Limits...
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

Seeding density determines aggregate size of insulinoma and human islet cell aggregates. (A) INS-1E cell aggregates of 50, 100, and 500 cells after 7 days of culture inside agarose microwells (top panel) and after flushing out of the chip (bottom panel); scale bar = 200 μm. (B) Correlation between number of INS-1E and MIN6 cells per microwell and diameter of obtained aggregates after 7 days of culture. INS-1E and MIN6 cells were seeded at 10, 25, 50, 100, 250 and 500 cells per microwell. Each datapoint represents the average measurement of at least 50 aggregates per condition; error bars represent ±SD. (C) Primary human islets were dispersed and single islet cells were seeded at 100, 500 and 1000 cells per microwell. Figures represent human islet cell aggregates inside the microwells (top panel) and after flushing them out of the chip (bottom panel) after 7 days of culture; scale bar = 200 μm. (D) The diameter of human islet cell aggregates increased with increasing the number of cells seeded per well. Cells were seeded at 100, 500 and 1000 cells per microwell. At least 40 human islet cell aggregates were measured per condition. Data points represent the average values for three human donors; error bars represent ±SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549034&req=5

fig03: Seeding density determines aggregate size of insulinoma and human islet cell aggregates. (A) INS-1E cell aggregates of 50, 100, and 500 cells after 7 days of culture inside agarose microwells (top panel) and after flushing out of the chip (bottom panel); scale bar = 200 μm. (B) Correlation between number of INS-1E and MIN6 cells per microwell and diameter of obtained aggregates after 7 days of culture. INS-1E and MIN6 cells were seeded at 10, 25, 50, 100, 250 and 500 cells per microwell. Each datapoint represents the average measurement of at least 50 aggregates per condition; error bars represent ±SD. (C) Primary human islets were dispersed and single islet cells were seeded at 100, 500 and 1000 cells per microwell. Figures represent human islet cell aggregates inside the microwells (top panel) and after flushing them out of the chip (bottom panel) after 7 days of culture; scale bar = 200 μm. (D) The diameter of human islet cell aggregates increased with increasing the number of cells seeded per well. Cells were seeded at 100, 500 and 1000 cells per microwell. At least 40 human islet cell aggregates were measured per condition. Data points represent the average values for three human donors; error bars represent ±SD.

Mentions: Controlled aggregation using these microwells was further optimized using MIN6 clone B1 and INS-1E insulinoma cell lines. These insulin-producing cell lines are widely used as a model for primary beta cells since they resemble most native beta cells 31,32. Cells were seeded onto microwell chips at various densities to generate aggregates of 10, 25, 50, 100, 250 and 500 cells, resulting in aggregates with well-defined dimensions. The aggregates could easily be removed from the chips by flushing out and mild centrifugation (Fig.3A), after which they were used for further analysis. The average aggregate diameter correlates with the initial number of cells seeded per microwell, which ranged between 29 and 105 μm for INS-1E cells, and between 28 and 93 μm for MIN6 clone B1 cells, as shown in Figure3B. Increasing the number of cells per microwell to 1000 resulted in unstable cell aggregates that were not suitable for further use. Next to controlled aggregation of MIN6 and INS-1E cell lines, our microwell platform supports the controlled reassembly of primary human islet cells. Human donor islets of Langerhans were dissociated into a single cell suspension (>95% viability, data not shown) and seeded at 100, 500 and 1000 cells per microwell to induce controlled aggregation similar to the insuloma cell lines. The resulting primary islet cell aggregates were uniform in size and remained intact after harvesting from the chips (Fig.3C). After 7 days of culture, the islet cell aggregates had an average diameter of 80 ± 5.4, 105 ± 11.3 and 134 ± 15.3 μm respectively, depending on the initial cell number seeded (Fig.3D). To study cell assembly kinetics, representative images were taken at different time-points during aggregate formation. We observed that aggregation occurs in the first 2–4 days after cell seeding, and stable aggregates are obtained after 7–14 days of culture (Fig. S2). Since 200 μm diameter microwells cannot contain more than 1000 cells, we created wells with a diameter of 400 μm. Increasing the cell seeding density in these microwells to 2000, we observed that islet cells assembled uncontrollably into multiple smaller aggregates per well.


