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Humans are consistently exposed to thousands of untested chemicals that have been detected in the follicular fluid of the ovaries, and can disrupt reproductive health. Human granulosa cells (GCs) are the functional unit of the ovarian follicle with steroidogenic and signaling activities, and play a pivotal role in oocyte development. During follicle progression, GCs multiply to form a 3D avascular structure, and establish gap junction intercellular communication (GJIC) that is critical to maintaining optimal viability and function. We developed a high-throughput in vitro platform of human GCs for the screening of chemicals that can impact GJIC and estradiol (E2) production of human granulosa. Our granulosa 3D microtissues fabricated with human ovarian granulosa-like tumor KGN cells are multicell-layered structures that mimic the avascular granulosa layers surrounding the oocyte. These microtissues robustly expressed the steroidogenic CYP19 aromatase enzyme and GJIC intercellular membrane channel, connexin 43. Granulosa microtissues produced E2 at rates comparable to primary human GCs as previously reported. E2 production was suppressed by the CYP19 inhibitor, letrozole, and induced by CYP19 activators, bisphenol A at 100 µM, and genistein at 100 µM. Granulosa microtissues displayed active GJIC function, as demonstrated by the connexin 43-dependent diffusion of calcein fluorescent dye from microtissue surface to the core using high-throughput confocal microscopy in conjunction with our open-sourced automated image analysis tool. Overall, our 3D human granulosa screening platform is highly promising for predictive and efficient in vitro toxicity testing to screen for chemicals that contaminate follicular fluid and may affect fertility.

Tissue engineering has been largely confined to academic research institutions with limited success in commercial settings. To help address this issue, more work is needed to develop new automated manufacturing processes for tissue-related technologies. In this article, we describe the automation of the funnel-guide, an additive manufacturing method that uses living tissue rings as building units to form bio-tubes. We developed a method based on 96-well plates and a modified off-the-shelf liquid-handling robot to retrieve, perform real-time quality control, and transfer tissue rings to the funnel-guide. Cells seeded into 96-well plates containing specially designed agarose micromolds self-assembled and formed ring-shaped microtissues that could be retrieved using a liquid-handling robot. We characterized the effects of time, cell type, and mold geometry on the morphology of the ring-shaped microtissues to inform optimal use of the building parts. We programmed and modified an off-the-shelf liquid-handling robot to retrieve ring-shaped microtissues from the 96-well plates, and we fabricated a custom illuminated pipette to visualize each ring-shaped microtissue prior to deposit in the funnel guide. Imaging at the liquid-air interface presented challenges that were overcome by controlling lighting conditions and liquid curvature. Based on these images, we incorporated into our workflow a real-time quality control step based on visual inspection and morphological criteria to assess each ring prior to use. We used this system to fabricate bio-tubes of endothelial cells with luminal alignment.

Essentials Bleeding is a major source of morbidity during mechanical circulatory support. von Willebrand factor (VWF) multimer loss may contribute to bleeding. Different patterns of VWF multimer loss were seen with the two device types. This is the first report of total artificial heart associated VWF multimer loss.Background Bleeding remains a challenge during mechanical circulatory support and underlying mechanisms are incompletely understood. Functional von Willebrand factor (VWF) impairment because of loss of high-molecular-weight multimers (MWMs) produces acquired von Willebrand disease (VWD) after left ventricular assist device (LVAD). Little is known about VWF multimers with total artificial hearts (TAHs). Here, VWF profiles with LVADs and TAHs are compared using a VWD panel. Methods VWD evaluations for patients with LVAD or TAH (2013-14) were retrospectively analyzed and included: VWF activity (ristocetin cofactor, VWF:RCo), VWF antigen (VWF:Ag), ratio of VWF:RCo to VWF:Ag, and quantitative VWF multimeric analysis. Results Twelve patients with LVADs and 12 with TAHs underwent VWD evaluation. All had either normal (47.8%) or elevated (52.2%) VWF:RCo, normal (26.1%) or elevated (73.9%) VWF:Ag and 50.0% were disproportional (ratio ≤ 0.7). Multimeric analysis showed abnormal patterns in all patients with LVADs: seven with high MWM loss; five with highest MWM loss. With TAH, 10/12 patients had abnormal patterns: all with highest MWM loss. High MWM loss correlated with presence of LVAD and highest MWM loss with TAH. Increased low MWMs were detected in 22/24. Conclusion Using VWF multimeric analysis, abnormalities after LVAD or TAH were detected that would be missed with measurements of VWF level alone: loss of high MWM predominantly in LVAD, loss of highest MWM in TAH, and elevated levels of low MWM in both. This is the first study to describe TAH-associated highest MWM loss, which may contribute to bleeding.

