Acoustofluidic Interfaces for the Mechanobiological Secretome of MSCs

Figure: a Schematic of the formation of MSC aggregates via acoustofluidic assembly and N-cadherin mediated cell–cell interactions to enhance the MSC secretome. b The simulation results showed that a three-dimensional circular vortex tube generated by a high-intensity focal point at the center of the substrate functioned as a virtual wall, trapping and aggregating cells at the center of the droplet. c Illustration of the comparison between 2D monolayer MSC culture and acoustofluidic 3D MSC aggregation. d Fluorescence images of MSCs before/after the acoustofluidic assembly, and the morphology of MSC aggregates after 3 days of incubation. The green color indicates alive cells. The blue color indicates cell nuclei, and the red color indicates the cytoskeleton. n = 4 tests with similar results. Scale bar: 100 μm. e CyQUANT™ Cell Proliferation Assay measuring the DNA contents of different groups. The statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test. Data are graphed as the mean ± SD (n = 4, biological repeats). Source data are provided as a Source Data file.
While mesenchymal stem cells (MSCs) have gained enormous attention due to their unique properties of self-renewal, colony formation, and differentiation potential, the MSC secretome has become attractive due to its roles in immunomodulation, anti-inflammatory activity, angiogenesis, and anti-apoptosis. However, the precise stimulation and efficient production of the MSC secretome for therapeutic applications are challenging problems to solve. Here, we report on Acoustofluidic Interfaces for the Mechanobiological Secretome of MSCs: AIMS. We create an acoustofluidic mechanobiological environment to form reproducible three-dimensional MSC aggregates, which produce the MSC secretome with high efficiency. We confirm the increased MSC secretome is due to improved cell-cell interactions using AIMS: the key mediator N-cadherin was up-regulated while functional blocking of N-cadherin resulted in no enhancement of the secretome. After being primed by IFN-γ, the secretome profile of the MSC aggregates contains more anti-inflammatory cytokines and can be used to inhibit the pro-inflammatory response of M1 phenotype macrophages, suppress T cell activation, and support B cell functions. As such, the MSC secretome can be modified for personalized secretome-based therapies. AIMS acts as a powerful tool for improving the MSC secretome and precisely tuning the secretory profile to develop new treatments in translational medicine.
Acoustothermal transfection for cell therapy

Figure: Acoustothermal transfection for enhancing cell membrane and nuclear envelope permeabilities.
(A) Schematics for illustrating the mechanism of acoustothermal transfection showing our developed acoustothermal transfection device with 6 × 8 units for high-throughput transfection. Each unit consists of IDTs for generating SAWs, a PDMS well for hosting cells, and a 1-mm-thick PDMS layer on top of the IDTs for delivering acoustic energy to cells and enabling local temperature increases through viscous damping. The combination of SAW-induced acoustic and thermal effects can enhance the transient permeabilities of the cell membrane and nuclear envelope to achieve efficient transfection of hard-to-transfect cells. (B) Time-sequential fluorescence images showing the cell membrane permeability change induced by 10 s of acoustothermal treatment. Propidium iodide (PI; red) and calcein-AM (green) are for characterizing the cell permeability changes and live MCF-7 cells. (C) Time history plots of mean PI and calcein-AM fluorescence intensities were obtained by extracting the intensities from fluorescence images in (B). Twenty cells’ intensity changes were analyzed. The fluorescence intensity was normalized by the control group without acoustothermal treatment. a.u., arbitrary units. (D) Fluorescence images of CHMP4B-Cy5 foci (red) for validating the nuclear envelope permeability changes induced by 10 s of acoustothermal transfection. (E) The number of CHMP4B-Cy5 foci per cell for characterizing nuclear envelope rupture and resealing over time. Fluorescence areas larger than 5 μm2 with a gray level higher than 30 were counted as CHMP4B-Cy5 foci. Ten cells were counted for the CHMP4B foci statistical analysis. (F) Confocal microscopy images showing acoustothermal transfection enabled the delivery of CdTe quantum dots (red) into nuclei. (G) Fluorescence intensity changes over time for characterizing the accumulation of CdTe quantum dots in nuclei [blue: 4′,6-diamidino-2-phenylindole (DAPI)]. Ten cells’ intensity changes were presented. For all the statistical analysis, the experiment was repeated at least three times.
Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face a fundamental trade-off between transfection efficiency and cell viability; achieving both simultaneously remains a substantial challenge. This study presents an acoustothermal transfection method that leverages acoustic and thermal effects on cells to enhance the permeability of both the cell membrane and nuclear envelope to achieve safe, efficient, and high-throughput transfection of primary T cells and stem cells. With this method, two types of plasmids were simultaneously delivered into the nuclei of mesenchymal stem cells (MSCs) with efficiencies of 89.6 ± 1.2%. CXCR4-transfected MSCs could efficiently target cerebral ischemia sites in vivo and reduce the infarct volume in mice. Our acoustothermal transfection method addresses a key bottleneck in balancing the transfection efficiency and cell viability, which can become a powerful tool in the future for cellular and gene therapies.