A novel method for the coating of pancreatic beta-cell spheroids

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Standard

A novel method for the coating of pancreatic beta-cell spheroids. / Nikravesh, Niusha; Chouhan, Gurpreet; Williams, Richard; Ghag, Anita; Grover, Liam.

10th World Biomaterials Congress. World Biomaterials Congress, 2016.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Harvard

Nikravesh, N, Chouhan, G, Williams, R, Ghag, A & Grover, L 2016, A novel method for the coating of pancreatic beta-cell spheroids. in 10th World Biomaterials Congress. World Biomaterials Congress, 10th World Biomaterials Congress, Montréal, Canada, 17/05/16. https://doi.org/10.3389/conf.FBIOE.2016.01.02857

APA

Vancouver

Author

Bibtex

@inproceedings{937bafaefa2344d4b7a72e2c45e3c746,
title = "A novel method for the coating of pancreatic beta-cell spheroids",
abstract = "Introduction: Type 1 diabetes mellitus (T1DM) is an autoimmune disorder caused by the destruction of β-cells within pancreatic islets. Currently insulin therapy is the most common treatment, however, maintaining normal glucose level with this treatment requires several daily injections, and frequent monitoring of blood glucose level[1]. Transplantation of pancreatic islets is a promising method for treating T1DM. However, efficacy of this procedure is currently restricted by various challenges including the shortage of donors, poor revascularization, immune rejection, and the need for life-long immunosuppressive drugs[2]. Immunoisolation of β-cells in polymeric membranes can address some of these issues by providing a barrier between the host and the implanted cells thus eliminating the need for immunosuppressives. In this study, we have coated the surface of uniform β-cell spheroids with layers of anionic (alginate) and cationic polymers (Poly-L-Lysine; PLL) that interact through electrostatic forces. The method employed here has been designed to minimise the toxicity of the PLL and maximise viability of the encapsulated cells by reducing the diffusion path from the exterior of the shell to the embedded spheroid.Experimental Methods: Mouse Beta-TC-6 cells (ATCC-CRL 11506) were cultured in supplemented DMEM and incubated at 37 in 5% CO2. β-cells spheroids were prepared using 3D agarose micro-well dishes. After 5 days of culturing cells in wells, the deposition of polymers on cells surface occurred following treatment with 100mM of CaCl2. This step enabled the addition of alginate (1mg/ml) as the first coating layer, followed by the addition of PLL and alginate, respectively. Furthermore, the viability of coated β-cells spheroids were compared with conventional coating method of using the cationic polymer as the first layer. The functionality of the β-cell spheroids to secret insulin was then investigated using the glucose simulated insulin secretion (GSIS) assay.Results: The prepared spheroids had an average diameter of 150±9.8μm, which can be assumed as one islet equivalent (IEQ). In addition, images from scanning electron microscopy confirm the cell-cell interactions on β-cell spheroids surface. Moreover, labelling the cell spheroids showed the ability of cells to proliferate inside agarose wells without any negative effect. After coating the spheroids, fluorescent and confocal microscopy (figure-1) demonstrated that applying alginate as the first coating layer improved cell viability more than 20% over 7 days in culture when compared to coating with PLL as the first layer. Furthermore, the GSIS assay confirmed the ability of cell spheroids to secret insulin in response to change in glucose concentration. The secretion of insulin granules were also investigated using immunohistochemical studies. Conclusion: In this study we demonstrated a novel method to encapsulate and maintain viability of β-cells for 14 days. The viability and functionality analysis showed that treating β-cells with 100mM CaCl2 prior to coating with alginate then PLL allowed for enhanced viability when compared with the usual method of coating first with PLL. The protection of the islets with the CaCl2 ensured that the toxic effects caused by exposure of the cell membrane to the PLL molecules were minimised. In future studies we will seek to further enhance the viability of coated cells in-vivo by modifying the layered structure with an angiogenic agent.",
keywords = "microstructure, polymer, gel, Cell interaction",
author = "Niusha Nikravesh and Gurpreet Chouhan and Richard Williams and Anita Ghag and Liam Grover",
note = "Nikravesh N, Chouhan G, Williams R, Ghag A and Grover L (2016). A novel method for the coating of pancreatic beta-cell spheroids. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02857; 10th World Biomaterials Congress ; Conference date: 17-05-2016 Through 22-05-2016",
year = "2016",
month = mar,
day = "30",
doi = "10.3389/conf.FBIOE.2016.01.02857",
language = "English",
booktitle = "10th World Biomaterials Congress",
publisher = "World Biomaterials Congress",
url = "https://www.frontiersin.org/events/10th_World_Biomaterials_Congress/2893",

}

RIS

TY - GEN

T1 - A novel method for the coating of pancreatic beta-cell spheroids

AU - Nikravesh, Niusha

AU - Chouhan, Gurpreet

AU - Williams, Richard

AU - Ghag, Anita

AU - Grover, Liam

N1 - Nikravesh N, Chouhan G, Williams R, Ghag A and Grover L (2016). A novel method for the coating of pancreatic beta-cell spheroids. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02857

