Microcapsules and 3D customizable shelled microenvironments from laser direct-written microbeads.

Microcapsules are shelled 3D microenvironments, with a liquid core. These core-shelled structures enable cell-cell contact, cellular proliferation and aggregation within the capsule, and can be utilized for controlled release of encapsulated contents. Traditional microcapsule fabrication methods provide limited control of capsule size, and are unable to control capsule placement. To overcome these limitations, we demonstrate size and spatial control of poly-l-lysine and chitosan microcapsules, using laser direct-write (LDW) printing, and subsequent processing, of alginate microbeads. Additionally, microbeads were used as volume pixels (voxels) to form continuous 3D hydrogel structures, which were processed like capsules, to form custom shelled aqueous-core 3D structures of prescribed geometry; such as strands, rings, and bifurcations. Heterogeneous structures were also created with controlled initial locations of different cell types, to demonstrate the ability to prescribe cell signaling (heterotypic and homotypic) in co-culture conditions. Herein, we demonstrate LDW's ability to fabricate intricate 3D structures, essentially with "printed macroporosity," and to precisely control structural composition by bottom-up fabrication in a bead-by-bead manner. The structural and compositional control afforded by this process enables the creation of a wide range of new constructs, with many potential applications in tissue engineering and regenerative medicine. Biotechnol. Bioeng. 2016;113: 2264-2274. © 2016 Wiley Periodicals, Inc.

Synthetic, Chemically Defined Polymer-Coated Microcarriers for the Expansion of Human Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC), also called marrow stromal cells, are adult cells that have attracted interest for their potential uses in therapeutic applications. There is a pressing need for scalable culture systems due to the large number of cells needed for clinical treatments. Here, a tailorable thin polymer coating-poly(poly(ethylene glycol) methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) [P(PEGMEMA-r-VDM-r-GMA); PVG]-to the surface of commercially available polystyrene and glass microcarriers to create chemically defined surfaces for large-scale cell expansion is applied. These chemically defined microcarriers create a reproducible surface that does not rely on the adsorption of xenogenic serum proteins to mediate cell adhesion. Specifically, this coating method anchors PVG copolymer through ring opening nucleophilic attack by amine residues on poly-l-lysine that is pre-adsorbed to the surface of microcarriers. Importantly, this anchoring reaction preserves the monomer VDM reactivity for subsequent functionalization with an integrin-specific Arg-Gly-Asp peptide to enable cell adhesion and expansion via a one-step reaction in aqueous media. MSCs cultured on PVG-coated microcarriers achieve sixfold expansion-similar to the expansion achieved on PS microcarriers-and retain their ability to differentiate after harvesting.

Microcarriers with Synthetic Hydrogel Surfaces for Stem Cell Expansion.

Microcarriers are scalable support surfaces for cell growth that enable high levels of expansion, and are particularly relevant for expansion of human mesenchymal stem cells (hMSCs). The goal of this study is to develop a poly(ethylene glycol) (PEG)-based microcarrier coating for hMSC expansion. Commercially available microcarriers do not offer customizability of microcarrier surface properties, including elastic modulus and surface cell adhesion ligands. The lab has previously demonstrated that tuning these material properties on PEG-based hydrogels can modulate important cellular growth characteristics, such as cell attachment and expansion, which are important in microcarrier-based culture. Eosin-Y is adsorbed to polystyrene microcarriers and used as a photoinitiator for thiol-ene polymerization under visible light. Resultant PEG coatings are over 100 µm thick and localized to microcarrier surfaces. This thickness is relevant for cells to react to mechanical properties of the hydrogel coating, and coated microcarriers support hMSC attachment and expansion. hMSC expansion is highly favorable on coated microcarriers in serum-free media, with doubling times under 25 h in the growth phase, and retained osteogenic and adipogenic differentiation capacity after culture on microcarriers. These microcarriers with defined, synthetic coatings enable tailorable surfaces for cell expansion that may be suitable for a variety of biomanufacturing applications.

Stem cell bioprinting for applications in regenerative medicine.

Many regenerative medicine applications seek to harness the biologic power of stem cells in architecturally complex scaffolds or microenvironments. Traditional tissue engineering methods cannot create such intricate structures, nor can they precisely control cellular position or spatial distribution. These limitations have spurred advances in the field of bioprinting, aimed to satisfy these structural and compositional demands. Bioprinting can be defined as the programmed deposition of cells or other biologics, often with accompanying biomaterials. In this concise review, we focus on recent advances in stem cell bioprinting, including performance, utility, and applications in regenerative medicine. More specifically, this review explores the capability of bioprinting to direct stem cell fate, engineer tissue(s), and create functional vascular networks. Furthermore, the unique challenges and concerns related to bioprinting living stem cells, such as viability and maintaining multi- or pluripotency, are discussed. The regenerative capacity of stem cells, when combined with the structural/compositional control afforded by bioprinting, provides a unique and powerful tool to address the complex demands of tissue engineering and regenerative medicine applications.

Recent advances in bioprinting and applications for biosensing.

Future biosensing applications will require high performance, including real-time monitoring of physiological events, incorporation of biosensors into feedback-based devices, detection of toxins, and advanced diagnostics. Such functionality will necessitate biosensors with increased sensitivity, specificity, and throughput, as well as the ability to simultaneously detect multiple analytes. While these demands have yet to be fully realized, recent advances in biofabrication may allow sensors to achieve the high spatial sensitivity required, and bring us closer to achieving devices with these capabilities. To this end, we review recent advances in biofabrication techniques that may enable cutting-edge biosensors. In particular, we focus on bioprinting techniques (e.g., microcontact printing, inkjet printing, and laser direct-write) that may prove pivotal to biosensor fabrication and scaling. Recent biosensors have employed these fabrication techniques with success, and further development may enable higher performance, including multiplexing multiple analytes or cell types within a single biosensor. We also review recent advances in 3D bioprinting, and explore their potential to create biosensors with live cells encapsulated in 3D microenvironments. Such advances in biofabrication will expand biosensor utility and availability, with impact realized in many interdisciplinary fields, as well as in the clinic.