Evaluation of Compounds in Primary Human Islet Cell Culture

Deepika Walpita1, Bridget K. Wagner1

1 Center for the Science of Therapeutics, Broad Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140088
Online Posting Date:  September, 2014
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Abstract

The identification of novel small molecules that promote pancreatic beta‐cell proliferation is an important approach to therapeutic discovery for diabetes. Because human islets are not easy to culture, and attach poorly to plates, it had not been feasible to run high‐throughput phenotypic assays in these cells. Therefore, most laboratories have turned to rodent islets for ease of culture and accessibility. However, rodent islets are not physiologically similar to human islets, either in terms of islet architecture or endocrine cell interactions within the islet, and data generated in rodent islets do not typically translate to human islet biology. To address this challenge, we developed a human islet culture system for high‐throughput screening using a thymidine analog, EdU, to detect beta‐cell replication during screening. Simultaneous monitoring of EdU incorporation and beta cell numbers provides a robust assay for beta‐cell replication, and is now becoming a standard protocol enabling screening in human islets. Curr. Protoc. Chem. Biol. 6:157‐168 © 2014 by John Wiley & Sons, Inc.

Keywords: pancreatic beta cell; islets of Langerhans; primary cell culture system; high‐throughput screening; diabetes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing Plates Suitable for Culturing Human Islet Cells
  • Basic Protocol 2: Islet Dissociation
  • Basic Protocol 3: Human Beta‐Cell Proliferation Assay
  • Basic Protocol 4: Image Acquisition and Analysis of Islet Cell Cultures
  • Support Protocol 1: Adenovirus Infection of Dissociated Islets
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing Plates Suitable for Culturing Human Islet Cells

  Materials
  • HTB‐9 cells (ATTC, cat. no. 5637)
  • RPMI‐1640 medium (CellGro) supplemented with 10% FBS and 1× penicillin/streptomycin
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐023) with 1× penicillin/streptomycin for washing and storing fresh ECM plates
  • 0.5% trypsin‐EDTA (Corning CellGro, cat. no. 25‐053‐CI)
  • 20 mM NH 4OH in PBS (Sigma)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐023)
  • 15‐ and 50‐ml conical tubes
  • Centrifuge
  • 75‐cm2 (T‐75) cell culture flasks
  • Multidrop Combi plate filler (Thermo Scientific)
  • Optical (clear‐bottomed) black 96‐well plates (Costar, cat. no. 3904)
  • Optical black 384‐well plates (Corning, cat. no. 3712)
  • Plate washer (Biotek ELx405)
  • Multichannel pipettors and reservoirs

Basic Protocol 2: Islet Dissociation

  Materials
  • Human islets, available from a number of sources, such as the Integrated Islet Distribution Program (https://iidp.coh.org/)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐023)
  • Complete CMRL culture medium: CMRL 1066 medium (CellGro, cat. no. 15‐110‐CV) supplemented with 10% FBS, 1× L‐glutamine, and 1× penicillin/streptomycin
  • StemPro Accutase (Life Technologies, cat. no. A11105‐01)
  • 15‐ and 50‐ml conical centrifuge tubes
  • Untreated petri dishes (VWR, cat. no. 25384‐302)
  • Hemacytometer to count cells
  • Nylon mesh of appropriate pore size, e.g., 100 μm
  • 96‐ and 384‐well HTB‐9 ECM plates (prepared in protocol 1)

Basic Protocol 3: Human Beta‐Cell Proliferation Assay

  Materials
  • ECM plates treated with dissociated islets ( protocol 2)
  • Small‐molecule library of interest
  • Complete CMRL culture medium: CMRL 1066 (CellGro, cat. no. 15‐110‐CV) supplemented with 10% FBS, 1× L‐glutamine, and 1× penicillin/streptomycin
  • Click‐IT EdU HCS assay kit (Life Technologies, cat. nos. C10350 and C10351) including:
    • EdU (Component A)
    • Alexa Fluor azide (Component B)
    • Click‐iT EdU reaction buffer (Component C)
    • CuSO 4 (Component D)
    • Click‐iT EdU buffer additive (Component E)
    • Click‐iT reaction rinse buffer (Component F)
  • 16% paraformaldehyde (PFA) aqueous solution (VWR, cat. no. AA43368‐9M)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐023)
  • 0.2% (v/v) Triton X‐100 in PBS
  • Blocking buffer: 2% (w/v) bovine serum albumin (BSA) in PBS (store at 4°C and use within 2 to 3 weeks)
  • Primary C‐peptide antibody (Developmental Studies Hybridoma Bank at the University of Iowa, cat. no. GN‐ID4)
  • 1% (w/v) bovine serum albumin (BSA) in PBS
  • Secondary antibody: AlexaFluor 594 goat‐anti rat (Life Technologies, cat. no. A11007)
  • Hoechst 33342 nuclear stain (10 mg/ml stock solution from Life Technologies, cat. no. H3570)
  • Multi‐channel pipettors or Multidrop Combi
  • Optical (clear‐bottomed) black 96‐well plates (Costar, cat. no. 3904)
  • Optical black 384‐well plates (Corning, cat. no. 3712)
  • Reservoirs for working with individual 96‐well plates
  • Foil seals for plates

