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Controlled aggregation of primary human pancreatic islet cells leads to glucose-responsive pseudoislets comparable to native islets.

Hilderink J, Spijker S, Carlotti F, Lange L, Engelse M, van Blitterswijk C, de Koning E, Karperien M, van Apeldoorn A - J. Cell. Mol. Med. (2015)

Bottom Line: To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways.The re-associated human islet cells showed an a-typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets.The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell-cell interactions between insuloma and/or primary islet cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Bioengineering, University of Twente, Enschede, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of agarose microwell fabrication and cell aggregation.
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fig01: Schematic representation of agarose microwell fabrication and cell aggregation.

Mentions: Non-adherent agarose microwells were aseptically fabricated as described previously 30. Briefly, microwell chips containing 2865 microwells with a diameter of 200 μm and chips containing 1585 microwells with a diameter of 400 μm were fabricated by pouring a 3% (w/v) Ultrapure™ agarose (Invitrogen, Bleiswijk, the Netherlands) solution on negative moulds of polydimethylsiloxane (PDMS). After agarose solidification, the moulds were removed, covered with PBS and stored at 4°C until usage (see Fig.1). Before cell seeding, the agarose chips were pre-incubated in culture medium overnight at 37°C. For cell aggregate formation, single cells were resuspended in fresh medium and seeded onto agarose chips at various densities resulting in aggregates consisting of 10, 25, 50, 100, 250, 500 and 1000 cells. Immediately after seeding, agarose chips were briefly centrifuged at 300 × g to allow the cells to settle down in the microwells. As a control, 1 × 105 cells were seeded onto ultra-low attachment plastic to allow spontaneous cell aggregation. Medium was refreshed every 1–2 days. Cell aggregates were cultured up to 7 days after which they were removed from the chips by upside down centrifugation (1 min. at 300 × g) or by medium flush, and used for further analysis. To measure the average aggregate diameter, microscopic images were taken and aggregate diameter was quantified using ImageJ (NIH image). For INS-1E cell aggregates, at least 50 aggregates were measured. For human islet cell aggregates, at least 40 aggregates derived from islet preparations of three different human donors were analysed.


Controlled aggregation of primary human pancreatic islet cells leads to glucose-responsive pseudoislets comparable to native islets.

Hilderink J, Spijker S, Carlotti F, Lange L, Engelse M, van Blitterswijk C, de Koning E, Karperien M, van Apeldoorn A - J. Cell. Mol. Med. (2015)

Schematic representation of agarose microwell fabrication and cell aggregation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549034&req=5

fig01: Schematic representation of agarose microwell fabrication and cell aggregation.
Mentions: Non-adherent agarose microwells were aseptically fabricated as described previously 30. Briefly, microwell chips containing 2865 microwells with a diameter of 200 μm and chips containing 1585 microwells with a diameter of 400 μm were fabricated by pouring a 3% (w/v) Ultrapure™ agarose (Invitrogen, Bleiswijk, the Netherlands) solution on negative moulds of polydimethylsiloxane (PDMS). After agarose solidification, the moulds were removed, covered with PBS and stored at 4°C until usage (see Fig.1). Before cell seeding, the agarose chips were pre-incubated in culture medium overnight at 37°C. For cell aggregate formation, single cells were resuspended in fresh medium and seeded onto agarose chips at various densities resulting in aggregates consisting of 10, 25, 50, 100, 250, 500 and 1000 cells. Immediately after seeding, agarose chips were briefly centrifuged at 300 × g to allow the cells to settle down in the microwells. As a control, 1 × 105 cells were seeded onto ultra-low attachment plastic to allow spontaneous cell aggregation. Medium was refreshed every 1–2 days. Cell aggregates were cultured up to 7 days after which they were removed from the chips by upside down centrifugation (1 min. at 300 × g) or by medium flush, and used for further analysis. To measure the average aggregate diameter, microscopic images were taken and aggregate diameter was quantified using ImageJ (NIH image). For INS-1E cell aggregates, at least 50 aggregates were measured. For human islet cell aggregates, at least 40 aggregates derived from islet preparations of three different human donors were analysed.

Bottom Line: To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways.The re-associated human islet cells showed an a-typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets.The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell-cell interactions between insuloma and/or primary islet cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Bioengineering, University of Twente, Enschede, The Netherlands.

No MeSH data available.


Related in: MedlinePlus