Human Islet Applications Overview

General Approach

Recommended products

VitaCyte products that can be used for human islet isolation are listed in the table below with a recommended dose for use per isolation procedure. In the Clinical Islet Consortium trial, the dose was modified to calculate the activity units of collagenase or protease used per g of trimmed tissue. If you choose to dose per g of trimmed organ, consult specific literature reports for the recommended dose used for human islet isolation procedure.

Table 1: Recommended Products

Recommended method: Modified Ricordi Procedure

Nearly all human islet isolation methods are modified versions of the Ricordi procedure1. The modified Ricordi method most commonly performed today was developed by the Clinical Islet Transplantation (CIT) Consortium, a group funded by NIH to perform a number of different clinical trials designed to assess benefits of islet transplantation for management of adult, type I diabetes patients. This isolation method can be found by clicking on the hyperlink “Master Production Batch Record”.2 This paper cites 20 articles published by Ansite, et al. in CellR4 that provide more detail on the manufacturing documents.

The majority of protocols use about 2200 Wunsch Units (WU) per pancreas or about 20 WU/g tissue. The neutral protease activity can vary depending on the protocol and neutral protease used in the procedure. Consult the CIT protocol above for more details.

VitaCyte’s CIzyme HA product has been used to recover human islets from organ donors or for management of patients with chronic pancreatitis. In these situations, collagenase is used at a dose of 20 WU/g trimmed tissue in combination with an enriched Clostridium histolyticum neutral protease product. This “new enzyme mixture” gave higher islet yields than those obtained with purified C. histolyticum collagenase with thermolysin or with purified C. histolyticum collagenase from another supplier used with enriched Clostridium histolyticum neutral protease.

Recent studies using recombinant C. histolyticum collagenase or an enriched collagenase (Collagenase Gold 800) have shown that supplementation of collagenase with CIzyme BP Protease is effective in obtaining high numbers of human islets from research pancreata. The recommended amount for human islet isolation is 23,400 of VitaCyte’s neutral protease units per g of trimmed tissue.

Expected cell yield

As a reference value for a research human islet isolation, the expectation is to recover > 3500 islet equivalents (IEQ) per g of trimmed tissue with an islet purity > 70%. If your goal is to isolate a high purity islet population for use in diabetes research then this number is > 3000 IEQ per g tissue.

Please also consult Table 2, below, regarding factors to consider when focusing on enzymes used in isolation procedures.

Perspectives

It is difficult to rigorously assess factors that may influence human islet yield or function because of the high cost for performing these isolations and decreasing budgets for performing these experiments. The table below summarizes some of the factors raised in the scientific literature to consider when the focus is on the enzymes used in the isolation procedure. References associated with each issue are included in the statement.

Table 2: Factors to consider when focusing on enzymes used in isolation procedures

 

Resources & Reviews

There are several JoVE videos on human islet isolation20,21 and an annual islet isolation workshop sponsored by the European Pancreas and Islet Transplantation Association, a section of the European Society for Organ Transplantation.

References

  1. Ricordi C, Lacy PE, Scharp DW. Automated islet isolation from human pancreas. Diabetes 1989; 38 Suppl 1: 140-2.

  2. Consortium CIT. Master production batch record (Sep 2009) (pdf). 2009. http://www.ctsdmc.org/projects/cit/documents/MPBR,SOP3101,B01,Version04,Sep42009.pdf (accessed January 23, 2017 2017).

  3. Balamurugan AN, Breite AG, Anazawa T, et al. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplantation 2010; 89(8): 954-61.

  4. Brandhorst H, Kurfurst M, Johnson PR, Korsgren O, Brandhorst D. Comparison of Neutral Proteases and Collagenase Class I as Essential Enzymes for Human Islet Isolation. Transplantation direct 2016; 2(1): e47

  5. Dwulet FE, Ellis BB, Gill JF, Jacobsen LB, Smith ME, Waters DG, inventors; Purified mixture of collagenase I, collagenase II, and two other proteases patent US Patent # 5,753,485.

  6. Balamurugan AN, Green ML, Breite AG, et al. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation. Transplantation direct 2016; 2(1): e54.

  7. Wolters GH, Vos-Scheperkeuter GH, Lin HC, van Schilfgaarde R. Different roles of class I and class II Clostridium histolyticum collagenase in rat pancreatic islet isolation. Diabetes 1995; 44(2): 227-33.

  8. Fujio A, Murayama K, Yamagata Y, et al. Collagenase H is crucial for isolation of rat pancreatic islets. Cell transplantation 2014; 23(10): 1187-98.

  9. Mitchell WM. The contamination of purified collagenase preparations by clostridiopeptidase B (clostripain): the potential effect on studies utilizing collagenase as a highly specific structural tool. John Hopkins Medical Journal 1970; 127: 192-8.

  10. Brandhorst H, Friberg A, Andersson HH, et al. – The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation. – Transplantation 2009; 87: 370-5.

  11. Stahle M, Foss A, Gustafsson B, et al. Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation. Transplantation direct 2015; 1(5): e19.

  12. Brandhorst H, Friberg A, Nilsson B, et al. Large-scale comparison of Liberase HI and collagenase NB1 utilized for human islet isolation. Cell transplantation 2010; 19(1): 3-8.

  13. Qi M, Barbaro B, Wang S, Wang Y, Hansen M, Oberholzer J. Human pancreatic islet isolation: Part I: digestion and collection of pancreatic tissue. Journal of visualized experiments : JoVE 2009; (27).

  14. Qi M, Barbaro B, Wang S, Wang Y, Hansen M, Oberholzer J. Human pancreatic islet isolation: Part II: purification and culture of human islets. Journal of visualized experiments : JoVE 2009; (27).

  15. Lakey JR, Burridge PW, Shapiro AM. Technical aspects of islet preparation and transplantation. Transplant International 2003; 16(9): 613-32.

  16. Hawthorne WJ, Williams L, Chew YV. Clinical Islet Isolation. Advances in experimental medicine and biology 2016; 938: 89-122.