The recent development of 3D tissue spheroids aims to address current limitations with traditional 2D cell cultures in various studies, including cancer drug screening and environmental toxin testing. In these studies, measurements of cellular viability are commonly utilized to assess the effects of drug or toxins. Existing methods include live/dead assays, colorimetric assays, fluorescence calcium imaging, and immunohistochemistry. However, those methods involve the addition of histological stains, fluorescent proteins, or other labels to the sample; some methods also require sample fixation. Fixation-based methods preclude the possibility of longitudinal study of viability, and confocal fluorescence imaging-based methods suffer from insufficient delivery of labels near the center of 3D spheroids. Here, we demonstrate the use of label-free optical coherence tomography (OCT) for quantitative cellular viability imaging of 3D tissue spheroids. OCT intensity and decorrelation signals acquired from neurospheroids exhibited changes correlated with cellular viability as manipulated with ethanol. Interestingly, when we repeated the imaging while cells gradually became less viable, the intensity and decorrelation signals exhibited different time courses, suggesting that they may represent different cellular processes in cell death. More quantitative measurements of viability using dynamic light scattering optical coherence microscopy (DLS-OCM) will be also presented. DLS-OCM enables us to obtain 3D maps of the diffusion coefficient, and we found that the diffusion coefficient of intra-cellular motility correlated with cellular viability manipulated by changes in temperature and pH. Finally, applications of these novel methods to human-cell 3D spheroids will be discussed.

The field of tissue engineering is developing new additive manufacturing technologies to fabricate 3D living constructs for use as in vitro platforms for the testing of drugs and chemicals, or to restore lost function in vivo. In this article, we describe the funnel-guide (FG), a new additive manufacturing strategy for the noncontact manipulation and positioning of multicellular microtissues and we show that the FG can be used to build macrotissues layer by layer. We used agarose micromolds to self-assemble cells into toroid-shaped and honeycomb-shaped microtissues, and observed that when falling in cell culture medium, the microtissues spontaneously righted themselves to a horizontal orientation. We fabricated a funnel to guide these falling toroids and honeycombs into precise positions and stack them, wherein they fused to form tubular structures. We tested multiple cell types and toroid sizes, and ultimately used the FG to create a stack of 45 toroids that fused into a tube 5 mm long with an inner diameter of 600 μm. The FG is a new principle for the manipulation of microtissues and is a platform for the layer-by-layer positioning of microtissue building blocks to form macrotissues.

