Skip to main content
SpringerLink
Log in
Book cover

Islets of Langerhans pp 83–107Cite as

Mouse Islet Isolation

  • Reference work entry
  • First Online:

Abstract

Pancreatic islets transplantation is a therapeutic option for patients affected by type 1 diabetes. While it is always desirable to perform experiments directly with human islets, the limited availability of this precious tissue makes mouse islets a feasible option for experimental research in diabetes in many laboratories worldwide. This chapter summarizes from a practical perspective the main aspects of mouse islet isolation. We will discuss the technical factors that affect its success, as well as the practical issues (quality assessment, time, and cost) to take into account when selecting an islet isolation protocol. Finally, we will provide some guidelines on the necessary logistics for setting up this kind of methodology.

This is a preview of subscription content, log in via an institution.

Chapter
USD   29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   699.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD   549.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

References

  • Barbu A, Welsh N (2004) The use of tetrazolium salt-based methods for determination of islet cell viability in response to cytokines: a cautionary note. Diabetologia 47:2042–2043

    Article  PubMed  CAS  Google Scholar 

  • Barnett MJ, McGhee-Wilson D, Shapiro AM, Lakey JR (2004) Variation in human islet viability based on different membrane integrity stains. Cell Transplant 13:481–488

    Article  PubMed  CAS  Google Scholar 

  • Barnett MJ, Zhai X, LeGatt DF, Cheng SB, Shapiro AM, Lakey JR (2005) Quantitative assessment of collagenase blends for human islet isolation. Transplantation 80:723–728

    Article  PubMed  CAS  Google Scholar 

  • Benhamou PY, Stein E, Hober C, Miyamoto M, Watanabe Y, Nomura Y, Watt PC, Kenmochi T, Brunicardi FC, Mullen Y (1995) Ultraviolet light irradiation reduces human islet immunogenicity without altering islet function. Horm Metab Res 27:113–120

    Article  PubMed  CAS  Google Scholar 

  • Bensley RR (1911) Studies of the pancreas with the guinea pig. Am J Anat 12:297–388

    Article  Google Scholar 

  • Berney T, Molano RD, Cattan P, Pileggi A, Vizzardelli C, Oliver R, Ricordi C, Inverardi L (2001) Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis. Transplantation 71:125–132

    Article  PubMed  CAS  Google Scholar 

  • Berney T, Pileggi A, Molano RD, Ricordi C (2002) Epithelial cell culture: pancreatic islets. In: Atala A, Lanza RP (eds) Methods of tissue engineering. Academic, San Diego

    Google Scholar 

  • Bertera S, Balamurugan AN, Bottino R, He J, Trucco M (2012) Increased yield and improved transplantation outcome of mouse islets with bovine serum albumin. J Transplant 2012:856386

    Article  PubMed  PubMed Central  Google Scholar 

  • Bhonde RR, Parab PB, Sheorin VS (1999) An in vitro model for screening oral hypoglycemics. In Vitro Cell Dev Biol Anim 35:366–368

    Article  PubMed  CAS  Google Scholar 

  • Biarnés M, Montolio M, Nacher V, Raurell M, Soler J, Montanya E (2002) β-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia. Diabetes 51:66–72

    Article  PubMed  Google Scholar 

  • Bock T, Pakkenberg B, Buschard K (2005) Genetic background determines the size and structure of the endocrine pancreas. Diabetes 54:133–137

    Article  PubMed  CAS  Google Scholar 

  • Boyd V, Cholewa OM, Papas KK (2008) Limitations in the use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and cell permeable nucleic acid stains for viability measurements of isolated islets of Langerhans. Curr Trends Biotechnol Pharm 2:66–84

    PubMed  PubMed Central  Google Scholar 

  • Buchwald P, Wang X, Khan A, Bernal A, Fraker C, Inverardi L, Ricordi C (2009) Quantitative assessment of islet cell products: estimating the accuracy of the existing protocol and accounting for islet size distribution. Cell Transplant 18:1223–1235

    Article  PubMed  Google Scholar 

  • Cabrera O, Berman DM, Kenyon NS, Ricordi C, Berggren PO, Caicedo A (2006) The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl Acad Sci U S A 103:2334–2339

