Background We report the ability to extend lung preservation up to 24 hours (24H) by using autologous whole donor blood circulating within an ex vivo lung perfusion (EVLP) system. This approach facilitates donor lung reconditioning in a model of extended normothermic EVLP. We analyzed comparative responses to cellular and acellular perfusates to identify these benefits. Methods Twelve pairs of swine lungs were retrieved after cardiac arrest and studied for 24H on the Organ Care System (OCS) Lung EVLP platform. Three groups (n=4 each) were differentiated by perfusate: (1) isolated red blood cells (RBCs) (current clinical standard for OCS); (2) whole blood (WB); and (3) acellular buffered dextran-albumin solution (BDAS, analogous to STEEN solution). Results Only the RBC and WB groups met clinical standards for transplantation at 8 hours; our primary analysis at 24H focused on perfusion with WB versus RBC. The WB perfusate was superior (vs. RBC) for maintaining stability of all monitored parameters, including the following mean 24H measures: pulmonary artery pressure (6.8 vs. 9.0 mmHg), reservoir volume replacement (85 vs. 1607 mL), and PaO2:FiO2 ratio (541 vs. 223). Acellular perfusion was limited to 6 hours on the OCS system due to prohibitively high vascular resistance, edema, and worsening compliance. Conclusions The use of an autologous whole donor blood perfusate allowed 24H of preservation without functional deterioration and was superior to both RBC and BDAS for extended lung preservation in a swine model using OCS Lung. This finding represents a potentially significant advance in donor lung preservation and reconditioning.

Donation after circulatory death (DCD) is an underused source of donor lungs. Normothermic cellular ex vivo lung perfusion (EVLP) is effective in preserving standard donor lungs but may also be useful in the preservation and assessment of DCD lungs. Using a model of DCD and prolonged EVLP, the effects of donor warm ischemia and postmortem ventilation on graft recovery were evaluated. Adult male swine underwent general anesthesia and heparinization. In the control group (n = 4), cardioplegic arrest was induced and the lungs were procured immediately. In the four treatment groups, a period of agonal hypoxia was followed by either 1 h of warm ischemia with (n = 4) or without (n = 4) ventilation or 2 h of warm ischemia with (n = 4) or without (n = 4) ventilation. All lungs were studied on an EVLP platform for 24 h. Hemodynamic measures, compliance, and oxygenation on EVLP were worse in all DCD lungs compared with controls. Hemodynamics and compliance normalized in all lungs after 24 h of EVLP, but DCD lungs demonstrated impaired oxygenation. Normothermic cellular EVLP is effective in preserving and monitoring of DCD lungs. Early donor postmortem ventilation and timely procurement lead to improved graft function.

Cardiovascular disease is the leading cause of death worldwide and is associated with approximately 17.9 million deaths each year. Musculoskeletal conditions affect more than 1.71 billion people globally and are the leading cause of disability. These two areas represent a massive global health burden that is perpetuated by a lack of functionally restorative treatment options. The fields of regenerative medicine and tissue engineering offer great promise for the development of therapies to repair damaged or diseased tissues. Decellularized tissues and extracellular matrices are cornerstones of regenerative biomaterials and have been used clinically for decades and many have received FDA approval. In this review, we first discuss and compare methods used to produce decellularized tissues and ECMs from cardiac and skeletal muscle. We take a focused look at how different biophysical properties such as spatial topography, extracellular matrix composition, and mechanical characteristics influence cell behavior and function in the context of regenerative medicine. Lastly, we describe emerging research and forecast the future high impact applications of decellularized cardiac and skeletal muscle that will drive novel and effective regenerative therapies.

