Pseudoislets in stirred-suspension culture exhibit enhanced cell survival, propagation and insulin secretion

Lock, Lye T.; Laychock, Suzanne G.; Tzanakakis, Emmanuel S.

doi:10.1016/j.jbiotec.2010.12.015

Cited by 30 publications

(42 citation statements)

References 49 publications

(85 reference statements)

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“…In contrast to monolayer β-cells, PIs represent a more native structure for β cell cultures capable of improving β cell function. This improvement is partially attributed to enhanced cell-cell contact leading to increased glucose sensing, insulin production, and insulin release [4], [5], [6], [7], [8], [9]. Despite their usefulness, PI formation requires specialized culture conditions and extensive cell manipulations.…”

Section: Discussionmentioning

confidence: 99%

“…The formation of 3-D β cell aggregates, or pseudoislets (PIs), is useful for the study of β cell biology. β-cells in PIs show improved function as measured by increased insulin production and improved glucose-stimulated insulin secretion (GSIS) [4], [5], [6], [7], [8], [9], [10]. These effects are mediated in part by the formation of a 3-D configuration which enhances β-cell – cell contact [5], [9], increases calcium signaling [11], and preserves extracellular matrix (ECM) proteins [12].…”

Section: Introductionmentioning

confidence: 99%

“…Despite their usefulness, PI generation requires extensive cell handling and may take several days to form (7–14 d). Current methods for induction of PIs include the use of mechanical manipulations such as stirred cell suspension cultures [8], culturing of β-cells on gelatin coated plates [4], and hanging drop cell cultures [13].…”

Section: Introductionmentioning

confidence: 99%

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In Vitro Formation of β Cell Pseudoislets Using Islet-Derived Endothelial Cells

et al. 2013

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β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In Vitro Formation of β Cell Pseudoislets Using Islet-Derived Endothelial Cells

et al. 2013

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“…This technique is described in literature [16]–[19], [31]–[33], and was slightly modified to accommodate spheroid formation of β-TC6 cells. For all conditions, β-TC6 cells were first cultured and expanded in adherent cultures described above, until enough cells were obtained to reach the required (total n = 12) numbers for 250 ml stirred bioreactors (Corning, Corning, NY).…”

Section: Methodsmentioning

confidence: 99%

Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids

et al. 2013

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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.

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“…However, it seems likely that an additional factor limiting pseudoislet growth in vitro is hypoxia, a common consequence of culturing cell spheroids in static cultures. This view is supported by the ability of MIN6 mouse β-cell pseudoislets cultured in bioreactor with continuous stirring to grow continuously for two wk without exhibiting any signs of hypoxia, reduced functionality, or growth arrest[50]. In vivo 1.1B4 cells pseudoislets were able to quickly muster a blood supply which allowed proliferation of the cells.…”

Section: Discussionmentioning

confidence: 99%

Implanting 1.1B4 human β-cell pseudoislets improves glycaemic control in diabetic severe combined immune deficient mice

Green¹,

Vasu²,

McClenaghan³

et al. 2016

WJD

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AIMTo investigate the potential of implanting pseudoislets formed from human insulin-releasing β-cell lines as an alternative to islet transplantation.METHODSIn this study, the anti-diabetic potential of novel human insulin releasing 1.1B4 β-cells was evaluated by implanting the cells, either as free cell suspensions, or as three-dimensional pseudoislets, into the subscapular region of severe combined immune deficient mice rendered diabetic by single high-dose administration of streptozotocin. Metabolic parameters including food and fluid intake, bodyweight and blood glucose were monitored throughout the study. At the end of the study animals were given an intraperitoneal glucose tolerance test. Animals were then culled and blood and tissues were collected for analysis. Insulin and glucagon contents of plasma and tissues were measured by insulin radioimmunoassay and chemiluminescent enzyme-linked immunosorbance assay respectively. Histological analyses of pancreatic islets were carried out by quantitative fluorescence immunohistochemistry staining.RESULTSBoth pseudoislet and cell suspension implants yielded well vascularised β-cell masses of similar insulin content. This was associated with progressive amelioration of hyperphagia (P < 0.05), polydipsia (P < 0.05), body weight loss (P < 0.05), hypoinsulinaemia (P < 0.05), hyperglycaemia (P < 0.05 - P < 0.001) and glucose tolerance (P < 0.01). Islet morphology was also significantly improved in both groups of transplanted mice, with increased β-cell (P < 0.05 - P < 0.001) and decreased alpha cell (P < 0.05 - P < 0.001) areas. Whereas mice receiving 1.1B4 cell suspensions eventually exhibited hypoglycaemic complications, pseudoislet recipients displayed a more gradual amelioration of diabetes, and achieved stable blood glucose control similar to non-diabetic mice at the end of the study.CONCLUSIONAlthough further work is needed to address safety issues, these results provide proof of concept for possible therapeutic applicability of human β-cell line pseudoislets in diabetes.

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Pseudoislets in stirred-suspension culture exhibit enhanced cell survival, propagation and insulin secretion

Cited by 30 publications

References 49 publications

In Vitro Formation of β Cell Pseudoislets Using Islet-Derived Endothelial Cells

In Vitro Formation of β Cell Pseudoislets Using Islet-Derived Endothelial Cells

Nutrient Regulation by Continuous Feeding Removes Limitations on Cell Yield in the Large-Scale Expansion of Mammalian Cell Spheroids

Implanting 1.1B4 human β-cell pseudoislets improves glycaemic control in diabetic severe combined immune deficient mice

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