Apr 18, 2024

Public workspacePseudoislets for diabetes research: Purifying and selective re-aggregating human islet cells V.2

DOI
dx.doi.org/10.17504/protocols.io.5jyl85x7dl2w/v2
  • 1Columbia Center for Translational Immunology, Department of Medicine, Columbia University Medical Center, New York, NY, USA;
  • 2Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR, USA;
  • 3Columbia Center for Translational Immunology, Department of Surgery, Columbia University Medical Center, New York, NY, USA
  • Wei Liu: Current address: Department of Nephrology, The First Hospital of Jilin University, Changchun, Jilin, 130021, China;
Sandy Beshir
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl85x7dl2w/v2
Protocol Citation: Wei Liu, Craig Dorrell, Xiaojuan Chen 2024. Pseudoislets for diabetes research: Purifying and selective re-aggregating human islet cells. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl85x7dl2w/v2Version created by Sandy Beshir
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 18, 2024
Last Modified: April 18, 2024
Protocol Integer ID: 98375
Keywords: Pseudoislets, diabetes, human islet cells, HIRN
Funders Acknowledgement:
NIH
Grant ID: UC4 DK104207
Abstract
In vitro modeling of human islet cells for diabetes research utilizing purified and then selectively re-aggregated various combinations of human islet cells.

Note
Corresponding Author

Xiaojuan Chen, MD, PhD
Address: 650 West 168th Street, BB1701, New York, NY 10032, USA
Email: xc2248@cumc.columbia.edu
Tel: (212)342-3111
Fax: (212)342-6030


Guidelines
REFERENCES:

CITATION
1. Dorrell, C. et al. Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers. Stem Cell Research 1, 183-194 (2008).
LINK
https://doi.org/10.1016/j.scr.2008.04.001

CITATION
2. Dorrell, C. et al. Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers. Mol Cell Endocrinol 339, 144-150 (2011).
LINK
https://dx.doi.org/10.1016%2Fj.mce.2011.04.008

CITATION
3. Liu, W. et al. Abnormal regulation of glucagon secretion by human islet alpha cells in the absence of beta cells. EBioMedicine 50, 306-316 (2019).
LINK
https://doi.org/10.1016/j.ebiom.2019.11.018

CITATION
4. Ricordi, C. et al. National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities. Diabetes 65, 3418-3428.
LINK
https://doi.org/10.2337/db16-0234


Materials
ReagentCMRL-1066 supplemented CIT mediumCorningCatalog #98-304-CV
ReagentHeparinSagent PharmaceuticalsCatalog #25021-400-10
ReagentIGF-1 (Cell SciencesCat. #CRI500Cell SciencesCatalog #CRI500
Reagentfetal bovine serumGe Life SciencesCatalog #SH30088.03HI
ReagentTrypsinMillipore SigmaCatalog #SM-2004-C
ReagentDPBSGibco - Thermo FisherCatalog #70011044
ReagentGlucagonSigma AldrichCatalog #G2654
ReagentInsulinDakoCatalog #A0564
ReagentSomatostatin (SST ZYMED)Thermo Fisher ScientificCatalog #18-0078 (discontiued)
Reagentghrelin AbcamCatalog #ab209790
ReagentBovine serum albuminSigma AldrichCatalog #A6003
ReagentPicoGreen DNA assay kits Invitrogen - Thermo FisherCatalog #P7589
ReagentFlavin adenine dinucleotide (FAD) Sigma AldrichCatalog #F7378
ReagentPropidium Iodide (PI) Sigma AldrichCatalog #P4170
ReagentHuman Insulin ELISAMercodiaCatalog #10-1113-01
ReagentHuman Glucagon ELISAMercodiaCatalog #10-1271-01
Reagentsomatostatin EIA kitsPhoenix PharmaceuticalsCatalog #EK06003
Equipment
Ultra-Low Attachment plates
NAME
Corning
BRAND
3473
SKU
https://ecatalog.corning.com/life-sciences/b2c/US/en/Microplates/Assay-Microplates/96-Well-Microplates/Costar%C2%AE-Multiple-Well-Cell-Culture-Plates/p/3473
LINK

Equipment
confocal laser-scanning microscope
NAME
LSM410
TYPE
Zeiss
BRAND
None
SKU
https://www.olympus-lifescience.com/en/laser-scanning/fv3000/
LINK

Equipment
cell culture inserts
NAME
EMD Millipore
BRAND
PITP01250
SKU
https://www.emdmillipore.com/US/en/product/Millicell-Cell-Culture-Insert-12mm-polycarbonate-3.0m,MM_NF-PITP01250?ReferrerURL=https%3A%2F%2Fwww.google.com%2F
LINK













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 Temperature37 °C with 5% CO² 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 Temperature37 °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 Amount500 µL 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 Temperature37 °C , 5% CO²for 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 Temperature37 °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.