Robert McCarthy
When you are down to the last few bottles of your current lot of traditional crude or enriched collagenase, do you feel like Bill Murray in “Groundhog Day” since you know that sooner than later, you must repeat the process to qualify a new lot of collagenase. You will have to make a note to contact your supplier and have samples of available collagenase lots sent to you for evaluation in your cell isolation procedure. Collagenase lot qualification may be stressful depending on your experience finding a “good” lot. You know this is unproductive but essential work since a new lot must be assessed and approved by others before purchase of larger amount of a new lot of collagenase product that will last a year or more.
You can overcome the tyranny of Groundhog Day by using VitaCyte’s PD Collagenase 100 & 800 products. The enzyme composition of these products is identical to the DE Collagenase 100 & 800 products sold by VitaCyte in the past. The PD 100 & 800 package insert describes how you can use these two products to prepare intermediate enzyme formulations sold as the DE Collagenase 200, 400, and 600 products. The package insert also describes a method where these products can be used to replace any traditional or purified collagenase product. The benefits of this approach are:
- Avoid lot qualification; no more Groundhog days!
- New knowledge since you know the enzyme composition used to isolate your cells
- Ability to modify the above enzyme composition to make further improvements to your cell isolation method
How can I make this claim??? It is supported by VitaCyte’s working knowledge of how collagenase-protease enzyme mixtures digest tissue and the firm’s experience manufacturing three purified-defined enzyme formulations by applying these principles. This approach led to development of enzyme formulations to isolate human hepatocytes, rodent islets, and stromal vascular fraction cells from human lipoaspirate. The performance of each purified-defined enzyme mixture was equivalent and, in some cases, superior to those obtained with a lot of traditional collagenase.
The basis of preparing these enzyme formulations is applying a mechanistic model of enzyme-mediated tissue dissociation described in the literature.(1, 2) In an earlier blog post, this model was used to explain the results from a large series of human islet isolations performed using eight different enzyme mixtures. In brief, the key points of the model are:
- Collagen is the predominant protein in the extracellular matrix (ECM)
- Collagen is resistant to proteolytic degradation and provides a superstructure to protect other ECM proteins from degradation
- Cells are held within the matrix by ECM “cell anchoring proteins” that attach directly or indirectly to the cells
- Clostridium histolyticum collagenases’ collagen degradation activity (CDA) cuts nearly all forms of mammalian collagen
- Purified C. histolyticum class I (C1) and class II (C2) collagenase have restricted specificity, they cut native and denatured ( i.e., gelatin) collagen
- Collagen is an ECM protein, excess purified collagenase does not damage cells or affect their function
- C1 and C2 collagenases work synergistically to loosen the ECM, increasing the exposure of protease-sensitive sites on the ECM proteins, breaking the bonds that anchor the cells to the ECM, leads to release of cells from tissue
- The selection and dose of neutral protease(s) controls the speed of breaking these bonds between the ECM and cells
- Any adverse effects on cell yield, viability, and function are due to suboptimal use of neutral protease (i.e., using the wrong protease or using excess amounts of protease activity)
The golden rule for successful cell isolation is to prepare an enzyme mixture that has excess collagen degradation activity and an optimal neutral protease composition that releases viable and functional cells from tissue.
The steps to create a purified-defined collagenase-protease mixture that matches the collagenase and protease activities you use in your current collagenase products are described below. Additional information can be found on the PD Collagenase 100 & 800 package insert.
Step 1: Translate the collagenase and protease enzyme activities of the collagenase products you currently use for your application into VitaCyte’s collagen degradation activity and neutral protease activity units. These values are determined by using a kinetic, fluorescent microtiter plate assays.(3, 4) If you are using Collagenase Types 1, 2, 3, or 4 from Worthington Biochemicals or Roche’s Liberase Research Purified Enzyme Blends sold by Sigma, the PD Collagenase 100/800 package insert provides estimated values of the CDA/NPA ratios for these products. If you use any other traditional collagenase product, contact VitaCyte technical support for additional information. The biochemical variability for most collagenase products may require that you send VitaCyte a sample of your good lot of collagenase product to determine VitaCyte’s CDA and NP activities. There is a reasonable fee for this service. Contact VitaCyte for details.
Step 2: Consult the package insert, insert the CDA/NPA ratio determined in Step 1 into the algebraic equation: x = (10.47 – y)/9.16 where y is the CDA/NPA ratio. Solving for x will provide you with the amount of PD Collagenase 100 needed, while 1-x will provide the amount of PD 800 needed to prepare the enzyme mixture. The second step of the process is to determine how much of this mixture you need to prepare to perform your cell isolation procedure. The details are described in the package insert.
Step 3: Evaluate the performance of the purified-defined PD Collagenase enzyme mixture compared to your current lot of collagenase or incorporate it into your lot qualification process. If the results generated from the PD Collagenase enzyme mixtures do not perform to your expectations, it is likely that an additional neutral protease, clostripain, is needed. Details for using this product as a supplemental enzyme are found in the PD Collagenase package insert.
Step 4: Share your experience with using the PD Collagenase products. VitaCyte needs your feedback to address any technical issues with the goal of making further improvements to the method. VitaCyte is committed to practicing open science to ensure that others can repeat the results from research publications. Incorporating purified-defined enzyme mixtures into collagenase-mediated cell isolation is a gigantic step forward to improve the reproducibility of these procedures.
References
1. McCarthy RC BA, Green ML, Dwulet FE. Tissue dissociation enzymes for isolating human islets for transplantation: Factors to consider in setting enzyme acceptance criteria. Transplantation. 2011;91:137-45.
2. McCarthy RC, Green ML, Dwulet FE. Evolution of enzyme requirements for human islet isolation. OBM Transplantation [Internet]. 2018; 2:[1-30 pp.]. Available from: http://www.lidsen.com/journals/transplantation/transplantation-02-04-024.
3. McCarthy RC, Spurlin B, Wright MJ, Breite AG, Sturdevant LK, Dwulet CS, Dwulet FE. Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. Transplantation Proceedings. 2008;40:339-42.
4. Breite AG, Dwulet FE, McCarthy RC. Tissue Dissociation Enzyme Neutral Protease Assessment. Transplantation Proceedings. 2010;42:2052-4.