Mark D. Brigham

Nanowired three-dimensional cardiac patches

Engineered cardiac patches for treating damaged heart tissues after a heart attack are normally produced by seeding heart cells within three-dimensional porous biomaterial scaffolds. These biomaterials, which are usually made of either biological polymers such as alginate or synthetic polymers such as poly(lactic acid) (PLA), help cells organize into functioning tissues, but poor conductivity of these materials limits the ability of the patch to contract strongly as a unit. Here, we show that incorporating gold nanowires within alginate scaffolds can bridge the electrically resistant pore walls of alginate and improve electrical communication between adjacent cardiac cells. Tissues grown on these composite matrices were thicker and better aligned than those grown on pristine alginate and when electrically stimulated, the cells in these tissues contracted synchronously. Furthermore, higher levels of the proteins involved in muscle contraction and electrical coupling are detected in the composite matrices. It is expected that the integration of conducting nanowires within three-dimensional scaffolds may improve the therapeutic value of current cardiac patches.

Mechanically robust and bioadhesive collagen and photocrosslinkable hyaluronic acid semi-interpenetrating networks.

In this work, we present a class of hydrogels that leverage the favorable properties of the photo-cross-linkable hyaluronic acid (HA) and semi-interpenetrating collagen components. The mechanical properties of the semi-interpenetrating-network (semi-IPN) hydrogels far surpass those achievable with collagen gels or collagen gel-based semi-IPNs. Furthermore, the inclusion of the semi-interpenetrating collagen chains provides a synergistic mechanical improvement over unmodified HA hydrogels. Collagen-HA semi-IPNs supported fibroblast adhesion and proliferation and were shown to be suitable for cell encapsulation at high levels of cell viability. To demonstrate the utility of the semi-IPNs as a microscale tissue engineering material, cell-laden microstructures and microchannels were fabricated using soft lithographic techniques. Given their enhanced mechanical and biomimetic properties, we anticipate that these materials will be of value in tissue engineering and three-dimensional cell culture applications.

Blast-induced phenotypic switching in cerebral vasospasm

Vasospasm of the cerebrovasculature is a common manifestation of blast-induced traumatic brain injury (bTBI) reported among combat casualties in the conflicts in Afghanistan and Iraq. Cerebral vasospasm occurs more frequently, and with earlier onset, in bTBI patients than in patients with other TBI injury modes, such as blunt force trauma. Though vasospasm is usually associated with the presence of subarachnoid hemorrhage (SAH), SAH is not required for vasospasm in bTBI, which suggests that the unique mechanics of blast injury could potentiate vasospasm onset, accounting for the increased incidence. Here, using theoretical and in vitro models, we show that a single rapid mechanical insult can induce vascular hypercontractility and remodeling, indicative of vasospasm initiation. We employed high-velocity stretching of engineered arterial lamellae to simulate the mechanical forces of a blast pulse on the vasculature. An hour after a simulated blast, injured tissues displayed altered intracellular calcium dynamics leading to hypersensitivity to contractile stimulus with endothelin-1. One day after simulated blast, tissues exhibited blast force dependent prolonged hypercontraction and vascular smooth muscle phenotype switching, indicative of remodeling. These results suggest that an acute, blast-like injury is sufficient to induce a hypercontraction-induced genetic switch that potentiates vascular remodeling, and cerebral vasospasm, in bTBI patients.

Microporous Cell-laden Hydrogels for Engineered Tissue Constructs

In this article, we describe an approach to generate microporous cell‐laden hydrogels for fabricating biomimetic tissue engineered constructs. Micropores at different length scales were fabricated in cell‐laden hydrogels by micromolding fluidic channels and leaching sucrose crystals. Microengineered channels were created within cell‐laden hydrogel precursors containing agarose solution mixed with sucrose crystals. The rapid cooling of the agarose solution was used to gel the solution and form micropores in place of the sucrose crystals. The sucrose leaching process generated homogeneously distributed micropores within the gels, while enabling the direct immobilization of cells within the gels. We also characterized the physical, mechanical, and biological properties (i.e., microporosity, diffusivity, and cell viability) of cell‐laden agarose gels as a function of engineered porosity. The microporosity was controlled from 0% to 40% and the diffusivity of molecules in the porous agarose gels increased as compared to controls. Furthermore, the viability of human hepatic carcinoma cells that were cultured in microporous agarose gels corresponded to the diffusion profile generated away from the microchannels. Based on their enhanced diffusive properties, microporous cell‐laden hydrogels containing a microengineered fluidic channel can be a useful tool for generating tissue structures for regenerative medicine and drug discovery applications. Biotechnol. Bioeng. 2010; 106: 138–148.

Stimuli-responsive microwells for formation and retrieval of cell aggregates.

