Browsing by Author "Miller, Jordan S"

Now showing 1 - 5 of 5
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    Advanced Stereolithography for Translational Vascular 3D Bioprinting
    (2019-04-19) Grigoryan, Bagrat; Miller, Jordan S
    The field of tissue engineering aims to fulfill the great clinical need to repair, replace, or regenerate impaired tissues and organs by delivery of a combination of cells, biomaterials, and biochemical and physicochemical factors. Indeed, tremendous strides have been achieved over the past several decades to construct implantable avascularized constructs such as skin, cornea, and bladder towards treatment for diseases and/or injuries. However, obtaining functional, physiologically relevant tissues is still a major challenge in the field due to the necessity of a vasculature system to supply nutrients and remove waste in thick constructs. This challenge can be attributed to convective transport limitations which results in necrotic cells due to inadequate access to nutrients. Indeed, vascularization of engineered thick tissue constructs is the current impediment in tissue engineering. Additionally, lack of the ability to recreate the heterogeneous patterns of cells and matrix to obtain constructs with controlled shape and architectures has further hindered progress towards organ replacement. This thesis aims to address these limitations by developing, characterizing, and validating a light-based 3D printing platform towards realization of thick, functional tissue constructs containing physiologically relevant micro-architectures. Of key importance towards achieving this goal, novel photopolymerizable formulations were identified and demonstrated towards stereolithographic generation of thick, perfusable hydrogels. Our hardware and materials innovations were then applied towards generation of hydrogels (composed of ≥90% water) containing various unprecedented space-filling, interpenetrating vessel networks, a hallmark of advanced multicellular life. We demonstrated a plethora of biological utility of our approach by illustrating examples of intervascular interstitial transport, generation of viable and functioning in vitro models of lung and bone tissue, and construction of a therapeutic transplantation liver model. This work unlocks transformative opportunities to mimic, interrogate, and utilize intricate multivariate vascular architectures that are vital to advanced multicellular life.
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    Cell mediated remodeling of engineered microvasculature
    (2020-03-19) Calderon, Gisele Amanda; Miller, Jordan S
    Tissue engineering as a field aims to model and replicate human tissues and organs as substitutes for restoring or improving organ functionality or for studying tightly controlled niches in drug discovery, cancer therapeutics, or fundamental biology. We are in great need of engineered tissues to address the scant availability of organ donors compatible with recipient patients. Further, animal studies are not predictive of human physiology, so there is a tremendous need to quantitatively investigate human cells for drug screening and other therapeutics in a clinically relevant manner. However, current clinical successes have been limited to avascular and simple tissues such as the cornea or bladder. In order to engineer more complex tissue equivalents, we must incorporate essential blood supply or vasculature to provide long-term viability for clinical implantation or for modeled drug screening. Towards engineering more complex, solid organs, bioengineers must identify methods of achieving effective vasculature to permeate the high volume of cells while not compromising the organ’s function. We believe that in order to build solid and complex tissues, we are to include hierarchical vasculature that varies in dimension (ranging from centimeters to micrometers in diameter) to most effectively transport blood and nutrients and remove waste products. Some research groups have historically taken a bottom-up approach to create de novo capillaries by stimulating vascular endothelial cells and support cells to form a provisional capillary plexus in extracellular matrix (ECM) or ECM-like materials. By allowing cells to dictate the vascular organization, we can ensure physiologic relevance, but the technique is currently limited to forming only the smallest vessels found in humans. Further, capillary formation in this bottom-up approach is a slow process that may limit the scope of complexity that can be achieved with organ engineering. Other research groups have also developed top-down, 3D printing (3DP) techniques to control the creation of larger diameter vessels with precise control over every x, y, and z position. With 3DP, open channel geometries can be incorporated into engineered tissues with a high level of control and speed. However, tissue engineers might not design the most optimized architectures and are constrained by the physical limitations of soft material engineering, such as the spatial resolution of the printer and an inability to reach the smallest scale of vessels. Few groups have been able to engineer tissues with vasculature that spans from micro- to macro- scale dimensions, which we identify as a critical step toward the actualization of a multiscale cellularized tissue. Therefore, we propose a combined bottom-up and top-down system that mimics the hierarchy of vasculature within tissues to overcome existing strategies that are limited to either small-scale microvessels or macro-scale physically patterned vessels. We expect this approach may have the capacity to create tissues which could allow rapid anastomosis to an existing vasculature in vivo. We propose to couple cell-based self-assembled capillaries with 3D printed channels to generate a multiscale vascular network which can more adequately approximate living human tissue structure. First, we will assess co-culture pre-vascularization strategies that promote vasculogenesis in our assembled capillaries. Next, we will utilize advanced soft material 3D printing and assess cellular activity in these engineered networks. Finally, we will apply our vascularized networks to therapeutic applications. This setup will allow us to study fundamental questions in vascular biology such as cellular remodeling in response to endothelial cells’ sensed shear stress from convective transport and cell-mediated, angiogenic sprouting and vasculogenic tube formation. We expect that our combined vascular engineering strategy will promote the fluidic union of vascular networks fabricated across multiple length scales. We believe this work will allow researchers to incorporate multi-scale vasculature in vitro for tissue engineered constructs for regenerative medicine or disease modeling applications, and may facilitate the investigation of cellular mechanisms of vascular homeostasis and vascular remodeling in both normal and pathologic settings.
