a-standardized-porcine-kidney-auto-transplant-protocol-for-studying

A Standardized Porcine Kidney Auto-transplant Protocol for Studying Delayed Graft Function

Delayed graft function (DGF) is commonly defined as the need for dialysis within 7 days after kidney transplantation; however, current definitions often ...

Duke University

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. In vitro models that mimic in vivo tumor neovascularization facilitate exploration of this role. Here we investigated lung adenocarcinoma cancer cells (344SQ) and endothelial and pericyte vascular cells encapsulated in cell-adhesive, proteolytically-degradable poly(ethylene) glycol-based hydrogels. 344SQ in hydrogels formed spheroids and secreted proangiogenic growth factors that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, we engineered a 2-layer hydrogel with 344SQ and vascular cell layers. Large, invasive 344SQ clusters (area > 5,000 μm(2), circularity < 0.25) developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration. Our findings suggest vascular cells contribute to tumor progression and establish this culture system as a platform for studying tumor vascularization.

A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior.

Emphysema affects millions of patients worldwide. Cell transplantation and tissue engineering are promising approaches for the regeneration of gas exchange tissue in vivo. A reproducible and resource-efficient animal model with relevant pathological and physiological features is critical to assess efficacy of novel therapies. Here, we share a method for rapid development of emphysema in an adaptive immune-deficient rat with <5% mortality, which is ideal for high-throughput human cell-based experimentation.

Characterization of an elastase-induced emphysema model in immune-deficient rats.

Many tools from the field of tissue engineering can be used to develop novel model systems to study cancer. We have utilized biomimetic synthetic hydrogels, based on poly(ethylene glycol) (PEG) modified with cell adhesive peptides (RGDS) and peptides sensitive to degradation by matrix metalloproteinases 2 and 9 (GGGPQGIWGQGK), as highly controlled 3D substrates for cell culture. We have previously shown that this hydrogel can support growth of tumor cells and also growth and assembly of microvascular networks. Based on this technology, a 3D in vitro tumor angiogenesis model was developed using a dual layer PEG-based hydrogel comprised of vascular cells (endothelial cells, pericytes) and lung adenocarcinoma cells in separate layers to support recapitulation of the vessel recruitment process as it occurs in vivo. This model was previously used to study highly metastatic murine 344SQ cells and in this paper was used to investigate 2 additional types of lung adenocarcinoma cells: nonmetastatic murine 393P cells and somewhat metastatic human A549 cells. All three cell types readily formed spheroid structures in the 3D hydrogels. When cultured in the dual layer format, where tumor cell spheroids were adjacent to a hydrogel layer with microvascular tubule networks, all three tumor cell types recruited vascular cells into the cancer cell layer. Interactions between vessels invading the cancer layer and the cancer cell structures was nearly twice as high for the highly metastatic 344SQ cells as for the other two cell types. Secretion of angiogenic growth factors by the tumor cells was evaluated. 344SQ cells produced the greatest amount of VEGF and FGFb, which probably accounts for the greater degree of vessel recruitment observed. Upon interaction with vessel structures, the 344SQ spheroids underwent a dramatic change in morphology, increasing in size and adopting highly irregular shapes, suggestive of invasive phenotype. This behavior was observed to a much lesser degree for A549 cells and 393P cells.

Lung Adenocarcinoma Cell Responses in a 3D in Vitro Tumor Angiogenesis Model Correlate with Metastatic Capacity.

