Pseudoislets for diabetes research: Purifying and selective re-aggregating human islet cells

Human islet culture and single cell preparation

Islets isolated from non-diabetic deceased human donors within 2-4 days post-isolation will be used in experiments. Islets and the dispersed cells are cultured at 37°C with 5% CO_² in a CMRL-1066 supplemented CIT medium (Cellgro, Cat. #98-304-CV) with 10 IU/ml Heparin (Sagent Pharmaceuticals, Cat. #25021-400-10), 1 µg/ml IGF-1 (Cell Sciences,Cat. #CRI500), and 10% fetal bovine serum (GE Life Sciences, Cat. #SH30088.03HI). For single cell preparations[1-3], islets are incubated with 0.025-0.05% trypsin (Millipore Sigma, Cat. #SM-2004-C) for approximately 6 min at 37°C . Undispersed material is excluded using a 40 µm cell strainer. The dispersed cells are re-suspended in CMRL-1066 supplemented CIT medium with 2% FBS.

Fluorescence-activated cell sorting (FACS)

The dispersed cells are incubated with two monoclonal antibodies, HIC1-2B4-APC (https://apps.ohsu.edu/research/tech-portal/technology/view/269524) and HIC3-2D12-PE (https://apps.ohsu.edu/research/tech-portal/technology/view/3848191) at a 1:100 dilution. After incubation on ice for 30-40 minutes, the cells are washed with cold DPBS (Gibco, Cat. #70011-044), re-suspended in CMRL with 2% FBS, and sorted using the BD Influx sorter at the Flow Cytometry Core of Columbia Center for Translational Immunology (CCTI). A two-step sorting process, using the enrich mode followed by the pure sort mode, is performed to eliminate contamination from other pancreatic cell types.

Sorted human islet cell purity and culture

Sorted α-, β-, δ, and/or ε-cells are counted and cultured in 500µL of CMRL-1066 with 10% FBS and 5.6 mM glucose for a period of 3-7 days in Corning Ultra-Low Attachment plates (Corning/Costar, Ithaca, NY). Each well contains at least 20,000 sorted islet cells, calculated based on the number of cells determined by flow cytometry. The cells are given 3-7 days to aggregate in culture. Intact human islets are cultured under the same conditions for control purposes.

Samples of the sorted cells are concentrated on glass slides by cytocentrifugation, fixed in 2.5% paraformaldehyde for 10 mins, and then immunofluorescence stained for human glucagon (Sigma, Cat. #G2654), insulin (Dako, Cat. #A0564), Somatostatin (SST, ZYMED, Cat. #18-0078) and ghrelin (Abcam, Cat. #ab209790) to confirm purity. Secondary antibodies conjugated with Dylight-dyes (Jackson ImmunoResearch) are used for fluorescence detection. Digital images of fluorescence-labeled cells are acquired using a Zeiss fluorescence axial microscope attached with a digital camera or with a Zeiss LSM410 confocal laser-scanning microscope.

In vitro glucose challenge

Duplicate or triplicate samples from each donor are tested for each treatment condition. Cells from each donor are used as their own controls when testing and comparing function under the various treatment conditions.

After the 3-7 day culture, the cell aggregates and control intact islets are subjected to glucose challenge using a static incubation approach described for intact islets[4] with modifications. The cell aggregates or islets are carefully transferred into individual cell culture inserts with a pore size of 3 µm (EDM Millipore, Cat. #PITP01250) in 24-well plates (Non-tissue culture treated, Corning). The cells are first equilibrated in 2.0 mM glucose-containing HEPES-balanced Krebs-Ringer bicarbonate buffer (KRB) with 2 mg/ml Bovine serum albumin (Sigma-Aldrich, Cat. #A6003,) at 37°C , 5% CO_²for 1 hr. Following this, the inserts are removed from the wells, drained of any residual media and transferred to new wells containing KRB with 2.0 mM glucose and incubated at 37°C for one hour. Afterwards, the inserts are transferred to new wells containing KRB supplemented with various concentrations of glucose and growth factors or compounds (e.g., Glibenclamide) for another hour. The cells used in the high-to-low glucose challenge are first incubated in KRB with high concentration glucose for one hour and then transferred to KRB containing 2.0 mM glucose for another hour. The supernatants from each of the one hour-incubations of the two step-glucose challenge assay are collected for islet hormone measurements. The cells are lysed for cell number quantification at the end of the experiment using the PicoGreen DNA assay kits (Invitrogen, Cat. #P7589) according to the manufacturer’s protocol. In addition, subgroups of cells are also evaluated for viability using FAD (Sigma, Cat. #F7378) and PI (Sigma, Cat. #P4170) staining, and for glucagon content assessment by acid ethanol extraction (1.5% HCL in 75% ethanol) followed by freezing and neutralization steps prior to glucagon concentration measurement.

Islet endocrine hormone measurements

The supernatant collected are analyzed for insulin and glucagon concentrations using the human insulin and glucagon ELISA kits (Mercodia, Cat. #10-1113-01 and #10-1271-01) and somatostatin EIA kits (Phoenix Pharmaceuticals, Cat. #EK06003) according to the manufacturers' instructions. The hormone released by each group of cells was expressed as the ratio of the hormone released during the second step incubation divided by that released in the first step incubation of the two-step glucose challenge assay, as described above. The ratio represents the function of cells in each sample in a cell-number independent manner. In addition, in order to compare the absolute amount of hormone secretion by a given number of cells under different experimental conditions, glucagon or insulin release was standardized to cellular DNA content measured as described above.