Basic Protocol 4: Image Acquisition and Analysis of Islet Cell Cultures

  Materials
  • 96‐ or 384‐well plates of islet cells, treated, fixed, and stained ( protocol 3)
  • ImageXpress Micro automated microscopy system (Molecular Devices)
  • MetaXpress analysis software (Molecular Devices)

Support Protocol 1: Adenovirus Infection of Dissociated Islets

  Materials
  • Islet cultures ( protocol 2)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 10010‐023)
  • Serum‐free CMRL medium: CMRL 1066 medium (CellGro, cat. no. 15‐110‐CV) supplemented with 1× L‐glutamine, and 1× penicillin/streptomycin, but no serum
  • CDK6 adenovirus (provided by Andrew Stewart; Fiaschi‐Taesch et al., )
  • CyclinD1 adenovirus (provided by Andrew Stewart; Fiaschi‐Taesch et al., )
  • Fetal bovine serum (FBS)
  • Complete CMRL culture medium: CMRL 1066 medium (CellGro, cat. no. 15‐110‐CV) supplemented with 10% FBS, 1× L‐glutamine, and 1× penicillin/streptomycin
  • EdU (Component A in Click‐IT EdU HCS assay kit; Life Technologies)
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Figures

Videos

Literature Cited

Literature Cited
  Beattie, G.M., Cirulli, V., Lopez, A.D., and Hayek, A. 1997. Ex vivo expansion of human pancreatic endocrine cells. J. Clin. Endocrinol. Metab. 82:1852‐1856.
  Daoud, J., Petropavlovskaia, M., Rosenberg, L., and Tabrizian, M. 2010. The effect of extracellular matrix components on the preservation of human islet function in vitro. Biomaterials 31:1676‐1682.
  Efrat, S. 2008. Ex‐vivo expansion of adult human pancreatic beta‐cells. Rev. Diabet. Stud. 5:116‐122.
  Fiaschi‐Taesch, N., Bigatel, T.A., Sicari, B., Takane, K.K., Salim, F., Velazquez‐Garcia, S., Harb, G., Selk, K., Cozar‐Castellano, I., and Stewart, A.F. 2009. Survey of the human pancreatic β‐cell G1/S proteome reveals a potential therapeutic role for Cdk‐6 and Cyclin D1 in enhancing human β‐cell replication and function in vivo. Diabetes 58:882‐893.
  Kolb, H.C., Finn, M.G., and Sharpless, K.B. 2001. Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40:2004‐2021.
  Lee, Y.C. and Nielsen, J.H. 2009. Regulation of beta cell replication. Mol. Cell. Endocrinol. 297:18‐27.
  Otonkoski, T., Banerjee, M., Korsgren, O., Thornell, L.E., and Virtanen, I. 2008. Unique basement membrane structure of human pancreatic islets: Implications for beta‐cell growth and differentiation. Diab. Obes. Metab. 10:119‐127.
  Ouziel‐Yahalom, L., Zalzman, M., Anker‐Kitai, L., Knoller, S., Bar, Y., Glandt, M., Herold, K., and Efrat, S. 2006. Expansion and redifferentiation of adult human pancreatic islet cells. Biochem. Biophys. Res. Commun. 341:291‐298.
  Salic, A. and Mitchison, T.J. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U.S.A. 105:2415‐2420.
  Scholzen, T. and Gerdes, J. 2000. The Ki‐67 protein: From the known and the unknown. J. Cell. Physiol. 182:311‐322.
  Walpita, D., Hasaka, T., Spoonamore, J., Vetere, A., Takane, K.K., Fomina‐Yadlin, D., Fiaschi‐Taesch, N., Shamji, A., Clemons, P.A., Stewart, A.F., Schreiber, S.L., and Wagner, B.K. 2012. A human islet cell‐culture system for high‐throughput screening. J. Biomol. Screen. 17:509‐518.
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