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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I6 - Evaluation of Extracellular Vesicle and Mitochondria Supplemented Organ Storage Strategies Using Precision Cut Tissue Slices as a 3D Model

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Sophie Clarke (she/her/hers)

Undergraduate student
Carnegie Mellon University
Pittsburgh, Pennsylvania, United States

Primary Investigator(s)

PC

Phil Campbell

Research Professor in the Department of Biomedical Engineering
Carnegie Mellon University, United States

Co-Author(s)

Bingda Li (he/him/his)

PhD Candidate in Biomedical Engineering
Carnegie Mellon University
Pittsburgh, Pennsylvania, United States

Introduction: Due to the high demand and low availability of organs for transplantation, researchers are examining the use of organs from Extended Criteria Donors; however, these marginal organs have an increased susceptibility to immunological rejection and ischemia-reperfusion injury (IRI), structural and functional damage when blood flow is reinstated after ischemic conditions. Treating organs for transplantation with extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) and regulatory T-cells (Tregs) and mitochondria that have been isolated from muscle tissue are theorized to decrease immunological rejection and reduce IRI, among other possible effects that increase implantation success. Current research has proven the efficacy of using either type of EV to benefit implantation; however, synergistic outcomes of combining different types of EVs, mechanisms of action, the effects of temperature, and using both EVs and mitochondria have not yet been examined for organ transplantation. In this study, the addition of EVs and mitochondria to organ preservation solution will be explored with precision-cut tissue slices (PCTS) to mimic the in-vivo tissue-matrix environment. Preliminary results indicate that there is a synergistic effect when MSC and Treg EVs are combined; however, mitochondrial effects have yet to be explored.

Materials and

Methods: In order to observe the effects of lack of oxygen in organs removed from the body during transplantation, hypoxia is induced with cobalt chloride in Precision Cut Tissue Slices (PCTSs). An excess of cobalt chloride inhibits prolyl hydroxylases (PHDs) that normally degrade HIF-ɑ under normal oxygen conditions. Accumulated HIF-ɑ activates a cell signaling pathway that is associated with hypoxic conditions.

PCTSs from mouse lung and kidney are used as a 3D model of extracellular vesicle and mitochondria uptake. Each organ is sliced along the z-axis with a vibratome to produce 300 µm thick PCTSs utilizing custom-made 3D printed platforms. Lung and kidney cells remain in their in vivo positions within the extracellular matrix (ECM) in PCTSs, allowing for metabolic activity and overall homeostasis of the tissue to be preserved during experimentation. Overall, this yields a more precise replica of the in vivo environment.

Extracellular vesicles from mesenchymal stem cells (MSC) and regulatory T (Treg) cells are utilized during experimentation. MSC and Treg EVs are chosen for potential tissue regenerative, tissue-repair, and anti-inflammatory effects as a therapeutic. Furthermore, mitochondria are isolated from mouse skeletal muscles and are studied as a therapeutic to increase aerobic respiration in hypoxic conditions through increasing their availability.

Alamar Blue and Lactate Dehydrogenase assays are used to measure cell viability and death respectively where Alamar Blue introduces resazurin dye to be metabolized to undergo a redox reaction, producing a fluorescent product, and Lactate Dehydrogenase levels are quantified, displaying a directly proportional relationship between cellular damage and death.

Results, Conclusions, and Discussions: MSC and Treg EVs have been shown to have synergistic effects in decreasing cell death when combined under hypoxic treatment at 4C. Additionally, MSC EVs have been shown to have a protective effect when incubated in coculture with cobalt chloride at 4C. 3D printed platforms decrease variation between tissue slices, insuring a uniform thickness; additionally, these custom platforms increase the ease of slicing, which allows for more PCTSs to be produced per organ. Mitochondria can be successfully isolated from mouse skeletal tissue to be utilized to study as a therapeutic under hypoxic conditions. However, further experimentation is necessary to determine how mitochondria affect tissue viability and cellular death under hypoxic conditions. Potential implications from these results include the supplementation of organ preservation solutions with MSC and Treg EVs to decrease immunological rejection and ischemia-reperfusion injury.

Acknowledgements (Optional): I would like to thank Professor Phil Campbell for his guidance and continuous encouragement. I would also like to thank Bingda Li for his mentorship and support.