Paper-Based 3D Scaffold for Multiplexed Single Cell Secretomic Analysis.

Despite rapid progresses in single-cell analysis technologies, efforts to control the three-dimensional microenvironment for single cell measurements have been lacking. Here, we report a simple method to incorporate three-dimensional scaffolds, including polyvinylidene fluoride (PVDF) membranes and PVDF membrane replicated analog polydimethylsiloxane, into multiplexed single cell secretomic analysis platforms (including a microwell array and a single cell barcode microchip) to mimic the extracellular physical matrix and mechanical support for single cells. Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in three-dimensional (3D) microenvironments. This finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other three-dimensional cell culture scaffolds or other single cell assays and may become a broadly applicable three-dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.

Construction of a high-throughput aorta smooth muscle-on-a-chip for thoracic aortic aneurysm drug screening.

Thoracic aortic aneurysm (TAA), in which arteries enlarge asymptomatically over time until dissection or rupture occurs, is a serious health risk. The mainstay of TAA treatment remains surgical repair due to the lack of effective drugs. The complex etiology and pathogenesis of TAA, including hemodynamic alterations and genetic factors, lead to inaccuracies in preclinical models for drug screening. Previously, our group designed an aorta smooth muscle-on-a-chip to emulate human aorta physiology and pathophysiology and screened three promising therapeutic drugs targeting mitochondrial dynamics in TAA. On this foundation, we updated the one-channel chip to an eighteen-well chip platform with four polydimethylsiloxane layers. Benefiting from this high-throughput chip, we rapidly screened multiple drugs simultaneously using distinct cell lines in vitro. In addition, we observed the abnormal activation of hypoxia-inducible factor 1-alpha (HIF-1alpha) in aortas from TAA patients by Western blot and bioinformatics analyses. Intriguingly, this phenomenon was replicated only when smooth muscle cells (SMCs) were strained on the chip. We then screened seven specific HIF-1alpha inhibitors and selected the two most effective drugs (2-methoxyestradiol and digoxin) by quantitative PCR and colorimetric methods. The results demonstrated that these two drugs can improve respiratory chain function and rescue the SMC contractile phenotype, showing applicability for the clinical treatment of TAA. This high-throughput aorta smooth muscle-on-a-chip will become a potential preclinical model for TAA drug screening.

Rapid prototyping of PDMS microdevices viaµPLAT on nonplanar surfaces with flexible hollow-out mask.

A major goal of polydimethylsiloxane (PDMS) microfabrication is to develop a simple and inexpensive method for rapid fabrication. Despite the recent advancements in this field, facile PDMS microfabrication on non-planar surfaces remains elusive. Here we report a facile method for rapid prototyping of PDMS microdevices viaµPLAT (microscale plasma-activated templating) on non-planar surfaces through micropatterning of hydrophilic/hydrophobic (HL/HB) interface by flexible polyvinyl chloride (PVC) hollow-out mask. This mask can be easily prepared with flexible PVC film through a cutting crafter and applied as pattern definer during the plasma treatment for microscale HL/HB interface formation on different substrates. The whole process requires low inputs in terms of time as well as toxic chemicals. Inspired by liquid molding, we demonstrated its use for rapid prototyping of PDMS microstructures. Following the proof-of-concept study, we also demonstrated the use of the flexible hollow-out mask to facilitate cell patterning on curved substrates, which is difficult to realize with conventional methods. Collectively, our work utilizes flexible and foldable PVC film as mask materials for facile microscale HL non-planar surface modification to establish a useful tool for PDMS prototyping and cell patterning.

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution.

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.