Extracellular matrix composition and organization play a crucial role in numerous biological processes ranging from cell migration, differentiation, survival and metastasis. Consequently, there have been significant efforts towards the development of biomaterials and in vitro models that recapitulate the complexity of native tissue architecture. Here, we demonstrate an approach to fabricating highly aligned cell-derived tissue constructs via the self-assembly of human dermal fibroblasts. By optimizing mold geometry, cell seeding density, and media composition we can direct human dermal fibroblasts to adhere to one another around a non-adhesive agarose peg to facilitate the development of cell-mediated circumferential tension. By removing serum and adding ascorbic acid and l-proline, we tempered fibroblast contractility to enable the formation of stable tissue constructs. Similarly, we show that the alignment of cells and the ECM they synthesize can be modulated by changes to seeding density and that constructs seeded with the lowest number of cells have the highest degree of fibrillar collagen alignment. Finally, we show that this highly aligned, tissue engineered construct can be decellularized and that when re-seeded with fibroblasts, it provides instructive cues which enable cells to adhere to and align in the direction of the remaining collagen fiber network. Statement of Significance Cell and extracellular matrix organization is directly related to biological function including cell signaling and tissue mechanics. Changes to this organization are often associated with injury or disease. The majority of in vitro tissue engineering models investigating cell and matrix organization rely on the addition of stress-shielding exogenous proteins and polymers and, or the application of external forces to promote alignment. Here we present a completely cell-based approach that relies on the development of cell-mediated tension to direct anisotropic cellular alignment and matrix synthesis using human dermal fibroblasts. A major challenge with this approach is excessive cellular contractility that results in necking and failure of the tissue construct. While other groups have tried to overcome this challenge by simply adding more cells, here we show that matrix alignment is inversely related to cell seeding density. To engineer tissue constructs with the highest degree of alignment, we optimized media components to reduce cellular contractility and promote collagen synthesis such that fibroblast toroids remained stable for at least 28 days in culture. We subsequently showed that these collagen-rich tissue constructs could be decellularized while maintaining their collagen microstructure and that cells adhered to and responded to the decellularized cell-derived matrix by aligning and elongating along the collagen fibers. The complexity of cell-derived matrices has been shown to better recapitulate in vivo tissue architecture and composition. This study provides a straight-forward approach to fabricating instructive cell-derived matrices with a high degree of uniaxial alignment generated purely by cell-mediated tension.

Accurately predicting the human response to new compounds is critical to a wide variety of industries. Standard screening pipelines (including both in vitro and in vivo models) often lack predictive power. Three-dimensional (3D) culture systems of human cells, a more physiologically relevant platform, could provide a high-throughput, automated means to test the efficacy and/or toxicity of novel substances. However, the challenge of obtaining high-magnification, confocal z stacks of 3D spheroids and understanding their respective quantitative limitations must be overcome first. To address this challenge, we developed a method to form spheroids of reproducible size at precise spatial locations across a 96-well plate. Spheroids of variable radii were labeled with four different fluorescent dyes and imaged with a high-throughput confocal microscope. 3D renderings of the spheroid had a complex bowl-like appearance. We systematically analyzed these confocal z stacks to determine the depth of imaging and the effect of spheroid size and dyes on quantitation. Furthermore, we have shown that this loss of fluorescence can be addressed through the use of ratio imaging. Overall, understanding both the limitations of confocal imaging and the tools to correct for these limits is critical for developing accurate quantitative assays using 3D spheroids.

Purpose Patients with advanced heart failure and cardiorenal syndrome can experience significantly elevation in blood urea nitrogen (BUN) levels. We evaluated the impact of preoperative elevated BUN levels on bleeding and survival in patients who underwent continuous flow - left ventricular assist device (CF-LVAD) implantation. Methods We performed a retrospective review of 737 patients who underwent implantation of CF-LVADs from December 1999 through August 2017 at our center. Preoperative hemodynamics, perioperative characteristics, and postoperative outcomes were compared between patients with BUN ≥60 mmol/L and those Results Of the 664 patients included in the study (73 were excluded due to incomplete data), 53 patients had a BUN ≥60 mmol/L (mean: 83±28) and 611 patients had a BUN Conclusion Preoperative elevation in BUN above 60 mmol/L was associated with higher mortality. Additional studies are warranted to determine whether preoperative optimization of the uremia with medical therapy and/or dialysis can impact the increased risk of mortality.

There is a high demand for in vitro models of the central nervous system (CNS) to study neurological disorders, injuries, toxicity, and drug efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional two-dimensional culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry through both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the CNS in vitro.