Introduction: Current bioinks for 3D bioprinting, such as gelatin-methacryloyl, are generally low viscosity fluids at room temperature, requiring specialized systems to create complex geometries. Methods and Results: Adding decellularized extracellular matrix microparticles derived from porcine tracheal cartilage to gelatin-methacryloyl creates a yield stress fluid capable of forming self-supporting structures. This bioink blend performs similarly at 25 °C to gelatin-methacryloyl alone at 15 °C in linear resolution, print fidelity, and tensile mechanics. Conclusion: This method lowers barriers to manufacturing complex tissue geometries and removes the need for cooling systems.

Two‐dimensional (2D) cell culture models fail to recapitulate the complex tissue architectures, extracellular matrices, and cellular crosstalk that occur in vivo. Three‐dimensional models (3D), including scaffold‐free organoids, derived from a single human induced pluripotent stem cell (hiPSC) cell source can more accurately model cell‐cell and cell‐matrix interactions to study specific genetic and organ‐level diseases that are difficulty to study in vitro. Additionally, 3D culture methods utilizing simulated microgravity environments have been shown to improve (iPSC)‐derived cardiomyocyte (CM) maturity, differentiation efficiency, and function, while promoting self‐organization in models that include multiple cardiac cell types. However, current published models have incorporated only one or two cell types and have used cells from a mixture of primary and hiPSC‐derived sources, primarily due to inefficient hiPSC differentiation protocols, especially for epicardial cells (epiC) and cardiac fibroblasts (CF). This study investigates and compares the cellular organization and function of cardiac organoids consisting of all four predominant cardiac cell types (CM, epiC, endothelial, and CF) differentiated from a single hiPSC source using two differentiation methods for cardiac fibroblasts and two 3D culture techniques. hiPSCs were differentiated into cardiomyocytes, endothelial cells (EC), and epiC using established methods. hiPSC‐CF were differentiated two ways: differentiation of hiPSC‐epiC to hiPSC‐CF and isolation of vimentin‐positive, cardiac troponin t‐negative cells from standard CM differentiation. All cells were singularized with Accutase, mixed at a 5:2:2:1 ratio (CM:CF:epiC:EC) as proposed by the developing cardiac model, and cultured in a non‐adherent spheroid plate (Aggrewell, StemCell Tech) for two days. The spheroids were then cultured for seven days in standard gravity (suspension) or simulated microgravity (rotating bioreactor) environment, with media change every two days. After seven days the organoids were analyzed for organization through immunofluorescence, maximum pacing frequency, calcium transients, and motion contraction velocities. Cardiac organoids containing cells from both hiPSC‐CF differentiation methods were compact and viable, but organoids did not form when no CF was included. This suggests non‐CM cells (cardiac troponin t negative) from CM differentiations are functional cardiac fibroblasts for organoid formation and ECM production. Organoid formation was further supported by the positive staining of cTnT, CD31, WT1, Cx‐43, and alpha‐SMA. Therefore, this study demonstrates a method of generating cardiac organoids containing all four cardiac cell types derived from the same hiPSC with tissue‐level organization and function for the in vitro study of genetic diseases targeting cardiac tissue.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Augmenting adaptive immunity is a critical goal for developing next-generation cancer therapies. T and B cells infiltrating the tumor dramatically influence cancer progression through complex interactions with the local microenvironment. Cancer cells evade and limit these immune responses by hijacking normal immunologic pathways. Current experimental models using conventional primary cells, cell lines, or animals have limitations for studying cancer-immune interactions directly relevant to human biology and clinical translation. Therefore, engineering methods to emulate such interplay at local and systemic levels are crucial to expedite the development of better therapies and diagnostic tools. In this review, we discuss the challenges, recent advances, and future directions toward engineering the tumor-immune microenvironment (TME), including key elements of adaptive immunity. We first offer an overview of the recent research that has advanced our understanding of the role of the adaptive immune system in the tumor microenvironment. Next, we discuss recent developments in 3D in-vitro models and engineering approaches that have been used to study the interaction of cancer and stromal cells with B and T lymphocytes. We summarize recent advancement in 3D bioengineering and discuss the need for 3D tumor models that better incorporate elements of the complex interplay of adaptive immunity and the tumor microenvironment. Finally, we provide a perspective on current challenges and future directions for modeling cancer-immune interactions aimed at identifying new biological targets for diagnostics and therapeutics.