PY - 2016/3/30

Y1 - 2016/3/30

N2 - Introduction: Type 1 diabetes mellitus (T1DM) is an autoimmune disorder caused by the destruction of β-cells within pancreatic islets. Currently insulin therapy is the most common treatment, however, maintaining normal glucose level with this treatment requires several daily injections, and frequent monitoring of blood glucose level[1]. Transplantation of pancreatic islets is a promising method for treating T1DM. However, efficacy of this procedure is currently restricted by various challenges including the shortage of donors, poor revascularization, immune rejection, and the need for life-long immunosuppressive drugs[2]. Immunoisolation of β-cells in polymeric membranes can address some of these issues by providing a barrier between the host and the implanted cells thus eliminating the need for immunosuppressives. In this study, we have coated the surface of uniform β-cell spheroids with layers of anionic (alginate) and cationic polymers (Poly-L-Lysine; PLL) that interact through electrostatic forces. The method employed here has been designed to minimise the toxicity of the PLL and maximise viability of the encapsulated cells by reducing the diffusion path from the exterior of the shell to the embedded spheroid.Experimental Methods: Mouse Beta-TC-6 cells (ATCC-CRL 11506) were cultured in supplemented DMEM and incubated at 37 in 5% CO2. β-cells spheroids were prepared using 3D agarose micro-well dishes. After 5 days of culturing cells in wells, the deposition of polymers on cells surface occurred following treatment with 100mM of CaCl2. This step enabled the addition of alginate (1mg/ml) as the first coating layer, followed by the addition of PLL and alginate, respectively. Furthermore, the viability of coated β-cells spheroids were compared with conventional coating method of using the cationic polymer as the first layer. The functionality of the β-cell spheroids to secret insulin was then investigated using the glucose simulated insulin secretion (GSIS) assay.Results: The prepared spheroids had an average diameter of 150±9.8μm, which can be assumed as one islet equivalent (IEQ). In addition, images from scanning electron microscopy confirm the cell-cell interactions on β-cell spheroids surface. Moreover, labelling the cell spheroids showed the ability of cells to proliferate inside agarose wells without any negative effect. After coating the spheroids, fluorescent and confocal microscopy (figure-1) demonstrated that applying alginate as the first coating layer improved cell viability more than 20% over 7 days in culture when compared to coating with PLL as the first layer. Furthermore, the GSIS assay confirmed the ability of cell spheroids to secret insulin in response to change in glucose concentration. The secretion of insulin granules were also investigated using immunohistochemical studies. Conclusion: In this study we demonstrated a novel method to encapsulate and maintain viability of β-cells for 14 days. The viability and functionality analysis showed that treating β-cells with 100mM CaCl2 prior to coating with alginate then PLL allowed for enhanced viability when compared with the usual method of coating first with PLL. The protection of the islets with the CaCl2 ensured that the toxic effects caused by exposure of the cell membrane to the PLL molecules were minimised. In future studies we will seek to further enhance the viability of coated cells in-vivo by modifying the layered structure with an angiogenic agent.

AB - Introduction: Type 1 diabetes mellitus (T1DM) is an autoimmune disorder caused by the destruction of β-cells within pancreatic islets. Currently insulin therapy is the most common treatment, however, maintaining normal glucose level with this treatment requires several daily injections, and frequent monitoring of blood glucose level[1]. Transplantation of pancreatic islets is a promising method for treating T1DM. However, efficacy of this procedure is currently restricted by various challenges including the shortage of donors, poor revascularization, immune rejection, and the need for life-long immunosuppressive drugs[2]. Immunoisolation of β-cells in polymeric membranes can address some of these issues by providing a barrier between the host and the implanted cells thus eliminating the need for immunosuppressives. In this study, we have coated the surface of uniform β-cell spheroids with layers of anionic (alginate) and cationic polymers (Poly-L-Lysine; PLL) that interact through electrostatic forces. The method employed here has been designed to minimise the toxicity of the PLL and maximise viability of the encapsulated cells by reducing the diffusion path from the exterior of the shell to the embedded spheroid.Experimental Methods: Mouse Beta-TC-6 cells (ATCC-CRL 11506) were cultured in supplemented DMEM and incubated at 37 in 5% CO2. β-cells spheroids were prepared using 3D agarose micro-well dishes. After 5 days of culturing cells in wells, the deposition of polymers on cells surface occurred following treatment with 100mM of CaCl2. This step enabled the addition of alginate (1mg/ml) as the first coating layer, followed by the addition of PLL and alginate, respectively. Furthermore, the viability of coated β-cells spheroids were compared with conventional coating method of using the cationic polymer as the first layer. The functionality of the β-cell spheroids to secret insulin was then investigated using the glucose simulated insulin secretion (GSIS) assay.Results: The prepared spheroids had an average diameter of 150±9.8μm, which can be assumed as one islet equivalent (IEQ). In addition, images from scanning electron microscopy confirm the cell-cell interactions on β-cell spheroids surface. Moreover, labelling the cell spheroids showed the ability of cells to proliferate inside agarose wells without any negative effect. After coating the spheroids, fluorescent and confocal microscopy (figure-1) demonstrated that applying alginate as the first coating layer improved cell viability more than 20% over 7 days in culture when compared to coating with PLL as the first layer. Furthermore, the GSIS assay confirmed the ability of cell spheroids to secret insulin in response to change in glucose concentration. The secretion of insulin granules were also investigated using immunohistochemical studies. Conclusion: In this study we demonstrated a novel method to encapsulate and maintain viability of β-cells for 14 days. The viability and functionality analysis showed that treating β-cells with 100mM CaCl2 prior to coating with alginate then PLL allowed for enhanced viability when compared with the usual method of coating first with PLL. The protection of the islets with the CaCl2 ensured that the toxic effects caused by exposure of the cell membrane to the PLL molecules were minimised. In future studies we will seek to further enhance the viability of coated cells in-vivo by modifying the layered structure with an angiogenic agent.

KW - microstructure

KW - polymer

KW - gel

KW - Cell interaction

U2 - 10.3389/conf.FBIOE.2016.01.02857

DO - 10.3389/conf.FBIOE.2016.01.02857

M3 - Conference contribution

BT - 10th World Biomaterials Congress

PB - World Biomaterials Congress

T2 - 10th World Biomaterials Congress

Y2 - 17 May 2016 through 22 May 2016

ER -