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Cantarelli E, Citro A, Marzorati S, Melzi R, Scavini M, Piemonti L (2013) Murine animal models for preclinical islet transplantation: no model fits all (research purposes). Islets 5:79–86

    Article  PubMed  Google Scholar 

  • Carlson FD, Brink F Jr, Bronk DW (1950) A continuous flow respirometer utilizing the oxygen cathode. Rev Sci Instrum 21:923–932

    Article  PubMed  CAS  Google Scholar 

  • Carter JD, Dula SB, Corbin KL, Wu R, Nunemaker CS (2009) A practical guide to rodent islet isolation and assessment. Biol Proced Online 11:3–31

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Chadwick DR, Robertson GS, Rose S, Contractor H, James RF, Bell PR, London NJ (1993a) Storage of porcine pancreatic digest prior to islet purification. The benefits of UW solution and the roles of its individual components. Transplantation 56:288–293

    Article  PubMed  CAS  Google Scholar 

  • Chadwick DR, Robertson GS, Toomey P, Contractor H, Rose S, James RF, Bell PR, London NJ (1993b) Pancreatic islet purification using bovine serum albumin: the importance of density gradient temperature and osmolality. Cell Transplant 2:355–361

    PubMed  CAS  Google Scholar 

  • Colton CK, Papas KK, Pisania A, Rappel MJ, Powers DE, O’Neil JJ, Omer A, Weir G, Bonner-Weir S (2007) Characterization of islet preparations. In: Halberstadt CR (ed) Cellular transplantation: from laboratory to clinic. Elsevier, Oxford, UK, pp 85–133

    Chapter  Google Scholar 

  • Conget JI, Sarri Y, González-Clemente JM, Casamitjana R, Vives M, Gomis R (1994) Deleterious effect of dithizone-DMSO staining on insulin secretion in rat and human pancreatic islets. Pancreas 9:157–160

    Article  PubMed  CAS  Google Scholar 

  • De Duve C (1971) Tissue fraction-past and present. J Cell Biol 50:20

    Article  PubMed  PubMed Central  Google Scholar 

  • De Groot M, de Haan BJ, Keizer PP, Schuurs TA, van Schilfgaarde R, Leuvenink HG (2004) Rat islet isolation yield and function are donor strain dependent. Lab Anim 38:200–206

    Article  PubMed  Google Scholar 

  • De Haan BJ, Faas MM, Spijker H, van Willigen JW, de Haan A, de Vos P (2004) Factors influencing isolation of functional pancreatic rat islets. Pancreas 29:e15–e22

    Article  PubMed  Google Scholar 

  • Dionne KE, Colton CK, Yarmush ML (1993) Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans. Diabetes 42:12–21

    Article  PubMed  CAS  Google Scholar 

  • Goto M, Holgersson J, Kumagai-Braesch M, Korsgren O (2006) The ADP/ATP ratio: a novel predictive assay for quality assessment of isolated pancreatic islets. Am J Transplant 6:2483–2487

    Article  PubMed  CAS  Google Scholar 

  • Gotoh M, Maki T, Kiyoizumi T, Satomi S, Monaco AP (1985) An improved method for isolation of mouse pancreatic islets. Transplantation 40:437–438

    Article  PubMed  CAS  Google Scholar 

  • Gotoh M, Maki T, Satomi S, Porter J, Monaco AP (1986) Immunological characteristics of purified pancreatic islet grafts. Transplantation 42:387–390

    Article  PubMed  CAS  Google Scholar 

  • Holmes MA, Clayton HA, Chadwick DR, Bell PR, London NJ, James RF (1995) Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media. Transplantation 60:854–860

    Article  PubMed  CAS  Google Scholar 

  • Jahr H, Bretzel RG, Wacker T, Weinand S, Brandhorst H, Brandhorst D, Lau D, Hering BJ, Federlin K (1995) Toxic effects of superoxide, hydrogen peroxide, and nitric oxide on human and pig islets. Transplant Proc 27:3220–3221

    PubMed  CAS  Google Scholar 

  • Jahr H, Pfeiffer G, Hering BJ, Federlin K, Bretzel RG (1999) Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll. J Mol Med (Berl) 77:118–120