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    Computational and experimental models of vascular transport in engineered tissues
    (2018-04-19) Paulsen, Samantha Jean; Miller, Jordan S; Dickinson, Mary E
    As tissue engineering advances from developing simple two-dimensional (2D) constructs towards the development of thick three-dimensional (3D) tissues on the scale of human organs, the transport of oxygen and nutrients to cells via functional vasculature becomes a paramount engineering challenge. Our field lacks methodologies to fabricate the requisite architecture, while quantitative workflows to predict and evaluate the effectiveness of a given design are also lacking. We and others are adapting 3D printing technologies to generate complex and bioinspired vascular geometries that can support the transport needs of large 3D tissues. We applied computational tools and linked them to experimental analyses of convective and diffusive transport provided by three-dimensional vascular networks. Human vasculature is multiscale with fractal complexity; to begin to approach this complexity we designed and studied mimics of specific aspects of vascular anatomy such as branching blood vessel networks and intravascular bicuspid valves. Our perfusable vessels supported arterial pressures, so we further investigated the feasibility of surgically connecting our constructs directly to host vasculature in small and large animal studies. The objective of this work is to close the loop between computational and experimental models involving blood flow and mass transport in vascular networks, allowing scientists to more effectively design and fabricate vascularized tissues. This work provides a quantitative roadmap for the design of vascular networks and the evaluation of their function within 3D tissue constructs.
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    Engineered tissues supported by convection and diffusion through dendritic vascular networks
    (2020-10-22) Kinstlinger, Ian S.; Miller, Jordan S
    Metabolic function in mammalian tissues is sustained by the delivery of oxygen and nutrients as well as the removal of waste through complex, three-dimensional (3D) networks of hierarchically organized blood vessels. However, fabrication of such 3D vascular networks within soft hydrogels remains one of the greatest challenges in tissue engineering. Sacrificial templates have proven useful for patterning perfusable vascular networks in engineered tissues, but such templates have been constrained in architectural complexity by limitations in the techniques which have been used to fabricate them. We hypothesized that these architectural limitations could be overcome by creating sacrificial vascular templates via selective laser sintering (SLS), an additive manufacturing process which uses a laser to fabricate solid structures from powdered raw materials. We developed an open-source SLS system and demonstrated its capacity to pattern biomimetic scale models of vascular topology. To adapt SLS fabrication for biocompatible and water-soluble materials which could be used sacrificially in the presence of cells, we identified carbohydrate powders formulations which are compatible with SLS and demonstrated laser sintering of carbohydrates into elaborate branched structures, including algorithmically-generated biomimetic branching networks which we term dendritic networks. Laser sintered carbohydrate templates were used to pattern perfusable vascular networks in a range of materials including natural and synthetically-derived biocompatible hydrogels, which can support cells in both the lumenal and parenchymal spaces. We leveraged this methodology to establish a complete pipeline encompassing generative vascular design, additive fabrication, perfusion culture, and volumetric spatial analysis of tissue performance. We identify heterogeneous zones of metabolic activity that emerge in perfused cell-laden hydrogels and we demonstrate that dendritic vascular networks can sustain cell metabolism deep within model tissues greater than 1 cm thick. We also seed endothelial cells, characterize convective transport through dendritic networks, and explore strategies to modulate the dynamics of changing cell densities within perfused gels. Finally, we demonstrate that perfusion culture through dendritic networks can support the survival and function of primary hepatocyte cultures. This approach for rapid design and biofabrication of engineered volumetric tissues offers an experimental strategy for interrogating the relationship between vascular network architecture, metabolite transport, and tissue function.
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    Production and Characterization of Uniform and Heterogeneous Cancer Multicellular Aggregates for Longitudinal Studies of Epithelial-To-Mesenchymal Transition
    (2018-01-22) Albritton, Jacob L; Miller, Jordan S
    Epithelial-to-mesenchymal transition (EMT) is a proposed mechanism for initial metastatic invasion. Tumors are highly heterogeneous mixtures of tumor cells, and heterogeneous multicellular aggregates (MCAs) have emerged as in vitro surrogates for heterogeneous tumors. We custom-modified a commercial laser cutter to produce microwells by laser ablation of poly(dimethylsiloxane) (PDMS), which could generate MCAs with EMT phenotype. Tumor invasion is a dynamic process, but methods for longitudinal characterization of heterogeneous MCAs are lacking. We propose improvements to quantifying 1) MCA size using 2D maximum Feret diameter measured by automated image analysis; 2) Cell sub-population ratio determined from Imaris-mediated nuclei counting of cells with nuclear localized fluorescent proteins; and 3) Segmentation efficiency based on cell population overlap volume. Finally, we discuss directions for future longitudinal studies of EMT. Improvements to MCA production throughput and longitudinal characterization methods will improve studies of EMT with heterogeneous MCAs.