Brian M. Gillette

In situ collagen assembly for integrating microfabricated three-dimensional cell-seeded matrices

Microscale fabrication of three-dimensional (3D) extracellular matrices (ECMs) can be used to mimic the often inhomogeneous and anisotropic properties of native tissues and to construct in vitro cellular microenvironments. Cellular contraction of fibrous natural ECMs (such as fibrin and collagen I) can detach matrices from their surroundings and destroy intended geometry. Here, we demonstrate in situ collagen fibre assembly (the nucleation and growth of new collagen fibres from preformed collagen fibres at an interface) to anchor together multiple phases of cell-seeded 3D hydrogel-based matrices against cellular contractile forces. We apply this technique to stably interface multiple microfabricated 3D natural matrices (containing collagen I, Matrigel, fibrin or alginate); each phase can be seeded with cells and designed to permit cell spreading. With collagen-fibre-mediated interfacing, microfabricated 3D matrices maintain stable interfaces (the individual phases do not separate from each other) over long-term culture (at least 3 weeks) and support spatially restricted development of multicellular structures within designed patterns. The technique enables construction of well-defined and stable patterns of a variety of 3D ECMs formed by diverse mechanisms (including temperature-, ion- and enzyme-mediated crosslinking), and presents a simple approach to interface multiple 3D matrices for biological studies and tissue engineering.

Dynamic Hydrogels: Switching of 3D Microenvironments Using Two-Component Naturally Derived Extracellular Matrices

2010 WILEY-VCH Verlag Gm In native tissues, cells respond to changes in local threedimensional (3D) microenvironments during diverse processes such as tissue morphogenesis, wound healing and cancer progression. A number of methods have been developed to dynamically control cell spreading and migration on 2D surfaces. Few techniques, however, are available to dynamically modify the structural properties of natural 3D extracellular matrix (ECM), after the initial properties of the matrix are established. Recently, a technique has also been developed to spatially and temporally control cell spreading in 3D synthetic ECM materials after gelling, using ultraviolet radiation-induced degradation of synthetic polymers. In many instances, however, cellular behavior follows native processes more closely inside 3D naturally derived ECM, which contain native ligands for cell adhesion and signaling, than in synthetic polymers. This paper describes a method to dynamically and reversibly modify the structure of a 3D ECM, in order to permit temporal switching of properties such as rate of solute transport and cell spreading and mobility. Rather than using ECM consisting primarily of a single component that either permits or restricts cell spreading and migration, we hypothesized that the structural properties of a two-component ECM – in which one component acts as a stable structural element and another component gels or dissolves – could be dynamically modified (Fig. 1A). Such a concept could allow reversible switching of 3D microenvironments in a structurally solid gel, made of naturally derived ECM. Specifically, we explored the use of collagen I, a fibrous and cell-adhesive matrix that has a microporous structure (on the order of 1 to 20 micrometers), and alginate, a polysaccharide that lacks cell adhesion sites and has a nanoporous structure (on the orders of tens of nanometers). The state of crosslinking of the alginate component can be controlled by adding or removing divalent cations (Ca2þ, Zn2þ, Co2þ, Ba2þ, Ni2þ, Mn2þ, Pb2þ, Cu2þ, Cd2þ, Sr2þ), with Ca2þ being the most commonly used. In this manner, we sought to control the structure of the two-component 3D ECM by switching the state of alginate crosslinking. We pipetted a mixture of collagen and alginate into a chamber, and covered the solution with a membrane (either aluminum oxide or cellulose) that was permeable to small molecular-weight solutes but not alginate polymer chains (Fig. 1B). Compared to our previous work, this configuration robustly permitted multiple (rather than a single) rounds of reagent addition to permit reversible switching of the alginate crosslinking state. To form the composite gel, we first gelled the collagen component, and then crosslinked the alginate by placing a CaCl2 solution on top of the membrane. Subsequently, we could uncrosslink the alginate component by adding sodium citrate (a chelator of Ca2þ). To examine whether changes in the crosslinking state of alginate influence transport characteristics the gels, we first characterized diffusivity in two-component matrices (with single-component matrices as controls) using fluorescence recovery after photobleaching (FRAP) of fluorescein isothiocyanate (FITC)-tagged dextrans of 3, 70 and 500 kD. Lines were bleached through the center of a sample, and fluorescence recovery curves orthogonal to the bleach line were fit as described previously (see Supporting Information Fig. S1). Collagen alone did not appreciably restrict diffusion of any dextran (92 to 96% of measured diffusion coefficient in water), whereas pure alginate appreciably hindered the diffusion of 70 and 500 kD dextrans (Table 1). The diffusion coefficients of all three dextrans increased upon crosslinking the alginate, with significant (p< 0.05) differences in diffusion coefficients between the crosslinked and uncrosslinked states for 70 and 500 kD dextrans (see Supporting Methods). Importantly, after crosslinking and uncrosslinking, the diffusion coefficients returned to baseline values (i.e., the non-crosslinked state) for all three dextrans. These results suggested that the transport characteristics of the gels could indeed be reversibly switched, by adding calcium or citrate to the two-component ECM. In our model of composite ECM (Fig. 1), it is possible that changes to the state of the modulatory ECM component (alginate) could affect the structural component (collagen). To examine whether crosslinking of the alginate altered collagen fiber structure, we visualized the fine structure of the collagen fiber network using confocal reflectance microscopy on gels that were not fixed and without cells (since fixation and cell contraction alters fiber morphology). We then performed image segmentation to obtain estimates of collagen mesh size (Supporting Information Fig. S2). This analysis revealed that collagen fibers