Lung regeneration is dependent on the availability of progenitor lung cells. Large numbers of self-renewing, patient-specific induced pluripotent stem cell-derived alveolar epithelial cells (iPSC-AECs) are needed to adequately recellularize whole-organ constructs. Prior methods to generate functional iPSC-AECs are not feasible for large-scale cell production. We present a novel protocol to produce iPSC-AECs, which is scalable for whole-organ regeneration. Differentiation of iPSCs was performed with genetically modified iPSCs with fluorescent reporters, which underwent differentiation in a stepwise protocol mimicking lung development. Cells were purified, sorted, and embedded in a liquid Matrigel precursor either to form adherent droplets or to form cell-laden Matrigel spheroids, which were subsequently transferred to spinner flasks with media as floating droplets. After culture, monolayer spheres of iPSC-AECs were isolated to form single cell suspensions. Equal numbers of iPSC-AECs from the two culture conditions were seeded into decellularized lung scaffolds. IPSC-AECs cultured in floating droplets were significantly more proliferative than those in adherent droplets, with significantly higher total cell counts and Ki67 expression. Equivalent expression of the distal lung markers was observed for both culture conditions. Lungs recellularized from both culture groups had similar histological appearance. Media changes took significantly less time with the floating droplet method and was more cost effective. The floating droplet culture method demonstrated enhanced proliferative capacity, stable distal lung epithelial phenotype, and reduced resources compared with prior culture methods. In this study, we provide a means for iPSC-AEC production for regeneration of whole-lung constructs. Impact statement We describe a novel culture method for induced pluripotent stem cell-derived alveolar epithelial cell (AEC) expansion with enhanced proliferative capacity and reduced resource requirements compared with previously described methods. This method is scalable for human whole-lung regeneration bioengineering or could be automated for commercial cell production. This culture method may have implications for the differentiation of type I AECs from type II AECs.

High-Throughput Culture Method of Induced Pluripotent Stem Cell-Derived Alveolar Epithelial Cells.

Lung transplantation remains the only curative treatment for end-stage pulmonary disease. Lung ischemia-reperfusion injury (IRI) is a major contributor to primary allograft dysfunction and donor organ nonutilization. The alveolar macrophage is a key inflammatory mediator in IRI.  lung perfusion (EVLP) has been investigated to rehabilitate lungs before transplant but has failed to provide significant improvements after IRI. We hypothesized that liquid ventilation (LV) could be utilized for  lung reconditioning in a rat IRI model. We compared EVLP with LV in an isolated  rat lung with an aqueous ventilant using quantitative physiological and immunological parameters. We observed improved physiological parameters and mechanical clearance of alveolar macrophages and cytokines halting the propagation of the inflammatory response in IRI. While the wide applicability to large animal or human transplantation have yet to be explored, these findings represent a method for lung reconditioning in the setting of significant IRI that could widen the lung organ donation pool and limit morbidity and mortality associated with ischemia-induced primary graft dysfunction.

Liquid Ventilation Reconditions Isolated Rat Lungs Following Ischemia-Reperfusion Injury.

The COVID-19 pandemic in parallel with concerns about bias in grading resulted in many medical schools adopting pass/fail clinical grading and relying solely on narrative assessments. However, narratives often contain bias and lack specificity. The purpose of this project was to develop asynchronous faculty development to rapidly educate/re-educate > 2000 clinical faculty spread across geographic sites and clinical disciplines on components of a well-written narrative and methods to minimize bias in the assessment of students.

Creation of an asynchronous faculty development curriculum on well-written narrative assessments that avoid bias.

Partial heart transplantation is a new approach to deliver growing heart valve implants. Partial heart transplants differ from heart transplants because only the part of the heart containing the necessary heart valve is transplanted. This allows partial heart transplants to grow, similar to the valves in heart transplants. However, the transplant biology of partial heart transplantation remains unexplored. This is a critical barrier to progress of the field. Without knowledge about the specific transplant biology of partial heart transplantation, children with partial heart transplants are empirically treated like children with heart transplants because the valves in heart transplants are known to grow. In order to progress the field, an animal model for partial heart transplantation is necessary. Here, we contribute our surgical protocol for partial heart transplantation in growing piglets. All aspects of partial heart transplantation, including the donor procedure, the recipient procedure, and recipient perioperative care are described in detail. There are important nuances in the conduct of virtually all aspects of open heart surgery that differs in piglets from humans. Our surgical protocol, which is based on our experience with 34 piglets, will allow other investigators to leverage our experience to seek fundamental knowledge about the nature of partial heart transplants. This is significant because the partial heart transplant model in piglets is complex and very resource intensive.