    Article  CAS  Google Scholar 

  • Janjic D, Wollheim CB (1992) Islet cell metabolism is reflected by the MTT (tetrazolium) colorimetric assay. Diabetologia 35:482–485

    Article  PubMed  CAS  Google Scholar 

  • Juang JH, Kuo CH, Wu CH, Juang C (2008) Exendin-4 treatment expands graft β-cell mass in diabetic mice transplanted with a marginal number of fresh islets. Cell Transplant 17:641–647

    Article  PubMed  Google Scholar 

  • Jun Y, Kim MJ, Hwang YH, Jeon EA, Kang AR, Lee SH, Lee DY (2013) Microfluidics-generated pancreatic islet microfibers for enhanced immunoprotection. Biomaterials 34:8122–8130

    Article  PubMed  CAS  Google Scholar 

  • Kelly CB, Blair LA, Corbett JA, Scarim AL (2003) Isolation of islets of Langerhans from rodent pancreas. Methods Mol Med 83:3–14

    PubMed  Google Scholar 

  • Kemp CB, Knight MJ, Scharp DW, Lacy PE, Ballinger WF (1973) Transplantation of isolated pancreatic islets into the portal vein of diabetic rats. Nature 244:447

    Article  PubMed  CAS  Google Scholar 

  • King A, Lock J, Xu G, Bonner-Weir S, Weir GC (2005) Islet transplantation outcomes in mice are better with fresh islets and exendin-4 treatment. Diabetologia 48:2074–2079

    Article  PubMed  CAS  Google Scholar 

  • Lacy PE (1967) The pancreatic β cell. Structure and function. N Engl J Med 276:187–195

    Article  PubMed  CAS  Google Scholar 

  • Lacy PE, Kostianovsky M (1967) Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16:35–39

    PubMed  CAS  Google Scholar 

  • Latif ZA, Noel J, Alejandro R (1988) A simple method of staining fresh and cultured islets. Transplantation 45:827–830

    Article  PubMed  CAS  Google Scholar 

  • Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ (2009) A protocol for islet isolation from mouse pancreas. Nat Protoc 4:1649–1652

    Article  PubMed  CAS  Google Scholar 

  • Lindall A, Steffes M, Sorenson R (1969) Immunoassayable insulin content of subcellular fractions of rat islets. Endocrinology 85:218–223

    Article  PubMed  CAS  Google Scholar 

  • London NJ, Swift SM, Clayton HA (1998) Isolation, culture and functional evaluation of islets of Langerhans. Diabetes Metab 24:200–207

    PubMed  CAS  Google Scholar 

  • McCall MD, Maciver AH, Pawlick R, Edgar R, Shapiro AM (2011) Histopaque provides optimal mouse islet purification kinetics: comparison study with Ficoll, iodixanol and dextran. Islets 3:144–149

    Article  PubMed  Google Scholar 

  • Melzi R, Battaglia M, Draghici E, Bonifacio E, Piemonti L (2007) Relevance of hyperglycemia on the timing of functional loss of allogeneic islet transplants: implication for mouse model. Transplantation 83:167–173

    Article  PubMed  Google Scholar 

  • Moore GE, Gerner RE, Franklin HA (1967) Culture of normal human leukocytes. JAMA 199:519–524

    Article  PubMed  CAS  Google Scholar 

  • Morini S, Brown ML, Cicalese L, Elias G, Carotti S, Gaudio E, Rastellini C (2007) Revascularization and remodelling of pancreatic islets grafted under the kidney capsule. J Anat 210:565–577

    Article  PubMed  PubMed Central  Google Scholar 

  • Moskalewski S (1965) Isolation and culture of the islets of Langerhans of the guinea pig. Gen Comp Endocrinol 44:342–353

    Article  PubMed  CAS  Google Scholar 

  • Nano R, Clissi B, Melzi R, Calori G, Maffi P, Antonioli B, Marzorati S, Aldrighetti L, Freschi M, Grochowiecki T, Socci C, Secchi A, Di Carlo V, Bonifacio E, Bertuzzi F (2005) Islet isolation for allotransplantation: variables associated with successful islet yield and graft function. Diabetologia 48:906–912