Generating cell aggregates is beneficial for various applications ranging from biotechnology to regenerative therapies. Previously, poly(ethylene glycol) (PEG) microwells have been demonstrated as a potentially useful method for generating controlled-size cell aggregates. In addition to controlling cell aggregate size and homogeneity, the ability to confine cell aggregates on glass adhesive substrates and subsequently retrieve aggregates from microwells for further experimentation and analysis could be beneficial for various applications. However, it is often difficult to retrieve cell aggregates from these microwells without the use of digestive enzymes. This study describes the stable formation of cell aggregates in responsive microwells with adhesive substrates and their further retrieval in a temperature dependent manner by exploiting the stimuli responsiveness of these microwells. The responsive polymer structure of the arrays can be used to thermally regulate the microwell diameters causing a mechanical force on the aggregates, subsequently facilitating the retrieval of cell aggregates from the microwells with high efficiency compared to PEG arrays. This approach can be potentially integrated into high-throughput systems and may become a versatile tool for various applications that require aggregate formation and experimentation, such as tissue engineering, drug discovery, and stem cell biology.

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9–15 weeks, followed by 4–5 weeks of validation.

Cell confinement in patterned nanoliter droplets in a microwell array by wiping

Cell patterning is useful for a variety of biological applications such as tissue engineering and drug discovery. In particular, the ability to localize cells within distinct fluids is beneficial for a variety of applications ranging from microencapsulation to high-throughput analysis. However, despite much progress, cell immobilization and maintenance within patterned microscale droplets remains a challenge. In particular, no method currently exists to rapidly seed cells into microwell arrays in a controllable and reliable manner. In this study, we present a simple wiping technique to localize cells within arrays of polymeric microwells. This robust method produces cell seeding densities that vary consistently with microwell geometry and cell concentration. Moreover, we develop a simple theoretical model to accurately predict cell seeding density and seeding efficiency in terms of the design parameters of the microwell array and the cell density. This short-term cell patterning approach is an enabling tool to develop new high-throughput screening technologies that utilize microwell arrays containing cells for screening applications.

Preventing cardiac remodeling: The combination of cell-based therapy and cardiac support therapy preserves left ventricular function in rodent model of myocardial ischemia

OBJECTIVE Cellular and mechanical treatment to prevent heart failure each holds therapeutic promise but together have not been reported yet. The goal of the present study was to determine whether combining a cardiac support device with cell-based therapy could prevent adverse left ventricular remodeling, more than either therapy alone. METHODS The present study was completed in 2 parts. In the first part, mesenchymal stem cells were isolated from rodent femurs and seeded on a collagen-based scaffold. In the second part, myocardial infarction was induced in 60 rats. The 24 survivors were randomly assigned to 1 of 4 groups: control, stem cell therapy, cardiac support device, and a combination of stem cell therapy and cardiac support device. Left ventricular function was measured with biweekly echocardiography, followed by end-of-life histopathologic analysis at 6 weeks. RESULTS After myocardial infarction and treatment intervention, the ejection fraction remained preserved (74.9-80.2%) in the combination group at an early point (2 weeks) compared with the control group (66.2-82.8%). By 6 weeks, the combination therapy group had a significantly greater fractional area of change compared with the control group (69.2% ± 6.7% and 49.5% ± 6.1% respectively, P = .03). Also, at 6 weeks, the left ventricular wall thickness was greater in the combination group than in the stem cell therapy alone group (1.79 ± 0.11 and 1.33 ± 0.13, respectively, P = .02). CONCLUSIONS Combining a cardiac support device with stem cell therapy preserves left ventricular function after myocardial infarction, more than either therapy alone. Furthermore, stem cell delivery using a cardiac support device is a novel delivery approach for cell-based therapies.

Fabrication of microchannels in methacrylated hyaluronic acid hydrogels

Culturing cells in a vascularized three-dimensional (3D) hydrogel scaffold has significant applications ranging from tissue engineering to drug discovery. In many large 3D scaffolds, mass transport and nutrient exchange leads to cell necrosis, limiting functionality. Here we present a technique for fabricating microfluidic channels in cell-laden methacrylated hyaluronic acid (MeHA) hydrogels. Using standard soft lithographic techniques, MeHA pre-polymer was molded against a PDMS master and cross-linked using UV light. A second UV cross-linking step generated sealed channels. Channels of different dimensions and geometric complexity demonstrated that MeHA, though highly porous, is a suitable material for microfluidics. Cells embedded within the microfluidic molds were well distributed and media pumped through the channels allowed the exchange of nutrients and waste products. Through repeated stacking and crosslinking steps, we were able to form multiple layers of 3D MeHA channels to form a highly perfuse microchannel network. Incorporating collagen into the MeHA to form a semi-interpenetrating network enabled endothelial cell attachment to the interior of the channels. Further development of this technique may lead to the generation of biomimetic synthetic vasculature for tissue engineering and drug screening.

Author Correction: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

In the published version of this paper, Step 64 of the Procedure reads, “Refer to Steps 37–39 for NGS analysis of the sgRNA distribution.” This step should refer the reader to Steps 35–39. This text has not been corrected in the original paper.