Surgical Protocol for Partial Heart Transplantation in Growing Piglets.

Partial heart transplantation (PHT) is a novel surgical approach that involves transplantation of only the part of the heart containing a valve. The rationale for this approach is to deliver growing heart valve implants that reduce the need for future re-operations in children. However, prior to clinical application of this approach, it was important to assess it in a preclinical model. To investigate PHT short-term outcomes and safety, we performed PHT in a piglet model. Yorkshire piglets (n = 14) were used for PHT of the pulmonary valve. Donor and recipient pairs were matched based on blood types. The piglets underwent PHT at an average age of 44 days (range 34-53). Post-operatively, the piglets were monitored for a period of two months. Of the 7 recipient piglets, one mortality occurred secondary to anesthesia complications while undergoing a routine echocardiogram on post-operative day 19. All piglets had appropriate weight gain and laboratory findings throughout the post-operative period indicating a general state of good health and rehabilitation after undergoing PHT. We conclude that PHT has good short-term survival in the swine model. PHT appears to be safe for clinical application.

Survival after partial heart transplantation in a piglet model.

Traditional heart valve replacement options lack growth potential, limiting their long-term effectiveness in pediatric patients. Partial heart transplantation is an emerging approach that offers growth-capable valves by transplanting only the valve-containing segment of the heart. We report on the effects of extended cold storage on the viability and growth potential of partial heart transplantation grafts.

Partial heart transplant valves demonstrate growth in piglets despite prolonged cold storage.

Existing mass casualty incident (MCI) simulations rely on high-fidelity patient simulators, which are cost-prohibitive and often exclude pediatric patients. To address the need for deployable, low-fidelity pediatric MCI simulations, we developed and evaluated a cost-conscious model to teach the principles of JumpSTART, the pediatric variation of the Simple Triage and Rapid Treatment (START) algorithm.

Low-Fidelity, In Situ, Accessible Pediatric Mass Casualty Incident Simulation to Evaluate and Improve Pediatric Readiness.

To assess real-world use of adjuvant nivolumab in patients with esophageal/gastroesophageal junction cancer.

Article	Year
A 3D Poly(ethylene glycol)-based Tumor Angiogenesis Model to Study the Influence of Vascular Cells on Lung Tumor Cell Behavior. Scientific reports\| PubMed ID: 27596933	2016
Characterization of an elastase-induced emphysema model in immune-deficient rats. European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery\| PubMed ID: 33141186	2020
Lung Adenocarcinoma Cell Responses in a 3D in Vitro Tumor Angiogenesis Model Correlate with Metastatic Capacity. ACS biomaterials science & engineering\| PubMed ID: 33418731	2018
High-Throughput Culture Method of Induced Pluripotent Stem Cell-Derived Alveolar Epithelial Cells. Tissue engineering. Part C, Methods\| PubMed ID: 34751582	2021
Liquid Ventilation Reconditions Isolated Rat Lungs Following Ischemia-Reperfusion Injury. Tissue engineering. Part A\| PubMed ID: 35946070	2022
Creation of an asynchronous faculty development curriculum on well-written narrative assessments that avoid bias. BMC medical education\| PubMed ID: 37060081	2023
Surgical Protocol for Partial Heart Transplantation in Growing Piglets. World journal for pediatric & congenital heart surgery\| PubMed ID: 38780414	2024
Survival after partial heart transplantation in a piglet model. Scientific reports\| PubMed ID: 38811656	2024
Partial heart transplant valves demonstrate growth in piglets despite prolonged cold storage. The Journal of thoracic and cardiovascular surgery\| PubMed ID: 40154775	2025
Low-Fidelity, In Situ, Accessible Pediatric Mass Casualty Incident Simulation to Evaluate and Improve Pediatric Readiness. MedEdPORTAL : the journal of teaching and learning resources\| PubMed ID: 40583862	2025
Real-world use of adjuvant nivolumab among patients with esophageal and gastroesophageal junction cancer at a large integrated health system. JTCVS open\| PubMed ID: 41473061	2025

Sydney Jeffs

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