A grand challenge of tissue engineering is the fabrication of large constructs with a high density of living cells. By adapting the principles of pick-and-place machines used in the high-speed assembly of electronics, we have developed an innovative instrument, the Bio-Pick, Place, and Perfuse (Bio-P3), which picks up large complex multicellular building parts, transports them to a build area, and precisely places the parts at desired locations while perfusing the parts. These assembled parts subsequently fuse to form a larger contiguous tissue construct. Multicellular microtissues were formed by seeding cells into nonadhesive micro-molds, wherein cells self-assembled scaffold-free parts in the shape of spheroids, toroids, and honeycombs. After removal from the molds, the parts were gripped, transported (using an x, y, z controller), and released using the Bio-P3 with little to no effect on cell viability or part structure. As many as 16 toroids were stacked over a 170 μm diameter post where they fused over the course of 48 h to form a single tissue. Larger honeycomb parts were also gripped and stacked onto a build head that, like the gripper head, provided fluid suction to hold and perfuse the parts during assembly. Scaffold-free building parts help to address several of the engineering and biological challenges to large tissue biofabrication, and the Bio-P3 described in this article is a novel instrument for the controlled gripping, placing, stacking, and perfusing of living building parts for solid organ fabrication.

None of the ABCG2 inhibitors are effective clinically against multidrug resistant tumors overexpressing ABCG2. New in vitro models are needed to characterize inhibitors and discover new ones. We report a 3D spheroid model and image-based method to quantify ABCG2 action. Nonadhesive micro-molds were used to self-assemble spheroids overexpressing ABCG2; these spheroids were then incubated with the transporter substrate Hoechst 33342. Time-lapse fluorescent microscopy was used to determine the transporter-dependent efflux of Hoechst 33342 and dose response of three inhibitors (Ko143, Iressa, Elacridar). This 3D microtissue model was also used to determine the time to maximal effect as well as duration of effect after inhibitor removal. All acted within one hour and Elacridar had a surprisingly long duration of effect, active 5 hours after removal. This model can be used with multiple cell types, provides new insight into the pharmacokinetics of inhibitors, and can be adapted to high throughput analyses.

Substrate stiffness is known to alter cell behavior and drive stem cell differentiation, though most research in this area has been restricted to traditional, two-dimensional (2D) culture systems rather than more physiologically relevant, three-dimensional (3D) platforms. In this study, we utilized polymer-based, cell mimicking microparticles (CMMPs) to deliver distinct, stable mechanical cues to human adipose derived stem cells (ASCs) in 3D spheroid culture to examine changes in adipogenic differentiation response and mechanophenotype. After 21 days of adipogenic induction, spheroids containing CMMPs (composite spheroids) stiffened in accordance with CMMP elasticity such that spheroids containing the stiffest, ~10 kPa, CMMPs were over 27% stiffer than those incorporating the most compliant, ~0.25 kPa CMMPs. Adipogenically induced, cell-only spheroids were over 180% larger and 50% more compliant than matched controls. Interestingly, composite spheroids cultured without chemical induction factors dissociated when presented with CMMPs stiffer than ~1 kPa, while adipogenic induction factors mitigated this behavior. Gene expression for PPARG and FABP4 were upregulated more than 45-fold in adipogenically induced samples compared to controls but were unaffected by CMMP elasticity, attributed to insufficient cell-CMMP contacts throughout the composite spheroid. In summary, mechanically tuned CMMPs influenced whole-spheroid mechanophenotype and stability but minimally affected differentiation response.

Hundreds of commercially available fluorescent dyes are used to quantify a wide range of biological functions of cells in culture, and their use has been a mainstay of basic research, toxicity testing, and drug discovery. However, nearly all of these dyes have been optimized for use on cells cultured as two-dimensional monolayers. Three-dimensional culture systems more accurately recapitulate native tissues, but their size and complexity present a new set of challenges for the use of fluorescent dyes, especially with regards to accurate quantitation. We determined the most accurate method to quantify fluorescence as a function of whether cells were uniformly labeled with dye prior to spheroid formation or if the dye was diffused into the spheroid after its formation. Using multicellular spheroids labeled with calcein-AM via these two different staining methods, we performed time-lapse fluorescence microscopy. For uniformly labeled spheroids, fluorescence was best normalized to volume, whereas for spheroids labeled via dye diffusion, fluorescence was best normalized to surface area. This framework for evaluating dyes can easily be extended to other applications. Utilizing the appropriate size-based normalization strategy enhanced our ability to detect statistically significant differences between experimental conditions.