Direct patterning of composite biocompatible microstructures using microfluidics.

This study demonstrates a versatile and fast method for patterning three-dimensional (3D) monolithic microstructures made of multiple (up to 24 demonstrated) types of materials, all spatially aligned, inside a microchannel. This technique uses confocal scanning or conventional fluorescence microscopy to polymerize selected regions of a photocurable material, and microfluidics to automate the delivery of a series of washes and photocurable reagents. Upon completion of lithographic cycles, the aligned 3D microstructures are suitable for microfluidic manipulation and analysis. We demonstrated the fabrication of composite 3D microstructures with various geometries, size scales (up to 1 mm2), spatial resolution (down to 3 microm), and materials. For a typical multi-cycle process, the total fabrication time was tens of minutes, compared to tens of hours for conventional methods. In the case of 3D hydrogels, a potential use is the direct patterning of inhomogeneous 3D microenvironments for studying cell behavior.

Engineering extracellular matrix structure in 3D multiphase tissues

In native tissues, microscale variations in the extracellular matrix (ECM) structure can drive different cellular behaviors. Although control over ECM structure could prove useful in tissue engineering and in studies of cellular behavior, isotropic 3D matrices poorly replicate variations in local microenvironments. In this paper, we demonstrate a method to engineer local variations in the density and size of collagen fibers throughout 3D tissues. The results showed that, in engineered multiphase tissues, the structures of collagen fibers in both the bulk ECM phases (as measured by mesh size and width of fibers) as well as at tissue interfaces (as measured by density of fibers and thickness of tissue interfaces) could be modulated by varying the collagen concentrations and gelling temperatures. As the method makes use of a previously published technique for tissue bonding, we also confirmed that significant adhesion strength at tissue interfaces was achieved under all conditions tested. Hence, this study demonstrates how collagen fiber structures can be engineered within all regions of a multiphase tissue scaffold by exploiting knowledge of collagen assembly, and presents an approach to engineer local collagen structure that complements methods such as flow alignment and electrospinning.

Human Skin Constructs with Spatially Controlled Vasculature Using Primary and iPSC‐Derived Endothelial Cells

Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival, functionality, and viable engraftment. In this study, the latest microfabrication techniques are used and a novel bioengineering approach is established to micropattern spatially controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells. Using 3D printing technology makes it possible to control the geometry of the micropatterned vascular networks. It is verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function, which allows for the recapitulation of both topical and systemic delivery of drugs. In addition, the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice is examined and it is demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall, this innovative bioengineering approach can enable in vitro evaluation of topical and systemic drug delivery as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds.

