, Volume 35, Issue 8, pp 735–742 | Cite as

An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation

  • G. H. J. Wolters
  • G. H. Vos -Scheperkeuter
  • J. H. M. van Deijnen
  • R. van Schilfgaarde


Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99±10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36±1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2–5.0 μl islet tissue volume (3,300–3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.

Key words

Collagenase protease pancreas extracellular matrix islet isolation 


  1. 1.
    Burghen GA, Murrell LR (1989) Factors influencing isolation of islets of Langerhans. Diabetes 38 [Suppl 1]: 129–132Google Scholar
  2. 2.
    Sharp DW (1988) The elusive human islet: variables involved in its effective recovery. In: Van Schilfgaarde R, Hardy MA (eds) Transplantation of the endocrine pancreas in diabetes mellitus. Elsevier, Amsterdam, pp 97–107Google Scholar
  3. 3.
    Wolters GHJ, Van Suylichem PTR, Van Deijnen JHM, Van Schilfgaarde R (1990) Factors influencing the isolation process of islets of Langerhans. Horm Metab Res 25 [Suppl]: 20–26Google Scholar
  4. 4.
    Alejandro R, Strasser S, Zucker PF, Mintz DH (1990) Isolation of pancreatic islets from dogs; semi-automated purification on albumin gradients. Transplantation 50: 207–210Google Scholar
  5. 5.
    Ricordi C, Socci C, Davalli AM et al. (1990) Swine islet isolation and transplantation. Horm Metab Res 25 [Suppl]: 26–30Google Scholar
  6. 6.
    Warnock GL, Kneteman NM, Evans MG, Rajotte RV (1990) Isolation of purified large mammal and human islets of Langerhans. Horm Metab Res 25 [Suppl]: 37–44Google Scholar
  7. 7.
    Hering BJ, Bretzel RG, Federlin K (1988) Current status of clinical islet transplantation. Horm Metab Res 20: 537–545Google Scholar
  8. 8.
    Scharp DW, Lacy PE, Santiago JV et al. (1991) Results of our first nine intraportal islet allografts in type I, insulin-dependent diabetic patients. Transplantation 51: 76–85Google Scholar
  9. 9.
    Warnock GL, Kneteman NM, Ryan EA, Rabinovitch A, Rajotte RV (1992) Long-term follow-up after transplantation of insulin-producing pancreatic islets into patients with type 1 (insulin-dependent) diabetes mellitus. Diabetologia 35: 89–95Google Scholar
  10. 10.
    Tzakis AG, Ricordi C, Alejandro R et al. (1990) Pancreatic islet transplantation after upper abdominal exenteration and liver replacement. Lancet 336: 402–405Google Scholar
  11. 11.
    London NJM, Lake SP, Wilson J et al. (1990) A simple method for the release of islets by controlled collagenase digestion of the human pancreas. Transplantation 49: 1109–1113Google Scholar
  12. 12.
    Overholser MD (1925) The number of islets of Langerhans in the pancreas of the albino rat. Endocrinology 9: 493–504Google Scholar
  13. 13.
    Hellman B (1959) The numerical distribution of the islets of Langerhans at different ages in the rat. Acta Endocrinol 32: 63–77Google Scholar
  14. 14.
    Van Suylichem PTR, Wolters GHJ, Van Schilfgaarde (1992) Peri-insular presence of collagenase during islet isolation procedures. J Surg Res (in press)Google Scholar
  15. 15.
    Martinez-Hernandez A (1987) Electron immunohistochemistry of the extracellular matrix: an overview. In: Cunningham LW (ed), Methods in enzymology, Vol. 145, Structural and contractile proteins, Part E: Extracellular matrix. Academic Press, New York, pp 78–103Google Scholar
  16. 16.
    Bond MD, Van Wart HE (1984) Characterization of the individual collagenases from Clostridium histolyticum. Biochemistry 23: 3085–3091Google Scholar
  17. 17.
    Van Wart HE, Steinbrink DR (1985) Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum. Biochemistry 24: 6520–6526Google Scholar
  18. 18.
    Kessler E, Yaron A (1973) A novel aminopeptidase from Clostridium histolyticum. Biochem Biophys Res Commun 50: 405–412Google Scholar
  19. 19.
    Mitchell WM, Harrington WF (1968) Purification and properties of clostridiopeptidase B (clostripain). J Biol Chem 243: 4683–4692Google Scholar
  20. 20.
    McQuade AB, Crewther WG (1968) Peptide substrates for a proteinase of Clostridium histolyticum. Biochim Biophys Acta 167: 619–620Google Scholar
  21. 21.
    McShane P, Sutton R, Gray DWR, Morris PJ (1989) Protease activity in pancreatic islet isolation by enzymatic digestion. Diabetes 38 [Suppl 1]: 126–128Google Scholar
  22. 22.
    López-de León A, Rojkind M (1985) A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem 33: 737–743Google Scholar
  23. 23.
    Van Suylichem PTR, Pasma A, Wolters GHJ, Van SchilfgaardeR (1987) Microscopic aspects of the structure and collagen content of the pancreas from the perspective of islet isolation. Transplant Proc 19: 3958–3959Google Scholar
  24. 24.
    Romeis B (1989) Mikroskopische Technik, 17th edn. Urban und Schwarzenberg, Munich, Vienna, Baltimore, pp 441–624Google Scholar
  25. 25.
    Latif ZA, Noel J, Alejandro R (1988) A simple method of staining fresh and cultured islets. Transplantation 45: 827–830Google Scholar
  26. 26.
    Van Suylichem PTR, Wolters GHJ, Van Schilfgaarde R (1990) The efficacy of density gradients for islet purification: a comparison of seven density gradients. Transplant Int 3: 156–161Google Scholar
  27. 27.
    Hefley TJ (1987) Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone. J Bone Mineral Res 2: 505–516Google Scholar
  28. 28.
    Wolters GHJ, Konijnendijk W, Bouman PR (1977) Effects of fasting on insulin secretion, islet glucose metabolism and the cyclic adenosine 3′, 5′-monophosphate content of rat pancreatic islets in vitro. Diabetes 26: 530–537Google Scholar
  29. 29.
    Wolters GHJ, Konijnendijk W (1980) Relationship between insulin secretion, insulin content and dry weight of single rat pancreatic islets. Acta Endocrinologica 94: 365–370Google Scholar
  30. 30.
    Laemmli VK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227: 680–685Google Scholar
  31. 31.
    Heinegard D, Sommarin Y (1987) Isolation and characterization of proteoglycans. In: Cunningham LW (ed) Methods in enzymology, Vol. 144, Structural and contractile proteins, Part D: extra cellular matrix. Academic Press, New York, pp 319–372Google Scholar
  32. 32.
    Linares HA, Laros DL (1978) Proteoglycans and collagenase in hypertrophic scar formation. Plast Reconstr Surg 62: 589–593Google Scholar
  33. 33.
    Van Deijnen JHM, Hulstaert CE, Wolters GHJ, Van Schilfgaarde R (1992) Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat, dog, pig and man. Cell Tissue Res 267: 139–146Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • G. H. J. Wolters
    • 1
  • G. H. Vos -Scheperkeuter
    • 1
  • J. H. M. van Deijnen
    • 1
  • R. van Schilfgaarde
    • 1
  1. 1.Department of SurgeryUniversity of GroningenGroningenThe Netherlands

Personalised recommendations