We previously developed the Bio-Pick, Place, and Perfuse (Bio-P3) instrument to fabricate large perfusable tissue constructs by stacking and aligning scaffold-free living microtissues with integrated lumens. The Bio-P3 required an actuating mechanism to manipulate living microtissues of various sizes and shapes that are fragile, and must remain in an aqueous environment. The optical transparency of the Bio-P3 gripping device was essential to provide unobstructed visuals for accurate alignment of microtissues. We previously engineered a pilot fluid force-driven bio-gripper that can pick-and-place microtissue in planar position without causing cellular damage by pulling culture medium through track-etched membrane-integrated cell culture inserts. In this study, we invented a new flexible bio-gripper design that maximized the bio-gripper utilities. We utilized experimental approaches, multivariate analyzes, and theoretical modeling to elucidate how membrane characteristics (pore size, pore density, membrane thickness, membrane area, and surface chemistry) altered bio-gripper robustness and the flow rate (Q(c)) required for successful gripping. We devised two standardized tests and synthetic parts that mimicked microtissues, to systematically quantify bio-gripper performance. All thirteen syringe pump-driven bio-grippers except one successfully gripped and released synthetic parts with values of Q(c) that coincided with our mathematical simulation of the fluid mechanics of gripping. The bio-gripper could grip synthetic parts of various sizes, shapes and masses, demonstrating the robustness of the actuating mechanism. Multivariate analysis of experimental data indicated that both membrane porosity and thickness modulated Q(c), and in addition, revealed that membrane pore density determined membrane optical transparency. Fabricating large tissue constructs requires repeated stacking of microtissues. We showed that one bio-gripper could pick-and-place living microtissues thirty times with Q(c) corresponding to our simulation. Our bio-gripper was capable of stacking and aligning twenty microtissues. In summary, we successfully engineered a robust controllable fluid-driven bio-gripper to efficiently manipulate living microtissues and micro-objects in an aqueous environment.

Three-dimensional (3-D) in vitro platforms have been shown to closely recapitulate human physiology when compared with conventional two-dimensional (2-D) in vitro or in vivo animal model systems. This confers a substantial advantage in evaluating disease mechanisms, pharmaceutical drug discovery, and toxicity testing. Despite the benefits of 3-D cell culture, limitations in visualization and imaging of 3-D microtissues present significant challenges. Here we optimized histology and microscopy techniques to overcome the constraints of 3-D imaging. For morphological assessment of 3-D microtissues of several cell types, different time points, and different sizes, a two-step glycol methacrylate embedding protocol for evaluating 3-D microtissues produced using agarose hydrogels improved resolution of nuclear and cellular histopathology characteristic of cell death and proliferation. Additional immunohistochemistry, immunofluorescence, and in situ immunostaining techniques were successfully adapted to these microtissues and enhanced by optical clearing. Utilizing the ClearT2 protocol greatly increased fluorescence signal intensity, imaging depth, and clarity, allowing for more complete confocal fluorescence microscopy imaging of these 3-D microtissues compared with uncleared samples. The refined techniques presented here address the key challenges associated with 3-D imaging, providing new and alternative methods in evaluating disease pathogenesis, delineating toxicity pathways, and enhancing the versatility of 3-D in vitro testing systems in pharmacological and toxicological applications.

