Announcement: Bashoo Naziruddin will discuss the results from the report noted below as part of his webinar on 24 April 2025 on “Challenges facing the isolation of high-quality islets for total pancreatectomy with islet autotransplantation.”
Robert McCarthy
In a recently published report, Kuncha et al. provided additional insight into one factor that may explain low islet yields obtained after isolating islets from pancreata recovered as part of the total pancreatectomy-islet autotransplant procedure (TP-IAT) performed on chronic pancreatitis (CP) patients. In a prospective study of 29 TP-IAT procedures, they divided these patients into two groups based on islet yields: those with > 5000 IEQ/g tissue (high) or < 5000 IEQ/g tissue (low). They found that the low islet group had the highest laminin α 5 (LAMA5) concentrations in plasma collected on day 0 or day 1 after transplant. This difference was significant compared to LAMA5 concentrations in the high islet group. Normal patients had about 50% of the LAMA 5 concentration in the low islet yield group.
Further analysis of frozen tissue sections immunohistochemically stained for LAMA5 and other extracellular matrix protein markers found stronger LAMA5 area expression in both islet groups than healthy controls (i.e., normal organ donors). The LAMA5 expression ranged from 10 to 25% of the analyzed area. Increased LAMA5 area expression was negatively correlated with the IEQs delivered to the patient (p = 0.02). A positive correlation was found between increased LAMA5 area expression with fasting blood glucose levels (p = 0.0018) and HbA1c 3 months post-transplant (p= = 0.0039).
These notable observations highlight changes in the ECM that occur in CP patients. An obvious question is: Do the increased levels of LAMA 5 resist degradation by the enzymes commonly used for human islet isolation? The enzymes widely used for islet isolation are zinc metalloproteins that belong to the M4 protease family: Thermolysin, BP Protease, or C. histolyticum neutral protease. They share a common amino acid sequence in the active site of the catalytic domain.
I always believed that improvements in tissue dissociation enzymes must focus on the proteases used for isolation, not the C. histolyticum collagenase. For some, this may be a bold statement, but VitaCyte’s minimal lot variability of its purified collagenase products shown in the table below demonstrates that these enzymes can be consistently manufactured. All VitaCyte’s products at >95% pure with minimal contamination with clostripain (< 0.1% w/w) in the natural collagenase products.
Why Focus on Neutral Proteases?
In 2011, my colleagues and I proposed a hypothetical model of how collagenases and neutral proteases synergistically degrade the extracellular matrix (ECM), leading to the release of cell from tissue. Collagenase has a restricted specificity, it degrades nearly all forms of mammalian collagen and denatured collagen (i.e., gelatin). Excess purified collagenase should have no adverse effects on cell viability or function since collagen is an extracellular matrix (ECM) protein.
If collagen degradation activity is in excess, then collagenase thins the jungle of collagen fibrils/fibers while at the same time loosening the ECM, revealing protease sensitive sites on the ECM “anchoring proteins” that hold the cells to the matrix. Under these conditions, the speed of digestion and the potential adverse effects of tissue dissociation enzymes are dependent on the choice and dose of neutral protease(s) used in the cell isolation procedures.
An earlier blog post, “Occam’s razor guides the mechanistic model of tissue dissociation,” applied these concepts to explain the results from 94 human islet isolations performed at the University of Minnesota.
Why is this Relevant to Overcoming Lower Islet Yields in CP Patients with Increased LAMA 5 Expression?
One effective way to increase islet yields is to use multiple proteases with complementary specificities in collagenase containing, tissue dissociation enzyme mixtures. The sporadic reports of increased human islet yields when using enzyme mixtures containing collagenase, M4 protease, and clostripain illustrate the application of this concept. (1, 2) Clostripain is a trypsin-like, sulfhydl endoprotease found in C. histolyticum culture supernatants. It is a common contaminate of purified collagenase preparations. (3) Clostripain is not as active as trypsin, but like trypsin, it cuts on carboxy-terminal side of arginine or lysine residues. In contrast to the M4 proteases cut at the amino terminal side of hyphobic amino acid residues. (4)
The variability in clostripain increasing islet yields may reflect the oxidative state of the enzyme mixture since clostripain has maximal activity in reducing environments. Alternatively, an unknown, uncontrolled variable may be responsible for the lack of reproducibility of these results.
VitaCyte has two other proteases that complement the specificity of the M4 proteases used for human islet isolation. AM Protease is derived from Aspergillus melleus and has a specificity similar but not identical to chymotrypsin that cuts on the carboxyl side of aromatic and large hydrophobic amino acids. VitaCyte will soon launch a GMP Grade purified Elastase product purified from porcine pancreatin. Elastase cuts peptides on the carboxy-terminal side of small neutral amino acids such as alanine, glycine, or valine.
If you are interested in performing further research on using AM Protease or Elastase as a supplemental enzyme for human islet isolation, please contact me directly (rcmccarthy@vitacyte.com).
References
- Brandhorst H, Friberg A, Andersson HH, Felldin M, Foss A, Salmela K, et al. The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation. – Transplantation. 2009;87:370-5.
- Stahle M, Foss A, Gustafsson B, Lempinen M, Lundgren T, Rafael E, et al. Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation. Transplantation Direct. 2015 Jun;e19. PubMed PMID: 27500221.
- 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.
- McCarthy RC, Green ML, Dwulet FE. Evolution of enzyme requirements for human islet isolation. OBM Transplantation. 2018; 2(4):[1-30 pp.]. Available from: http://www.lidsen.com/journals/transplantation/transplantation-02-04-024.