perifusion and calcium imaging of human pancreatic tissue slices for pancreatic disease research

Slicing Human Pancreas for Islet Function Research

Studies on islet physiology are fundamental for understanding the pathogenesis of diabetes and developing new therapeutic approaches. So far, research has relied on isolated islets, which exposes the islets to mechanical and enzymatic stresses, likely causing changes in cell physiology; moreover, it is not possible to evaluate islet function in the context of their natural tissue environment, which is likely impacted by exocrine and vascular cells among others1. When studying pancreas from donors with T1D, there is the challenge that their islets are difficult to isolate and might become fragmented during the isolation, which might produce a selection effect on islets that may not represent the population in vivo2. Moreover, islets will be separated from their complex environment and cellular connections, especially from the infiltrating immune cells that are found in inflamed islets and are more abundant in the islet periphery. Thus, while isolated islets are a cornerstone tool in diabetes research, there are limitations. In response, we introduce a groundbreaking protocol for generating living pancreas slices, offering a solution to these challenges.
The recent development and adoption of pancreatic tissue slicing techniques is considered a breakthrough for our ability to explore the intricate biology and functions of the pancreas. This innovation has opened new avenues for dynamic studies of islet physiology and interactions among endocrine, exocrine, neural, vascular, and immune cells within their natural anatomical context. Unlike conventional approaches, this in vitro setting preserves much of the organ&#39;s cytoarchitecture, allowing for a closer approximation to its native biology. Initially developed in mice by Speier and Rupnik in 20033, this method has demonstrated its utility to assess calcium imaging, electrophysiology, and hormone secretion for both intra- and inter-cellular signaling4,5,6,7,8,9. The pancreas slice platform was then applied to the study of human pancreas tissue obtained via surgical biopsy4,10,11,12. Our group demonstrated the feasibility of obtaining and utilizing pancreas slices from cadaveric organ donors through the activity of the Network for Pancreatic Organ Donors with Diabetes (nPOD)13. nPOD provides pancreas tissues to approved investigators conducting research about human type 1 diabetes, and since adopting the pancreas slice platform nPOD routinely generates and distributes living pancreas slices14,15,16,17. Since the implementation of the pancreas slice platform in 2020, nPOD has successfully distributed tissue slices from 43 donors (including 12 donors with T1D) to numerous investigators. Using these slices, researchers performed groundbreaking research on critical aspects of islet function and explored the interplay between islets and the vasculature, nervous system, and immune cells in the context of T1D13,18,19,20,21,22,23,24. Numerous studies have highlighted the limitations of traditional approaches and underscored the significance of techniques that can capture the dynamic interplay within the pancreas25,26,27. The adaptability of slicing techniques from mouse to human pancreas, coupled with their integration into programs like the Network for Pancreatic Organ Donors with Diabetes (nPOD), exemplifies the growing acknowledgment of the method&#39;s potential to unlock valuable insights into diseases such as Type 1 diabetes.

Author Spotlight: Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology

Our focus is on type-one diabetes research, emphasizing therapies that regulate the immune system and restore beta/alpha cell functions. We have developed a unique study platform using living human pancreas slices from organ donations, complementing isolated islet research. This approach enables detailed analysis of vital cellular interactions and functions.Our collaboration with a network for pancreatic organ donors offers community investigators access to pancreatic tissue slices. However, culturing these slices presents challenges, as digestive enzymes can degrade the tissue and reduce viability. Therefore, there's a pressing need to establish culture conditions that maintain both viability and a physiological environment for accurate functional studies.Our protocol enables the collection of functional data on all cells within the pancreas, so investigators can use it for a variety of studies. Our own focus is on modeling type-one diabetes and evaluating mechanisms of actions for drugs. A key question for me is to address alpha cell function and their role in type-one diabetes.Our protocol not only overcomes the survivor bias associated with isolated islets but also uniquely facilitates the study of immune cells crucial in the context of type-one diabetes. By studying whole pancreas tissue, we can assess function and responses that more closely reflect the in-vivo environment. We're committed to sharing advancements in the pancreas slice platform with others.Through nPOD, our goal is to encourage collaboration, enabling more effective addressing of critical questions. By modeling disease mechanisms and testing potential therapeutic drugs, we aim to contribute significantly to the advancement on novel treatments.

Perifusion and Calcium Imaging of Human Pancreatic Tissue Slices for Pancreatic Disease Research

perifusion-calcium-imaging-human-pancreatic-tissue-slices-for

Research

JoVE Journal

Biology

218 Görüntüleme.  Arthur Riggs Diabetes and Metabolism Research Institute. Başlamak için perifüzyon makinesini açın. Perifüzyon yazılımını başlatın, oda sayısını girin, ardından giriş solüsyonlarını etiketleyin. Şimdi perifüzyon makinesinin giriş ve çıkış borularını bağlayın.Sonraki yer, stereo mikroskop altında üç agaroz gömülü insan pankreas dokusu dilimi. Dokuların agarozunu en aza indirmek için bir fırça ve neşter kullanın. Bir dilim odasının ızgarasına bir damla taban çizgisi arabelleği ekleyin.Her ...

Video: Pankreas Hastalığı Araştırmaları için İnsan Pankreas Dokusu Dilimlerinin Perifüzyon ve Kalsiyum Görüntülemesi

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the ...