Gas-modulating microcapsules for spatiotemporal control of hypoxia.

Oxygen is a vital molecule involved in regulating development, homeostasis, and disease. The oxygen levels in tissue vary from 1 to 14% with deviations from homeostasis impacting regulation of various physiological processes. In this work, we developed an approach to encapsulate enzymes at high loading capacity, which precisely controls the oxygen content in cell culture. Here, a single microcapsule is able to locally perturb the oxygen balance, and varying the concentration and distribution of matrix-embedded microcapsules provides spatiotemporal control. We demonstrate attenuation of hypoxia signaling in populations of stem cells, cancer cells, endothelial cells, cancer spheroids, and intestinal organoids. Varying capsule placement, media formulation, and timing of replenishment yields tunable oxygen gradients, with concurrent spatial growth and morphogenesis in a single well. Capsule containing hydrogel films applied to chick chorioallantoic membranes encourages neovascularization, providing scope for topical treatments or hydrogel wound dressings. This platform can be used in a variety of formats, including deposition in hydrogels, as granular solids for 3D bioprinting, and as injectable biomaterials. Overall, this platform's simplicity and flexibility will prove useful for fundamental studies of oxygen-mediated processes in virtually any in vitro or in vivo format, with scope for inclusion in biomedical materials for treating injury or disease.

Biomimetic Vasculatures by 3D-Printed Porous Molds.

Despite recent advances in biofabrication, recapitulating complex architectures of cell-laden vascular constructs remains challenging. To date, biofabricated vascular models have not yet realized four fundamental attributes of native vasculatures simultaneously: freestanding, branching, multilayered, and perfusable. In this work, a microfluidics-enabled molding technique combined with coaxial bioprinting to fabricate anatomically relevant, cell-laden vascular models consisting of hydrogels is developed. By using 3D porous molds of poly(ethylene glycol) diacrylate as casting templates that gradually release calcium ions as a crosslinking agent, freestanding, and perfusable vascular constructs of complex geometries are fabricated. The bioinks can be tailored to improve the compatibility with specific vascular cells and to tune the mechanical modulus mimicking native blood vessels. Crucially, the integration of relevant vascular cells (such as smooth muscle cells and endothelial cells) in a multilayer and biomimetic configuration is highlighted. It is also demonstrated that the fabricated freestanding vessels are amenable for testing percutaneous coronary interventions (i.e., drug-eluting balloons and stents) under physiological mechanical states such as stretching and bending. Overall, a versatile fabrication technique with multifaceted possibilities of generating biomimetic vascular models that can benefit future research in mechanistic understanding of cardiovascular diseases and the development of therapeutic interventions is introduced.

Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing.

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications.

Wound healing properties of magnesium mineralized antimicrobial nanofibre dressings containing chondroitin sulphate - a comparison between blend and core-shell nanofibres.

The development of antimicrobial nanofibre dressings that can protect the injured tissues from commensal pathogens while promoting tissue regeneration finds enormous potential in plastic and reconstructive surgery practices. To achieve this goal, we investigated the effect of chondroitin sulphate on the morphology, mechanical properties, wettability and biocompatibility of polydopamine crosslinked electrospun gelatin nanofibres containing mineralized magnesium. To extend the durability of dressings, we prepared composite dressings containing polycaprolactone (PCL) and gelatin as blend or core-shell nanofibres. Nanofibre blends presented greater tensile strength and stretchability, while core-shell nanofibres displayed superior photoluminescent properties. In a porcine model of cutaneous burn injury, both the blend and core-shell nanofibre dressings displayed improved re-epithelialization, wound closure and clinical outcome in comparison to untreated burns. Histology of the biopsied tissues indicated smooth regeneration and collagen organization of the burns treated with core-shell nanostructures than untreated burns. This study compared the physico-chemical and biological properties of composite nanofibres that are capable of accelerating burn wound healing and possess antimicrobial properties, highlighting their potential as wound dressings and skin substitutes.

A gel-free 3D microfluidic cell culture system.

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

Microfabricated silicon nitride membranes for hepatocyte sandwich culture.

