentiation media, where osteocalcin is shown in red and nuclei in blue. c Osteocalcin percentage difference between the acoustically patterned and non-patterned groups. Confocal images were analyzed using ImageJ to quantify the ratio of the osteocalcin signal area percentage to the total stained area (n = 3; ****p < 0.0001). Scale bar is 250 μm