Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel).

Simultaneous fabrication of PDMS through-holes for three-dimensional microfluidic applications.

Here we describe a simple yet efficient approach to making through-holes in a bound polydimethylsiloxane membrane for use in 3D microfluidic applications. Localized tearing of an elastomeric membrane is achieved by ripping an elastomeric stamp that is bound to the membrane by posts at desired regions. The tears in the membrane are confined by the underlying channel architecture of the substrate to which the membrane is bound. By varying the membrane thickness and channel dimensions, holes of different sizes can be obtained. This high-throughput method of generating through-holes will enable the design of complex microfluidic devices that require crossing of channel networks.

Optical stereolithography of antifouling zwitterionic hydrogels.

This paper reports the rapid 3D printing of tough (toughness, UT, up to 141.6 kJ m-3), highly solvated (φwater∼ 60 v/o), and antifouling hybrid hydrogels for potential uses in biomedical, smart materials, and sensor applications, using a zwitterionic photochemistry compatible with stereolithography (SLA). A Design of Experiments (DOE) framework was used for systematically investigating the multivariate photochemistry of SLA generally and, specifically, to determine an aqueous SLA system with an additional zwitterionic acrylate, which significantly increases the gelation rate, and the resilience of the resulting hybrid hydrogels relative to an equivalent non-ionic polyacrylamide hydrogel. Specifically, the resulting zwitterionic hybrid hydrogels (Z-gels) can be tuned over a large range of ultimate strains, ca. 0.5 < γult < 5.0, and elastic moduli, ca. 10 < E < 1000 kPa, while also demonstrating a high resilience under cyclic tensile loading. Importantly, unlike traditional chemistry, increasing the elastic modulus of the Z-gels does not necessarily reduce the ultimate strain. Moreover, the Z-gels can be rapidly printed using a desktop commercial SLA 3D printer, with relatively low photoirradiation dosages of visible light (135 to 675 mJ cm-2 per 50-100 µm layer). Compared with the counterpart polyacrylamide hydrogels, the Z-gels have greater antifouling properties and exhibit 58.2% less absorption of bovine serum albumin.

Rapid Manufacturing of Thin Soft Pneumatic Actuators and Robots.

This protocol describes a method for rapid manufacturing of soft pneumatic actuators and robots with an ultrathin form factor using a heat press and a laser cutter machine. The method starts with the lamination of thermoplastic polyurethane (TPU) sheets using a heat press for 10 min at the temperature of ~93 °C. Next, the parameters of the laser cutter machine are optimized to produce a rectangular balloon with maximum burst pressure. Using the optimized parameters, the soft actuators are laser cut/welded three times sequentially. Next, a dispensing needle is attached to the actuator, allowing it to be inflated. The effect of geometrical parameters on the deflection of the actuator are studied systematically by varying the channel width and length. Finally, the performance of the actuator is characterized using an optical camera and a fluid dispenser. Conventional fabrication methods of soft pneumatic actuators based on silicone molding are time consuming (several hours). They also result in strong but bulky actuators, which limits the actuator's applications. Moreover, microfabrication of thin pneumatic actuators is both time-consuming and expensive. The proposed manufacturing method in the current work resolves these issues by introducing a fast, simple, and cost-effective fabrication method of ultrathin pneumatic actuators.

In Vitro Validation of a Numerical Simulation of Leaflet Kinematics in a Polymeric Aortic Valve Under Physiological Conditions.

This paper describes a computational method to simulate the non-linear structural deformation of a polymeric aortic valve under physiological conditions. Arbitrary Lagrangian-Eulerian method is incorporated in the fluid-structure interaction simulation, and then validated by comparing the predicted kinematics of the valve's leaflets to in vitro measurements on a custom-made polymeric aortic valve. The predicted kinematics of the valve's leaflets was in good agreement with the experimental results with a maximum error of 15% in a single cardiac cycle. The fluid-structure interaction model presented in this study can simulate structural behaviour of a stented valve with flexible leaflets, providing insight into the haemodynamic performance of a polymeric aortic valve.

Laser Cutting as a Rapid Method for Fabricating Thin Soft Pneumatic Actuators and Robots.