We have developed a hepatocyte sandwich culture with improved mass transport properties based on ultra-thin microfabricated porous silicon nitride (Si(3)N(4)) membranes. The dimensions and uniformity of the membrane pores can be configurable, which confers more control over the mass transport. Instead of collagen gels used in conventional sandwich culture, we utilized galactose ligands immobilized on the Si(3)N(4) membranes to support hepatocyte attachment and function in the sandwich culture. Diffusion studies using FITC-dextrans confirmed that mass transport of the microfabricated Si(3)N(4) membrane based sandwich was significantly better than conventional collagen gel sandwich and can be configured by varying the porosity of the Si(3)N(4) membrane. Hepatocytes cultured in the microfabricated Si(3)N(4) membrane based sandwich culture exhibited earlier apical repolarization and biliary excretion, improved differentiated functions and enhanced drug sensitivity compared to hepatocytes cultured in a collagen gel sandwich. The Si(3)N(4) membrane based sandwich culture allows for a systematic optimization of the mass transport properties of hepatocyte culture by changing the pore size and inter-pore distance. This will enable more effective drug testing applications where optimal mass transport is required for hepatocyte function maintenance and drug accessibility.

Application of a polyelectrolyte complex coacervation method to improve seeding efficiency of bone marrow stromal cells in a 3D culture system.

High seeding efficiency with homogenous distribution of limited cell sources such as bone marrow stromal cells (BMSCs) are of clinical relevance in scaffold-based tissue engineering. Therefore, considerable research efforts have been invested to ameliorate the seeding efficiency in 3D scaffolds. Preliminary data demonstrated that indeed BMSCs were viable and were able to proliferate in a model 3D scaffold, i.e. Cytomatrix scaffold. However, the eventual practical application of BMSCs in such 3D scaffolds is limited by the low seeding efficiency of the cells within the scaffold. Here, we demonstrated that the cell seeding efficiency of BMSCs in the Cytomatrix scaffold can be improved significantly (t-test, p<0.05) by means of macroencapsulating the scaffold via the complex coacervation of a methylated collagen and terpolymer. The thickness and density of the polyeletrolyte complex can be modulated by the contact time between the methylated collagen and terpolymer to balance between cell entrapment efficacy and mass transfer impedance imparted by the complex. Porcine BMSCs were macroencapsulated in Cytomatrix scaffolds using various polyelectrolyte contact time and cultured under both static and dynamic conditions. Throughout the range of contact time investigated, macroencapsulation did not affect the viability of the porcine BMSCs in dynamic culture. However, the viability of the cells under static cultures was compromised with longer polyelectrolyte contact time. Therefore, this proposed method of macroencapsulation enables customization to achieve enhanced seeding efficiency without mass transfer impedance for different culture configurations.

Scalable alignment of three-dimensional cellular constructs in a microfluidic chip.

There have been considerable efforts to engineer three-dimensional (3D) microfluidic environments to enhance cellular function over conventional two-dimensional (2D) cultures in microfluidic chips, but few involve topographical features, such as micro/nano-grooves, which are beneficial for cell types of cardiac, skeletal and neuronal lineages. Here we have developed a cost-effective and scalable method to incorporate micro-topographical cues into microfluidic chips to induce cell alignment. Using commercially available optical media as molds for replica molding, we produced large surface areas of polydimethylsiloxane (PDMS) micro-grooved substrates and plasma-bonded them to multiple microfluidic chips. Besides aligning a 2D monolayer of cells, the micro-grooved substrate can align 3D cellular constructs on chip. C2C12 mouse myoblasts were cultured three-dimensionally in a microfluidic chip with incorporated PDMS micro-grooved substrate remodeled into an aligned 3D cellular construct, where the actin cytoskeleton and nuclei were preferentially oriented along the micro-grooves. Cells within the 3D cellular constructs can align without being in direct contact with the micro-grooves due to synergism between topography and fluid shear stress. Aligned C2C12 3D cellular constructs showed enhanced differentiation into skeletal muscles as compared to randomly aligned ones. This novel method enables the routine inclusion of micro-topographical cues into 2D or 3D microfluidic cultures to generate relevant physiological models for studying tissue morphogenesis and drug screening applications.

Scalable cell alignment on optical media substrates.

Cell alignment by underlying topographical cues has been shown to affect important biological processes such as differentiation and functional maturation in vitro. However, the routine use of cell culture substrates with micro- or nano-topographies, such as grooves, is currently hampered by the high cost and specialized facilities required to produce these substrates. Here we present cost-effective commercially available optical media as substrates for aligning cells in culture. These optical media, including CD-R, DVD-R and optical grating, allow different cell types to attach and grow well on them. The physical dimension of the grooves in these optical media allowed cells to be aligned in confluent cell culture with maximal cell-cell interaction and these cell alignment affect the morphology and differentiation of cardiac (H9C2), skeletal muscle (C2C12) and neuronal (PC12) cell lines. The optical media is amenable to various chemical modifications with fibronectin, laminin and gelatin for culturing different cell types. These low-cost commercially available optical media can serve as scalable substrates for research or drug safety screening applications in industry scales.