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)

Seeding density determines aggregate size of insulinoma and human islet cell aggregates. (A) INS-1E cell aggregates of 50, 100, and 500 cells after 7 days of culture inside agarose microwells (top panel) and after flushing out of the chip (bottom panel); scale bar = 200 μm. (B) Correlation between number of INS-1E and MIN6 cells per microwell and diameter of obtained aggregates after 7 days of culture. INS-1E and MIN6 cells were seeded at 10, 25, 50, 100, 250 and 500 cells per microwell. Each datapoint represents the average measurement of at least 50 aggregates per condition; error bars represent ±SD. (C) Primary human islets were dispersed and single islet cells were seeded at 100, 500 and 1000 cells per microwell. Figures represent human islet cell aggregates inside the microwells (top panel) and after flushing them out of the chip (bottom panel) after 7 days of culture; scale bar = 200 μm. (D) The diameter of human islet cell aggregates increased with increasing the number of cells seeded per well. Cells were seeded at 100, 500 and 1000 cells per microwell. At least 40 human islet cell aggregates were measured per condition. Data points represent the average values for three human donors; error bars represent ±SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: Seeding density determines aggregate size of insulinoma and human islet cell aggregates. (A) INS-1E cell aggregates of 50, 100, and 500 cells after 7 days of culture inside agarose microwells (top panel) and after flushing out of the chip (bottom panel); scale bar = 200 μm. (B) Correlation between number of INS-1E and MIN6 cells per microwell and diameter of obtained aggregates after 7 days of culture. INS-1E and MIN6 cells were seeded at 10, 25, 50, 100, 250 and 500 cells per microwell. Each datapoint represents the average measurement of at least 50 aggregates per condition; error bars represent ±SD. (C) Primary human islets were dispersed and single islet cells were seeded at 100, 500 and 1000 cells per microwell. Figures represent human islet cell aggregates inside the microwells (top panel) and after flushing them out of the chip (bottom panel) after 7 days of culture; scale bar = 200 μm. (D) The diameter of human islet cell aggregates increased with increasing the number of cells seeded per well. Cells were seeded at 100, 500 and 1000 cells per microwell. At least 40 human islet cell aggregates were measured per condition. Data points represent the average values for three human donors; error bars represent ±SD.
Mentions: Controlled aggregation using these microwells was further optimized using MIN6 clone B1 and INS-1E insulinoma cell lines. These insulin-producing cell lines are widely used as a model for primary beta cells since they resemble most native beta cells 31,32. Cells were seeded onto microwell chips at various densities to generate aggregates of 10, 25, 50, 100, 250 and 500 cells, resulting in aggregates with well-defined dimensions. The aggregates could easily be removed from the chips by flushing out and mild centrifugation (Fig.3A), after which they were used for further analysis. The average aggregate diameter correlates with the initial number of cells seeded per microwell, which ranged between 29 and 105 μm for INS-1E cells, and between 28 and 93 μm for MIN6 clone B1 cells, as shown in Figure3B. Increasing the number of cells per microwell to 1000 resulted in unstable cell aggregates that were not suitable for further use. Next to controlled aggregation of MIN6 and INS-1E cell lines, our microwell platform supports the controlled reassembly of primary human islet cells. Human donor islets of Langerhans were dissociated into a single cell suspension (>95% viability, data not shown) and seeded at 100, 500 and 1000 cells per microwell to induce controlled aggregation similar to the insuloma cell lines. The resulting primary islet cell aggregates were uniform in size and remained intact after harvesting from the chips (Fig.3C). After 7 days of culture, the islet cell aggregates had an average diameter of 80 ± 5.4, 105 ± 11.3 and 134 ± 15.3 μm respectively, depending on the initial cell number seeded (Fig.3D). To study cell assembly kinetics, representative images were taken at different time-points during aggregate formation. We observed that aggregation occurs in the first 2–4 days after cell seeding, and stable aggregates are obtained after 7–14 days of culture (Fig. S2). Since 200 μm diameter microwells cannot contain more than 1000 cells, we created wells with a diameter of 400 μm. Increasing the cell seeding density in these microwells to 2000, we observed that islet cells assembled uncontrollably into multiple smaller aggregates per well.

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