The alignment and blend of extracellular matrix (ECM) proteins give a tissue its specific mechanical properties as well as its physiological function. Various tissue engineering methods have taken purified ECM proteins and aligned them into gels, sponges and threads. Although, each of these methods has created aligned ECM, they have had many limitations including loss of hierarchal collagen structure and poor mechanical performance. Here, we have developed a new method to control ECM synthesis using self-assembled cells. Cells were seeded into custom designed, scaffold-free, micro-molds with fixed obstacles that harnessed and directed cell-mediated stresses. Cells within the microtissue reacted to self-generated tension by aligning, elongating, and synthesizing an ECM whose organization was dictated by the strain field that was set by our micro-mold design. We have shown that through cell selection, we can create tissues with aligned collagen II or aligned elastin. We have also demonstrated that these self-assembled microtissues have mechanical properties in the range of natural tissues and that mold design can be used to further tailor these mechanical properties.

It is well-established that upregulation of drug efflux pumps leads to multi-drug resistance. Less is known about the role of the architecture of the tumor microenvironment in this process: how the location of pump expressing cells influences drug exposure to cancerous as well as non-cancerous cells. Here, we report a 3D in vitro model of spheroids with mixtures of cells expressing high and low levels of ABCG2, quantifying pump activity by the ability to reject the fluorescent dye Hoechst 33342. With respect to the organization of the mixed spheroids, three different architectures were observed: 1) high-expressing ABCG2 cells located in the spheroid core surrounded by low-expressing cells, 2) high-expressing ABCG2 cells intermixed with low-expressing cells and 3) high-expressing ABCG2 cells surrounding a core of low-expressing cells. When high-expressing ABCG2 cells were in the core or intermixed, Hoechst uptake was directly proportional to the percentage of ABCG2 cells. When high-expressing ABCG2 cell formed an outer coating surrounding spheroids, small numbers of ABCG2 cells were disproportionately effective at inhibiting uptake. Specific inhibitors of the ABCG2 transporter eliminated the effect of this coating. Confocal microscopy of spheroids revealed the location of high- and low-expressing cells, and Hoechst fluorescence revealed that the ABCG2-dependant drug concentration in the cancer microenvironment is influenced by pump expression level and distribution among the cells within a tissue. In addition to providing a 3D model for further investigation into multicellular drug resistance, these data show that the location of ABCG2-expressing cells can control drug exposure within the tumor microenvironment.

Tissue fusion, whereby two or more spheroids coalesce, is a process that is fundamental to biofabrication. We have designed a quantitative, high-throughput platform to investigate the fusion of multicellular spheroids using agarose micro-molds. Spheroids of primary human chondrocytes (HCH) or human breast cancer cells (MCF-7) were self-assembled for 24 h and then brought together to form an array comprised of two spheroids (one doublet) per well. To quantify spheroid fusogenicity, we developed two assays: (1) an initial tack assay, defined as the minimum amount of time for two spheroids to form a mechanically stable tissue complex or doublet, and (2) a fusion assay, in which we defined and tracked key morphological parameters of the doublets as a function of time using wide-field fluorescence microscopy over a 24 h time-lapse. The initial tack of spheroid fusion was measured by inverting the micro-molds and centrifuging doublets at various time points to assess their connectedness. We found that the initial tack between two spheroids forms rapidly, with the majority of doublets remaining intact after centrifugation following just 30 min of fusion. Over the course of 24 h of fusion, several morphological changes occurred, which were quantified using a custom image analysis pipeline. End-to-end doublet lengths decreased over time, doublet widths decreased for chondrocytes and increased for MCF-7, contact lengths increased over time, and chondrocyte doublets exhibited higher intersphere angles at the end of fusion. We also assessed fusion by measuring the fluorescence intensity at the plane of fusion, which increased over time for both cell types. Interestingly, we observed that doublets moved and rotated in the micro-wells during fusion and this rotation was inhibited by ROCK inhibitor Y-27632 and myosin II inhibitor blebbistatin. Understanding and optimizing tissue fusion is essential for creating larger tissues, organs, or other structures using individual microtissues as building parts.