Andrew D. Dias

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.

Single-step laser-based fabrication and patterning of cell-encapsulated alginate microbeads

Alginate can be used to encapsulate mammalian cells and for the slow release of small molecules. Packaging alginate as microbead structures allows customizable delivery for tissue engineering, drug release, or contrast agents for imaging. However, state-of-the-art microbead fabrication has a limited range in achievable bead sizes, and poor control over bead placement, which may be desired to localize cellular signaling or delivery. Herein, we present a novel, laser-based method for single-step fabrication and precise planar placement of alginate microbeads. Our results show that bead size is controllable within 8%, and fabricated microbeads can remain immobilized within 2% of their target placement. Demonstration of this technique using human breast cancer cells shows that cells encapsulated within these microbeads survive at a rate of 89.6%, decreasing to 84.3% after five days in culture. Infusing rhodamine dye into microbeads prior to fluorescent microscopy shows their 3D spheroidal geometry and the ability to sequester small molecules. Microbead fabrication and patterning is compatible with conventional cellular transfer and patterning by laser direct-write, allowing location-based cellular studies. While this method can also be used to fabricate microbeads en masse for collection, the greatest value to tissue engineering and drug delivery studies and applications lies in the pattern registry of printed microbeads.

Generating size-controlled embryoid bodies using laser direct-write

Embryonic stem cells (ESCs) have the potential to self-renew and differentiate into any specialized cell type. One common method to differentiate ESCs in vitro is through embryoid bodies (EBs), three-dimensional cellular aggregates that spontaneously self-assemble and generally express markers for the three germ layers, endoderm, ectoderm, and mesoderm. It has been previously shown that both EB size and 2D colony size each influence differentiation. We hypothesized that we could control the size of the EB formed by mouse ESCs (mESCs) by using a cell printing method, laser direct-write (LDW), to control both the size of the initial printed colony and the local cell density in printed colonies. After printing mESCs at various printed colony sizes and printing densities, two-way ANOVAs indicated that the EB diameter was influenced by printing density after three days (p = 0.0002), while there was no effect of the printed colony diameter on the EB diameter at the same timepoint (p = 0.74). There was no significant interaction between these two factors. Tukeys honestly significant difference test showed that high-density colonies formed significantly larger EBs, suggesting that printed mESCs quickly aggregate with nearby cells. Thus, EBs can be engineered to a desired size by controlling printing density, which will influence the design of future differentiation studies. Herein, we highlight the capacity of LDW to control the local cell density and colony size independently, at prescribed spatial locations, potentially leading to better stem cell maintenance and directed differentiation.

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.

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 LDWs 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.

3D Bioprinting and 3D Imaging for Stem Cell Engineering

Three-dimensional (3D) bio-printing, a technology to create 3D tissue through layer-by-layer approach, offers great capacity to engineer tissue with desired cells, growth factors and biomaterial scaffolds in spatial patterns to mimic the native tissue architecture. With its flexibility and power, the 3D bio-printing technology can also be used to control stem cell fate and creating 3D stem cell niches. Meanwhile, 3D bio-printed tissues often incorporate thick opaque scaffold, dense population of cells, and are often large in size (1–100 mm). Thus, there are significant difficulties in visualizing the biological events within thick tissue constructs using current microscopic techniques. To elucidate the interaction of stem cells with the microenvironment in tissue engineering applications, it is necessary to develop novel molecular imaging techniques to non-invasively observe stem cell fate, cell-cell interactions, and structural features of an engineered tissue in real time. In this chapter, we review the usage of bio-printing technologies in stem cell and tissue engineering application, and the most recent development in the optical molecular imaging techniques for thick tissue imaging.

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.

Engineering 2D and 3D Cellular Microenvironments Using Laser Direct Write

Advances in biology and medicine have created a number of applications that demand greater control over the features that comprise the cellular microenvironment. In areas such as tissue engineering, in vitro diagnostics, and directed stem cell differentiation, it is important to manipulate specific microenvironmental features using engineering approaches to influence cell–material (cell–substrate) and cell–cell interactions. One way by which this can be achieved is using a high-resolution, spatially precise cell printing technique that is compatible with engineered materials. A patterning approach of particular interest is laser direct-write (LDW), a forward transfer technique that has been further developed to print biological materials and viable cells with high spatial precision in customizable patterns in 2D, and more recently in 3D microenvironments. The flexibility of LDW (2D and/or 3D) combined with substrates of controllable features (e.g. hydrogels, electrospun fibers) grants unprecedented control in microenvironment fabrication, allowing novel applications and fundamental research questions to be explored.

Laser direct-write patterning influences early embryonic stem cell differentiation

An important objective in stem cell research is controlling differentiation of pluripotent stem cells to a desired fate. Previous research in this area has focused on directing differentiation by manipulating morphogens and substrate material/mechanics. However, cell-cell signaling, whether by contact or paracrine signaling, also influences differentiation. One promising direction by which cell-cell signaling can be manipulated is through cellular patterning. Using mouse embryonic stem cells (mESCs) as a model system, this study investigates patterning mESCs in colonies of controlled size and spacing, to examine the effect of patterning on differentiation. Laser direct-write was used to pattern mESCs in an array of small colonies, and cells were permitted to spontaneously differentiate for five days. Expression levels of seven select genes were compared to those of randomly seeded mESCs, and mESCs from conventional hanging drop culture. Analysis of variance showed significant differences in some genes examined, including mesoderm and ectoderm markers, indicating that the initial spatial arrangement of cells influences differentiation of pluripotent stem cells. A multivariate linear discriminant analysis was used to classify input populations, and suggested how genes may be affected by spatial patterning.

High-Throughput, Laser-based Alginate Microbead Fabrication and Patterning

Laser direct-write (LDW) is a biofabrication technique that was developed to produce cell patterns of high spatial resolution. We present an adaptation to this technique to rapidly create alginate microbeads in large, precise arrays, with and without encapsulated cells. It is further demonstrated that we maintain a high degree of spatial control in these large fabricated patterns. Rapid and precisely alginate microbead deposition on a planar surface, enabled by LDW, cannot be accomplished by other techniques. The spatial precision of this LDW will enable bottom-up tissue engineering fabrication and large biological studies based on the placement of cell-encapsulated microbeads.