nutrient-regulation-continuous-feeding-for-large-scale-expansion

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

University of Minnesota

Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. The limited human islet supply from cadavers and poor islet yield and quality remain substantial impediments to progress in the field. Use of porcine islets holds great promise for large-scale application of islet transplantation. Consistent isolation of porcine islets is dependent on advances in pancreas procurement, pancreas preservation, and islet isolation, requiring detailed knowledge of the porcine pancreatic anatomy. The primary aim of this study was to describe the vascular and ductal anatomy of the porcine pancreas to guide and improve organ preservation and enzyme perfusion.

Pig pancreas anatomy: implications for pancreas procurement, preservation, and islet isolation.

Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications.

Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids.

Porcine islet xenotransplantation is emerging as a potential alternative for allogeneic clinical islet transplantation. Optimization of porcine islet isolation in terms of yield and quality is critical for the success and cost-effectiveness of this approach. Incomplete pancreas distention and inhomogeneous enzyme distribution have been identified as key factors for limiting viable islet yield per porcine pancreas. The aim of this study was to explore the utility of magnetic resonance imaging (MRI) as a tool to investigate the homogeneity of enzyme delivery in porcine pancreata. Traditional and novel methods for enzyme delivery aimed at optimizing enzyme distribution were examined. Pancreata were procured from Landrace pigs via en bloc viscerectomy. The main pancreatic duct was then cannulated with an 18-g winged catheter and MRI performed at 1.5-T. Images were collected before and after ductal infusion of chilled MRI contrast agent (gadolinium) in physiological saline. Regions of the distal aspect of the splenic lobe and portions of the connecting lobe and bridge exhibited reduced delivery of solution when traditional methods of distention were utilized. Use of alternative methods of delivery (such as selective re-cannulation and distention of identified problem regions) resolved these issues, and MRI was successfully utilized as a guide and assessment tool for improved delivery. Current methods of porcine pancreas distention do not consistently deliver enzyme uniformly or adequately to all regions of the pancreas. Novel methods of enzyme delivery should be investigated and implemented for improved enzyme distribution. MRI serves as a valuable tool to visualize and evaluate the efficacy of current and prospective methods of pancreas distention and enzyme delivery.

Magnetic resonance imaging: a tool to monitor and optimize enzyme distribution during porcine pancreas distention for islet isolation.

Porcine islet xenotransplantation is a promising alternative to human islet allotransplantation. Porcine pancreas cooling needs to be optimized to reduce the warm ischemia time (WIT) following donation after cardiac death, which is associated with poorer islet isolation outcomes. This study examines the effect of four different cooling Methods on core porcine pancreas temperature (n = 24) and histopathology (n = 16). All Methods involved surface cooling with crushed ice and chilled irrigation. Method A, which is the standard for porcine pancreas procurement, used only surface cooling. Method B involved an intravascular flush with cold solution through the pancreas arterial system. Method C involved an intraductal infusion with cold solution through the major pancreatic duct, and Method D combined all three cooling Methods. Surface cooling alone (Method A) gradually decreased core pancreas temperature to <10 °C after 30 min. Using an intravascular flush (Method B) improved cooling during the entire duration of procurement, but incorporating an intraductal infusion (Method C) rapidly reduced core temperature 15-20 °C within the first 2 min of cooling. Combining all methods (Method D) was the most effective at rapidly reducing temperature and providing sustained cooling throughout the duration of procurement, although the recorded WIT was not different between Methods (P = 0.36). Histological scores were different between the cooling Methods (P = 0.02) and the worst with Method A. There were differences in histological scores between Methods A and C (P = 0.02) and Methods A and D (P = 0.02), but not between Methods C and D (P = 0.95), which may highlight the importance of early cooling using an intraductal infusion. In conclusion, surface cooling alone cannot rapidly cool large (porcine or human) pancreata. Additional cooling with an intravascular flush and intraductal infusion results in improved core porcine pancreas temperature profiles during procurement and histopathology scores. These data may also have implications on human pancreas procurement as use of an intraductal infusion is not common practice.

Temperature profiles of different cooling methods in porcine pancreas procurement.

Diabetes is a major health problem worldwide and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges and young pigs (3-6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (~3 months; 25 kg Yorkshire pigs) immediately following procurement, or after 24 hours of hypothermic machine perfusion preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology and in vivo function. Islet yields were highest from HMP preserved pancreata (2242±449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 µm), and purity was on average 63±6%. Morphologically, islets appeared as clusters on day-0, loosely disaggregated structures at day-1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day-6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 175±16 nmol/min/mgDNA and physiological function was intact by glucose stimulation after 6-8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation requires specialized conditions. These immature islets undergo plasticity in culture and form fully functional multi-cellular structures. Further development of this method for culturing immature porcine islets is expected to generate small pancreatic tissuederived organoids termed "pancreatites," as a therapeutic product from juvenile pigs for xenotransplantation and diabetes research.

Article	Year
Pig pancreas anatomy: implications for pancreas procurement, preservation, and islet isolation. Transplantation\| PubMed ID: 19077881	2008
Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin. Xenotransplantation\| PubMed ID: 23384163	2013
Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids. PloS one\| PubMed ID: 24204645	2013
Magnetic resonance imaging: a tool to monitor and optimize enzyme distribution during porcine pancreas distention for islet isolation. Xenotransplantation\| PubMed ID: 24986758	2014
Temperature profiles of different cooling methods in porcine pancreas procurement. Xenotransplantation\| PubMed ID: 25040217	2014
Plasticity and Aggregation of Juvenile Porcine Islets in Modified Culture: Preliminary Observations. Cell transplantation\| PubMed ID: 27109912	2016

Bradley P. Weegman

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