    Article  PubMed  CAS  Google Scholar 

  • Nolan AL, O’Dowd JF (2009) The measurement of insulin secretion from isolated rodent islets of Langerhans. Methods Mol Biol 560:43–51

    Article  PubMed  CAS  Google Scholar 

  • O’Dowd JF (2009) The isolation and purification of rodent pancreatic islets of Langerhans. Methods Mol Biol 560:37–42

    Article  PubMed  Google Scholar 

  • Olsson R, Carlsson PO (2005) Better vascular engraftment and function in pancreatic islets transplanted without prior culture. Diabetologia 48:469–476

    Article  PubMed  CAS  Google Scholar 

  • Papas KK, Colton CK, Nelson RA, Rozak PR, Avgoustiniatos ES, Scott WE, Wildey GM, Pisania A, Weir GC, Hering BJ (2007) Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice. Am J Transplant 7:707–713

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Perdrizet GA, Rewinski MJ, Bartus SA, Hull D, Schweizer RT, Scharp DW (1995) Albumin improves islet isolation: specific versus nonspecific effects. Transplant Proc 27:3400–3402

    PubMed  CAS  Google Scholar 

  • Piemonti L, Pileggi A (2013) 25 years of the Ricordi automated method for islet isolation. Cell R4 1:8–22

    Google Scholar 

  • Piemonti L, Bertuzzi F, Nano R, Leone BE, Socci C, Pozza G, Di Carlo V (1999) Effects of cryopreservation on in vitro and in vivo long-term function of human islets. Transplantation 68:655–662

    Article  PubMed  CAS  Google Scholar 

  • Piemonti L, Guidotti LG, Battaglia M (2010) Modulation of early inflammatory reactions to promote engraftment and function of transplanted pancreatic islets in autoimmune diabetes. Adv Exp Med Biol 654:725–747

    Article  PubMed  Google Scholar 

  • Rabinovitch A, Quigley C, Russell T, Patel Y, Mintz DH (1982) Insulin and multiplication stimulating activity (an insulin-like growth factor) stimulate islet (β-cell replication in neonatal rat pancreatic monolayer cultures. Diabetes 31:160–164

    Article  PubMed  CAS  Google Scholar 

  • Ramirez-Dominguez M, Castaño L (2014) Filtration is a time-efficient option to Histopaque providing good quality islets in mouse islet isolation. Cytotechnology. doi:10.1007/s10616-014-9690-7

    PubMed  Google Scholar 

  • Ricordi C, Lacy PE, Sterbenz K, Davie JM (1987) Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival. Proc Natl Acad Sci U S A 84:8080–8084

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW (1988) Automated method for isolation of human pancreatic islets. Diabetes 37:413–420

    Article  PubMed  CAS  Google Scholar 

  • Ricordi C, Gray DW, Hering BJ, Kaufman DB, Warnock GL, Kneteman NM, Lake SP, London NJ, Socci C, Alejandro R et al (1990) Islet isolation assessment in man and large animals. Acta Diabetol Lat 27:185–195

    Article  PubMed  CAS  Google Scholar 

  • Salvalaggio PR, Deng S, Ariyan CE, Millet I, Zawalich WS, Basadonna GP, Rothstein DM (2002) Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function. Transplantation 74:877–879

    Article  PubMed  CAS  Google Scholar 

  • Scharp DW, Murphy JJ, Newton WT, Ballinger WF, Lacy PE (1975) Transplantation of islets of Langerhans in diabetic rhesus monkeys. Surgery 77:100–105

    PubMed  CAS  Google Scholar 

  • Shapiro AM, Hao E, Rajotte RV, Kneteman NM (1996) High yield of rodent islets with intraductal collagenase and stationary digestion–a comparison with standard technique. Cell Transplant 5:631–638

    Article  PubMed  CAS  Google Scholar 

  • Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230–238

    Article  PubMed  CAS  Google Scholar 

  • Song Y, Wei Z, Song C, Xie S, Feng J, Fan J, Zhang Z, Shi Y (2013) Simulated microgravity combined with polyglycolic acid scaffold culture conditions improves the function of pancreatic islets. Biomed Res Int 2013:150739