Pneumatically actuated soft robots address many challenges with interfacing with delicate objects, but these actuators/robots are still bulky and require many hours to fabricate, limiting their widespread use. This article reports a novel design and manufacturing method for ultrathin soft robots and actuators (∼70 µm) using a laser-cutting machine that cuts/welds sheets of thermoplastic polyurethane (TPU) from a 2D CAD drawing. Using this method, five different soft actuators (e.g., bending, rotating, contracting) are designed, fabricated, and characterized with both planar and nonplanar motions. Furthermore, we show how stacking multiple sheets of TPU enables rapid fabrication of multifunctional actuators. Finally, a portable four-arm swimming robot is designed and fabricated without any assembly steps. This rapid fabrication method enables soft robots to go from concept to operational within minutes, and creates a new subclass of soft robots suitable for applications requiring a robot to be ultrathin, lightweight, and/or fit within small volumes.

Patient-specific design of a soft occluder for the left atrial appendage.

3D printing has been used to create a wide variety of anatomical models and tools for procedural planning and training. Yet, the printing of permanent, soft endocardial implants remains challenging because of the need for haemocompatibility and durability of the printed materials. Here, we describe an approach for the rapid prototyping of patient-specific cardiovascular occluders via 3D printing and static moulding of inflatable silicone/polyurethane balloons derived from volume-rendered computed tomography scans. We demonstrate the use of the approach, which provides custom-made implants made of high-quality, durable and haemocompatible elastomeric materials, in the fabrication of devices for occlusion of the left atrial appendage-a structure known to be highly variable in geometry and the primary source of stroke for patients with atrial fibrillation. We describe the design workflow, fabrication and deployment of patient-specific left atrial appendage occluders and, as a proof-of-concept, show their efficacy using 3D-printed anatomical models, in vitro flow loops and an in vivo large animal model.

Advanced biomaterials and microengineering technologies to recapitulate the stepwise process of cancer metastasis.

Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.

Buckling of Elastomeric Beams Enables Actuation of Soft Machines.

Soft, pneumatic actuators that buckle when interior pressure is less than exterior provide a new mechanism of actuation. Upon application of negative pneumatic pressure, elastic beam elements in these actuators undergo reversible, cooperative collapse, and generate a rotational motion. These actuators are inexpensive to fabricate, lightweight, easy to control, and safe to operate. They can be used in devices that manipulate objects, locomote, or interact cooperatively with humans.

A paper-based invasion assay: assessing chemotaxis of cancer cells in gradients of oxygen.

This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.

Polymer-based mesh as supports for multi-layered 3D cell culture and assays.

Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system-Cells-in-Gels-in-Mesh (CiGiM)-that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells-layer-by-layer-within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis-(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format.

High-density fabrication of normally closed microfluidic valves by patterned deactivation of oxidized polydimethylsiloxane.

The use of polydimethylsiloxane (PDMS) in microfluidic devices is extensive in academic research. One of the most fundamental treatments is to expose PDMS to plasma oxidation in order to render its surface temporarily hydrophilic and capable of permanent bonding. Here, we show that changes in the surface chemistry induced by plasma oxidation can spatially be counteracted very cleanly and reliably in a scalable manner by subsequent microcontact printing of residual oligomers from a PDMS stamp. We characterize the surface modifications through contact angle, atomic force microscopy, X-ray photoelectron spectroscopy, and bond-strength measurements. We utilize this approach for negating the bonding of a flexible membrane layer within an elastomeric valve and demonstrate its effectiveness by integration of over one thousand normally closed elastomeric valves within a single substrate. In addition, we demonstrate that surface energy patterning can be used for "open microfluidic" applications that utilize spatial control of surface wetting.

Transwells with microstamped membranes produce micropatterned two-dimensional and three-dimensional co-cultures.

This article describes a simple and rapid cell patterning method to form co-culture microarrays in commercially available Transwells. A thin poly(dimethylsiloxane) (PDMS) layer is printed on the underside of a Transwell using a PDMS stamp. Arbitrary cellular patterns are generated according to the geometric features of the thin PDMS layer through hydrodynamic forces that guide cells onto the membrane only over the PDMS-uncoated regions. Micropatterns of surface-adhered cells (we refer to this as two-dimensional) or non-surface-adhered clusters of cells (we refer to this as three-dimensional) can be generated depending on the surface treatment of the filter membrane. Additionally, co-cultures can be established by introducing different types of cells on the membrane or in the bottom chamber of the Transwell. We show that this co-culture method can evaluate mouse embryonic stem (mES) cell differentiation based on heterogeneous cell-cell interactions. Co-culture of mES cells and HepG2 cells decreased SOX17 expression of mES cells, and direct cell-cell contact further decreased SOX17 expression, indicating that co-culture with HepG2 cells inhibits endoderm differentiation through soluble factors and cell-cell contact. This method is simple and user-friendly and should be broadly useful to study cell shapes and cell-cell interactions.