Intercellular Mitochondrial Transfer Using 3D Bioprinting

Mitochondria are one of the most complex and vital organelles in eukaryotic cells. In recent years, it has been shown that through intercellular mitochondrial transfer, this important organelle provides a critical role in tissue homeostasis, damaged tissue repair, and tumor progression under physiological conditions. However, the mechanism of mitochondrial transfer and its effect on various cellular microenvironments has not yet been defined. Understanding the metabolic effects of mitochondrial transfer between cells and exploring the signaling leading to the intercellular mechanisms could provide advancements in both translational medicine and cell therapy for cancer progression and age-related diseases. Our group has studied the ability of the normal mammary microenvironment to redirect cancer cells to a normal mammary epithelial cell fate both in vivo and in vitro using our 3D bioprinting system. Therefore, we sought to determine if mitochondrial transfer may play a role in mammary epithelium induced redirection of cancer cells. We used MCF-7 breast cancer cells and MCF-12a epithelial breast cells for experimentation. Using a fluorescent GFP-MITO lentivirus, we were able to mark mitochondrial protein in the MCF-12a epithelial cells to track mitochondrial transfer activity. The MCF-7 cells were labeled red to distinguish the two cell types. The cells were then co-cultured in 2D tissue flasks and printed into hydrogels using the 3D bioprinter. Using fluorescent microscopy, mitochondrial protein was observed traveling from epithelial to mammary cancer cells. We hypothesize this is done for cancer cells to stabilize mitochondria and improve metabolic function and ATP production. Further research to establish mitochondrial transfer, its mechanism(s), and molecular effects could lead insight into how this cellular communication rescues and normalizes metabolic factors of the mammary and stem cell microenvironment leading to potential fate redirection and cellular revitalization.https://digitalcommons.odu.edu/gradposters2022_healthsciences/1010/thumbnail.jp

Combined 3D Bioprinting and Tissue-Specific ECM System Reveals the Influence of Brain Matrix on Stem Cell Differentiation

We have previously shown that human and murine breast extracellular matrix (ECM) can significantly impact cellular behavior, including stem cell fate determination. It has been established that tissue-specific extracellular matrix from the central nervous system has the capacity to support neuronal survival. However, the characterization of its influence on stem cell differentiation and its adaptation to robust 3D culture models is underdeveloped. To address these issues, we combined our 3D bioprinter with hydrogels containing porcine brain extracellular matrix (BMX) to test the influence of the extracellular matrix on stem cell differentiation. Our 3D bioprinting system generated reproducible 3D neural structures derived from mouse embryonic stem cells (mESCs). We demonstrate that the addition of BMX preferentially influences 3D bioprinted mESCs towards neural lineages compared to standard basement membrane (Geltrex/Matrigel) hydrogels alone. Furthermore, we demonstrate that we can transplant these 3D bioprinted neural cellular structures into a mouse’s cleared mammary fat pad, where they continue to grow into larger neural outgrowths. Finally, we demonstrate that direct injection of human induced pluripotent stem cells (hiPSCS) and neural stem cells (NSCs) suspended in pure BMX formed neural structures in vivo. Combined, these findings describe a unique system for studying brain ECM/stem cell interactions and demonstrate that BMX can direct pluripotent stem cells to differentiate down a neural cellular lineage without any additional specific differentiation stimuli

Protocol for a randomised controlled trial comparing warfarin with no oral anticoagulation in patients with atrial fibrillation on chronic dialysis: the Danish Warfarin-Dialysis (DANWARD) trial

INTRODUCTION: Atrial fibrillation is highly prevalent in patients on chronic dialysis. It is unclear whether anticoagulant therapy for stroke prevention is beneficial in these patients. Vitamin K-antagonists (VKA) remain the predominant anticoagulant choice. Importantly, anticoagulation remains inconsistently used and a possible benefit remains untested in randomised clinical trials comparing oral anticoagulation with no treatment in patients on chronic dialysis. The Danish Warfarin-Dialysis (DANWARD) trial aims to investigate the safety and efficacy of VKAs in patients with atrial fibrillation on chronic dialysis. The hypothesis is that VKA treatment compared with no treatment is associated with stroke risk reduction and overall benefit.METHODS AND ANALYSIS: The DANWARD trial is an investigator-initiated trial at 13 Danish dialysis centres. In an open-label randomised clinical trial study design, a total of 718 patients with atrial fibrillation on chronic dialysis will be randomised in a 1:1 ratio to receive either standard dose VKA targeting an international normalised ratio of 2.0-3.0 or no oral anticoagulation. Principal analyses will compare the risk of a primary efficacy endpoint, stroke or transient ischaemic attack and a primary safety endpoint, major bleeding, in patients allocated to VKA treatment and no treatment, respectively. The first patient was randomised in October 2019. Patients will be followed until 1 year after the inclusion of the last patient.ETHICS AND DISSEMINATION: The study protocol was approved by the Regional Research Ethics Committee (journal number H-18050839) and the Danish Medicines Agency (case number 2018101877). The trial is conducted in accordance with the Helsinki declaration and standards of Good Clinical Practice. Study results will be disseminated to participating sites, at research conferences and in peer-reviewed journals.TRIAL REGISTRATION NUMBERS: NCT03862859, EUDRA-CT 2018-000484-86 and CTIS ID 2022-502500-75-00.</p