Long-term culture of human pancreatic slices as a model to study real-time islet regeneration

Abstract

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas.

Functional Characterization of the Human Islet Microvasculature Using Living Pancreas Slices

“…We obtained human living pancreas slices from de-identified cadaveric donors (from the head of the pancreas, n = 6 non-diabetic individuals, male and female, ages from 20–59 years old; information on the donors used in this study is provided in Table S1 ) from the Network of Pancreatic Organ Donors with Diabetes (nPOD) or sliced locally at the Diabetes Research Institute (University of Miami). Slices produced by nPOD were shipped overnight from Gainesville to Miami and used 3 h after arrival, while slices produced locally were cultured overnight and used the day after ( 26 , 27 ).…”

“…Pancreatic slices were incubated with calcein-AM (to label live cells in green) and ethidium homodimer-1 (labels dead cells in red). Addition of both reagents was done according to the manufacturer’s recommendations as part of the Live/Dead viability/cytotoxicity kit for mammalian cells [Invitrogen, Carlsbad, CA, Cat# L3224; ( 27 )]. Endocrine cell calcium responses to KCl depolarization (25 mM; protocol of loading with calcium indicator is below) were used as an additional readout of tissue viability.…”

“…We have previously used this platform to monitor microvascular responses in mouse islets ex vivo ( 11 , 22 ). Similar to mouse pancreas slices, living human pancreas slices can also be produced from small pancreas pieces and used in physiological experiments acutely after slicing ( 26 ) or after long-term culture [at least 10 days ( 27 )]. We have recently performed dynamic hormone secretion assays and imaged intracellular calcium levels in living human pancreas slices to characterize endocrine cell function in individuals at different stages of type 1 diabetes ( 26 ).…”

Context Matters: NOTCH Signatures and Pathway in Cancer Progression and Metastasis

“…Altered Notch/EMT genes were also associated with increased genomic instability and mutation load ( Figure 4 b). The association of Notch mutations with TNBC is not novel, and has been independently observed and confirmed in several studies [ 40 , 107 ]. In this fashion, cell- and tissue-specific epithelial differentiation and hormone-regulated maturation, e.g., in BrCa, are opposed to de-differentiation, concomitant with increased tumor cell aggressiveness or invasion, EMT, and (possibly) metastasis.…”

“…It now appears that most NOTCH mutations found in BrCa represent GOF mutations, often linked to a simultaneous loss of hormone receptor functions [ 39 ]. This is specifically the case in the aggressive and often treatment-resistant triple-negative (TNBC) and basal-like carcinomas of the breast [ 40 ]. The aggressive behavior of these cancers may be further promoted by concomitant BRCA1 or BRCA2 mutations, cooperating with Notch signaling towards cancer progression.…”