    PubMed  PubMed Central  Google Scholar 

  • Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG (2012) Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease. J Vis Exp. doi:10.3791/4137

    PubMed  PubMed Central  Google Scholar 

  • Szot GL, Koudria P, Bluestone JA (2007) Murine pancreatic islet isolation. J Vis Exp (7)255

    Google Scholar 

  • Vaithilingam V, Kollarikova G, Qi M, Larsson R, Lacik I, Formo K, Marchese E, Oberholzer J, Guillemin GJ, Tuch BE (2014) Beneficial effects of coating alginate microcapsules with macromolecular heparin conjugates-in vitro and in vivo study. Tissue Eng Part A 20:324–334

    Article  PubMed  CAS  Google Scholar 

  • Venkatesan V, Chalsani M, Nawaz SS, Bhonde RR, Challa SS, Nappanveettil G (2012) Optimization of condition(s) towards establishment of primary islet cell cultures from WNIN/Ob mutant rat. Cytotechnology 64:139–144

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Vos-Scheperkeuter GH, van Suylichem PT, Vonk MW, Wolters GH, van Schilfgaarde R (1997) Histochemical analysis of the role of class I and class II Clostridium histolyticum collagenase in the degradation of rat pancreatic extracellular matrix for islet isolation. Cell Transplant 6:403–412

    Article  PubMed  CAS  Google Scholar 

  • Warnock GL, Rajotte RV, Evans MG, Ellis D, DeGroot T, Dawidson I (1987) Isolation of islets of Langerhans following cold storage of human pancreas. Transplant Proc 19:3466–3468

    PubMed  CAS  Google Scholar 

  • Wolters GH, van Suylichem PT, van Deijnen JH, van Schilfgaarde R (1990) Factors influencing the isolation process of islets of Langerhans. Horm Metab Res Suppl 25:20–26

    PubMed  CAS  Google Scholar 

  • Wolters GH, Vos-Scheperkeuter GH, van Deijnen JH, van Schilfgaarde R (1992) An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation. Diabetologia 35:735–742

    Article  PubMed  CAS  Google Scholar 

  • Wolters GH, Vos-Scheperkeuter GH, Lin HC, van Schilfgaarde R (1995) Different roles of class I and class II Clostridium histolyticum collagenase in rat pancreatic islet isolation. Diabetes 44:227–233

    Article  PubMed  CAS  Google Scholar 

  • Yesil P, Michel M, Chwalek K, Pedack S, Jany C, Ludwig B, Bornstein SR, Lammert E (2009) A new collagenase blend increases the number of islets isolated from mouse pancreas. Islets 1:185–190

    Article  PubMed  Google Scholar 

  • Zmuda EJ, Powell CA, Hai T (2011) A method for murine islet isolation and subcapsular kidney transplantation. J Vis Exp. doi:10.3791/2096

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant of the Basque Government for Groups of Excellence (IT-472-07) and a grant of the Department of Industry of the Basque Government (SAIO09-PE09BF01).

Author information

Authors and Affiliations

  1. Department of Pediatrics, Faculty of Medicine and Odontology, University of the Basque Country, UPV/EHU, University Hospital Cruces, Leioa, País Vasco, Spain

    Miriam Ramirez-Dominguez

  2. β Cell Biology Unit, S. Raffaele Scientific Institute, Diabetes Research Institute-DRI, Via Olgettina 60, 20132, Milan, Italy

    Simona Marzorati

Authors
  1. Simona Marzorati
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Miriam Ramirez-Dominguez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Miriam Ramirez-Dominguez .

Editor information

Editors and Affiliations

  1. Department of Clinical Sciences and Education, Sodersjukhuset, Karolinska Institutet, Stockholm, Sweden

    Md. Shahidul Islam

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media Dordrecht

About this entry

Cite this entry

Marzorati, S., Ramirez-Dominguez, M. (2015). Mouse Islet Isolation. In: Islam, M. (eds) Islets of Langerhans. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-6686-0_33

Download citation

Publish with us

Policies and ethics

Search

Navigation