Use of Purified-Defined Enzymes Reduces Costs, Enables Troubleshooting of the Isolation Process
Product Development
VitaCyte collaborated with Steve Strom’s lab at the University of Pittsburgh to develop a purified-defined collagenase-protease mixture for human hepatocyte isolation.(1) The enzyme formulation was similar to an enzyme composition found in a superior lot of Sigma Type XI Collagenase.
The biochemical analyses performed on three Type XI lots are shown on the right. The anion exchange chromatographic profiles, endotoxin contamination, and CDA and NP enzyme specific activities (U/mg) summarize the biochemical characteristics of these lots. The top profile summarizes the analyses of the “Reference Lot,” used to guide the formulation of the purified collagenase-protease enzyme mixture. The other two profiles below show the analyses of two other Type XI lots that were ineffective in human hepatocyte isolation. Lot 086K8626 has acceptable CDA but low NP activity, whereas Lot 118K8608 had low CDA and NP activity.
Product Application
VitaCyte’s Collagenase MA and BP Protease products have been used for over 10 years by numerous companies that sell human and rodent cryopreserved hepatocytes or by cell therapy companies that use hepatocytes as a therapeutic agent. For this application, Strom’s study targeted 2500 CDA U and 2200 NP U per mL of enzyme solution for human hepatocyte isolation. One company expanded the use of these products to rodent hepatocyte isolation. Here, they modified the formulation and used about 50% of the amount of protease for these isolation procedures. For additional details, see Rafal Witek’s webinar on mammalian hepatocyte isolation(2) or contact VitaCyte Technical Support.
VitaCyte’s Products & Benefits
The table below shows the product specifications and lot-to-lot variability of the Collagenase MA and BP Protease products. The CVs are < 7% and <13% for the collagenase and protease products, respectively. These products have low endotoxin contamination.
The table below summarizes the differences between Traditional and VitaCyte’s purified-defined collagenase products. For more detail, see the 4 minute video(3) comparing these products.
The primary benefit for using VitaCyte’s collagenase and protease enzyme for hepatocyte isolation is improved control of the cell isolation process. Tissue dissociation enzymes can be controlled whereas since nearly all other factors are uncontrollable.
Adoption of an optimal formulation of VitaCyte’s enzymes for your application rules out the enzymes as the cause of a failed isolation. The consistent manufacture and stability of these enzymes focus troubleshooting discussions to non-enzymatic factors such as organ quality, warm and cold ischemia time, perfusion of the enzyme in the tissue, etc…. If the enzyme formulation appears to be the cause, it can be modified to adjust to new needs.
References
- Gramignoli R et al. Cell Transplantation. 2012 21:1245-60.
- Witek RP Collagenase selection & process optimization for high yield hepatocyte isolation. May 2024
- https://www.vitacyte.com/resources/documents/traditional-vs-vitacyte-collagenase/