Building a microphysiological skin model from induced pluripotent stem cells

The discovery of induced pluripotent stem cells (iPSCs) in 2006 was a major breakthrough for regenerative medicine. The establishment of patient-specific iPSCs has created the opportunity to model diseases in culture systems, with the potential to rapidly advance the drug discovery field. Current methods of drug discovery are inefficient, with a high proportion of drug candidates failing during clinical trials due to low efficacy and/or high toxicity. Many drugs fail toxicity testing during clinical trials, since the cells on which they have been tested do not adequately model three-dimensional tissues or their interaction with other organs in the body. There is a need to develop microphysiological systems that reliably represent both an intact tissue and also the interaction of a particular tissue with other systems throughout the body. As the port of entry for many drugs is via topical delivery, the skin is the first line of exposure, and also one of the first organs to demonstrate a reaction after systemic drug delivery. In this review, we discuss our strategy to develop a microphysiological system using iPSCs that recapitulates human skin for analyzing the interactions of drugs with the skin.

Challenges and promises in modeling dermatologic disorders with bioengineered skin.

The tremendous cost of drug development is often attributed to the long time interval between identifying lead compounds in preclinical studies to assessing clinical efficacy in randomized clinical trials. Many candidate molecules show promise in cell culture or animal models, only to fail in late stage in human investigations. There is a need for novel technologies that allow investigators to quickly and reliably predict drug safety and efficacy. The advent of microtechnology has made it possible to integrate multiple microphysiologic organ systems into a single microfabricated chip. This review focuses on three-dimensional engineered skin, which has enjoyed a long history of uses both in clinical treatments of refractory ulcers and as a laboratory model. We discuss current biological and engineering challenges in construction of a robust bioengineered skin and provide a blueprint for its potential utility to model dermatologic disorders such as psoriasis or cutaneous drug reactions.

A direct tissue-grafting approach to increasing endogenous brown fat

There is widespread evidence that increasing functional mass of brown adipose tissue (BAT) via browning of white adipose tissue (WAT) could potentially counter obesity and diabetes. However, most current approaches focus on administration of pharmacological compounds which expose patients to highly undesirable side effects. Here, we describe a simple and direct tissue-grafting approach to increase BAT mass through ex vivo browning of subcutaneous WAT, followed by re-implantation into the host; this cell-therapy approach could potentially act synergistically with existing pharmacological approaches. With this process, entitled “exBAT”, we identified conditions, in both mouse and human tissue, that convert whole fragments of WAT to BAT via a single step and without unwanted off-target pharmacological effects. We show that ex vivo, exBAT exhibited UCP1 immunostaining, lipid droplet formation, and mitochondrial metabolic activity consistent with native BAT. In mice, exBAT exhibited a highly durable phenotype for at least 8 weeks. Overall, these results enable a simple and scalable tissue-grafting strategy, rather than pharmacological approaches, for increasing endogenous BAT and studying its effect on host weight and metabolism.

Microfluidics for Engineering 3D Tissues and Cellular Microenvironments

Cellular microenvironments in native tissues are three dimensional (3D), inhomogeneous, anisotropic, and dynamic in terms of their composition of cells, extracellular matrix components, soluble factors, and physical forces (e.g. fluid flow and mechanical stress). Therefore, methods to recapitulate and control various components of cellular microenvironments in vitro and in vivo are highly useful for both studying and engineering biological systems. Microfluidics-based technologies enable precision spatiotemporal control over mass transport and are thus well suited to control the assembly and dynamic culture of engineered cellular microenvironments. In this chapter, we highlight several different ways that microfluidics can be utilized in engineering 3D tissues and cellular microenvironments, including methods to microfabricate 3D tissue scaffolds using microfluidics, methods to assemble and dynamically culture 3D microenvironments within PDMS-based microfluidic devices, and methods to incorporate microfluidic channels directly within engineered 3D tissue scaffolds, for both perfusion of tissue constructs with media and for assembly of multiphase 3D tissues.