Rat Pancreatic Islet Size Standardization by the “Hanging Drop” Technique

Cavallari, Giuseppe; Zuellig, Richard A.; Lehmann, Roger; Weber, Markus; Moritz, Wolfgang

doi:10.1016/j.transproceed.2007.05.016

Cited by 60 publications

(73 citation statements)

References 9 publications

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“…Recognizing this requirement, different approaches have been developed that encourage or increase cell-cell contact for b-cells, especially before cell encapsulation. [13][14][15][16] In this work, we present a method for controlling the aggregation of MIN6 cells into well-defined cluster sizes using PEG microwell devices formed by standard contact photolithographic techniques. The bioinertness of PEG provides a noncytotoxic environment that can be easily manipulated, sterilized, and used for cell culture.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, a hanging-drop method has been used to reaggregate islets into relatively uniform spherical clusters of *100 mm in diameter. 15 However, this method has not shown the versatility needed to create stable aggregates of different sizes and is instead limited to a narrow size range for the aggregates. 15,16 To address some of these limitations, dielectrophoresis has been used to aggregate insulinoma cells into at least two different-sized three-dimensional (3D) constructs.…”

mentioning

confidence: 99%

“…15 However, this method has not shown the versatility needed to create stable aggregates of different sizes and is instead limited to a narrow size range for the aggregates. 15,16 To address some of these limitations, dielectrophoresis has been used to aggregate insulinoma cells into at least two different-sized three-dimensional (3D) constructs. Radio frequency voltages applied to specific electrodes generate dielectrophorectic forces, which, when properly matched to the cell membrane capacitance and conductance, can be used to condense a single-cell suspension of b-cells into cell clusters of defined sizes.…”

mentioning

confidence: 99%

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A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells

Bernard

Lin

Anseth

2012

Tissue Engineering Part C: Methods

119

138

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Cell-cell contact between pancreatic b-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between b-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) b-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D b-cell aggregates of defined sizes from 25 to 210 mm in diameter. Using this platform, mouse insulinoma 6 (MIN6) b-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability ( > 90%) over 7 days in culture, while the b-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single b-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional b-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells

Bernard

Lin

Anseth

2012

Tissue Engineering Part C: Methods

119

138

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“…However, equivalent studies have been performed more recently with either glucagon (alphaTC)-or somatostatin (TGP52)-secreting cells (Ishihara et al 2003, Brereton et al 2006, Kelly et al 2010b. The resulting rat or murine pseudoislet structures resembled rodent pancreatic islets in both size and morphology (Hauge-Evans et al 1999, Cavallari et al 2007, Kelly et al 2010a) and displayed enhanced secretory properties compared with respective monolayer cells as a result of homotypic cellular interactions (Bosco et al 1989, Hauge-Evans et al 1999, Luther et al 2006, Kitsou-Mylona et al 2008, Kelly et al 2010a. Thus, although glucagon and somatostatin are thought to be involved in the regulation of b-cell function in native islets, homologous b-cell interactions appear sufficient for normal insulin secretion within pseudoislets (Bosco et al 1989, Hauge-Evans et al 1999, Brereton et al 2006, Luther et al 2006.…”

Section: Introductionmentioning

confidence: 99%

Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility

Guo-Parke¹,

McCluskey²,

Kelly³

et al. 2012

Journal of Endocrinology

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Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human b-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 mm in diameter, corresponding to w6000 b cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1 . 7-to 12 . 5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human b cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.

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“…Islets were harvested from pancreata of male Wistar rats (250 to 300 g) by collagenase (NB8 collagenase, Serva, Heidelberg, Germany) followed by trypsin digestion to dissociate them into single cells, according to (13).…”

Section: Animal Experimentsmentioning

confidence: 99%

Deoxysphingolipids, a novel biomarker for type 2 diabetes, are cytotoxic for insulin-producing cells

Sonda

Züllig²,

Hornemann

et al. 2013

Pancreatology

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Irreversible failure of pancreatic -cells is the main culprit in the pathophysiology of diabetes mellitus, a disease that is now a major global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as novel biomarkers for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulinproducing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the RhoGTPase Rac1. Moreover, 1-deoxysphinganine selectively up-regulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides, without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified JNK and p38 MAPK as antagonistic effectors of cellular senescence. Our results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic -cells. Thus, targeting deoxy-sphingolipid synthesis may complement the currently available therapies of diabetes. Analyses of signaling pathways identified JNK and p38 MAPK as antagonistic effectors of cellular senescence. Our results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulinproducing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic β-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies of diabetes.

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Rat Pancreatic Islet Size Standardization by the “Hanging Drop” Technique

Cited by 60 publications

References 9 publications

A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells

A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells

Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility

Deoxysphingolipids, a novel biomarker for type 2 diabetes, are cytotoxic for insulin-producing cells

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