The specific communication of multiple cell types in the tumor microenvironment plays a critical role in cancer progression. Current engineering methods have failed to adequately replicate the complexities of the tumor microenvironment (TME). In particular, generating engineered tissue-like environments with multiple TME cell types has remained challenging. Here we demonstrate the capability to pattern complex single cell circuit configurations, using a novel microfluidic bioprinting method, to study cell-cell communication in the early TME. A microfluidic dispenser (Biopixlar, Fluicell AB) was optimized to determine the delivery pressure (5 – 80 mbar), internal vacuum (0 – 80 -mbar), and external vacuum (0 – 80 -mbar) to enable highly controllable deposition of single cells suspended in complete media supplemented with polyethylene glycol (15 mg/mL, 1:1) at 1 × 106 cells/mL. Flow conditions were optimized for human cells: MDA-MB-231, MCF7, PC3, breast epithelial cells (MCF10a), fibroblasts, cancer associated fibroblasts, THP-1 derived macrophages, CD4+ T cells, CD8+ T cells, human umbilical vein endothelial cells (HUVECs), and mesenchymal stem cells. As proof of concept, the optimized settings were used to replicate a 2D tumor biopsy region of interest with high spatial precision. Next, cell-cell communication circuits were fabricated with cancer cells (PC3 or MDA-MB-231) and HUVECs. Communication circuits were bioprinted as 4 by 4 cell arrays, with 100 µm spacing between each cell, equal number of HUVECs and cancer cells, and three different cellular arrangements: alternating cell types, like cell types grouped, and groups of four like cell types. The circuits were live cell imaged for up to 30 hours to observe cell migration patterns, proliferation, and morphological changes as a function of cell-cell communication circuit arrangements. Optimal printing parameters were identified as 80 mbar delivery pressure, -25 mbar internal vacuum, and -55 mbar external vacuum. These parameters maintained >99% cell viability and ±10 µm spatial precision of printed cells. Live cell imaging of circuits containing PC3s or MDA-MB-231s with HUVECs on collagen substrates revealed changes in migration patterns, proliferation, and morphology depending on the surrounding cellular arrangement. HUVECs were highly migratory throughout the duration of the experiment, frequently extended protrusions towards nearby HUVECs, but did not display the same level of interaction with PC3s as they did with MDA-MB-231s. In MDA-MB-231 circuits, irrespective of patterning, we identified clear tendencies of HUVECs to herd MDA-MB-231s, travel overtop of MDA-MB-231s, collect and carry visible particles released from MDA-MB-231s, and maintain dendritic morphology instead of undergoing the expected vascular tubulogenesis. We found that HUVECs had the best morphology when clustered in groups of four and proliferated most when surrounded by MDA-MB-231s (alternating pattern). We found that MDA-MB-231s only proliferated when surrounded by HUVECs and had the least displacement when surrounded by like cells. These results demonstrate a method to precisely bioprint single cell circuits, enabling the investigation of cellular spatial organization and composition within the tumor microenvironment as it relates to tumor initiation and progression. Citation Format: Haylie R. Helms, Alexander E. Davies, Rebekka Duhen, Joshua M. Moreau, Ellen M. Langer, Luiz E. Bertassoni. Single cell bioprinted cell circuits for the systematic assessment of cell-cell communication in the early tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB161.

We illustrate SLA 3d-printing printing methods to produce COVID-19 nasopharyngeal testing swabs, proving that they can be produced in a manner that is safe and clean for the users. 3D printing is an effective and efficient tool that can be used for a wide variety of applications, especially for a quick turn around in response to a shortage, and specifically for this application, this method was created in response to the swab shortage in early summer 2020.