Robert McCarthy
“The most damaging phrase in the language is ‘We’ve always done it this way.’”
Grace Hopper, US Navy Rear Admiral, Computer Scientist, developed COBOL
Updates to an Earlier Blog Post
An earlier blog post, “Are you tired of lot qualification, try this,” offered a novel approach to enable you to convert the traditional crude or enriched collagenase products used for cell isolation to a comparable purified enzyme formulation using VitaCyte’s products. The benefits for using VitaCyte’s purified-defined enzyme products are summarized in the earlier blog post.
The earlier method required that you know the ratio of your current supplier’s collagenase’s collagen degradation activity to neutral protease activity (CDA/NPA) expressed in VitaCyte’s enzyme units. An earlier version of the PD Collagenase 100/800 package insert provided these ratios for the Roche Liberase Research products sold by Sigma and for Worthington Biochemicals Collagenase Types 1, 2, 3, or 4. However, there were several limitations of this method.
- The supplier’s enzyme activity units had to be converted to VitaCyte’s enzyme units
- VitaCyte has limited knowledge of the CDA/NPA ratios in traditional crude or enriched collagenase products manufactured by Sigma, Nordmark, or other suppliers
- If VitaCyte could not provide a conversion factor into VitaCyte’s enzyme units, then you would need to submit a sample and pay a service fee for VitaCyte to perform these enzymatic analysis
Development of the Rational Design Method
A new insight for solving this problem occurred when I began revising the content for the updated VitaCyte website (launched 9 September 2024). Based on the principles of the mechanistic model of enzyme mediated tissue dissociation described in the earlier blog post, I realized the problem could be simplified by making two reasonable assumptions. The first assumption is that VitaCyte can provide an estimate of the mass of collagenase required to ensure excess collagenase degradation activity to use for most specific cell isolation procedures. These data are obtained by reviewing the published literature and adjusting these values based on any additional information from VitaCyte’s internal knowledge for developing purified collagenase-protease mixtures. And second, VitaCyte’s experience with other investigators to develop purified enzyme mixtures showed that the amount of neutral protease activity contained in most traditional crude collagenase products is in excess. Since the Worthington Type 1 collagenase has been used successfully for digesting most tissues, then this amount of protease activity in this product should be sufficient for successfully digesting the tissue and isolating your cell of interest. Customer feedback has shown that the neutral protease activity can be reduced further without impacting cell yield or function.
VitaCyte’s rational design for formulating enzyme mixtures applies principles from a hypothetical model for enzyme mediated cell isolation described elsewhere. This model simplifies the optimization of collagenase-protease enzyme mixtures because it assumes that if collagenase’s collagen degradation activity (CDA) is in excess, then the focus of the optimization procedure should be on neutral protease activity. Under conditions of excess CDA, neutral protease activity impacts digestion time, cell yield, viability and detection of cell membrane markers used to characterize the cell population.
Four key assumptions of the enzyme-mediated cell isolation model are:
- Excess purified collagenase (with minimal protease contamination) will not have an adverse effect on cell viability or function
- Collagenase’s collagen degradation activity (CDA) must be in excess to ensure loosening of the extracellular matrix
- If CDA in excess, then neutral protease activity impacts the speed of tissue digestion, and yield of functional viable cells
- Neutral protease activity required for successful cell isolation is equivalent or less than the neutral protease activity found in Worthington Type 1 collagenase
The Rational Design Method for Optimizing Tissue Dissociation Enzymes
The infographic below presents an overview of the method. For specific details that illustrate the calculations, consult the PD Collagenase 100/800 package insert.
Note, the amount of neutral protease activity in PD Collagenase 100 & 800 products are similar to those found in a Worthington Type 1 Collagenase. The broad application of Type 1 collagenase to digest tissue means that this amount of protease activity is acceptable to use for any tissue.
Estimate the mass of collagenase in the product you currently or are planning to use in the cell isolation procedure by referring to the reference table below. This dose is referred to as the reference collagenase dose (RCD). The table does not include data for isolating rodent or human hepatocytes or islets or adipose derived stromal vascular fraction (ADSVF) cells from human lipoapirate since these protocols can be found under the Applications Tab.
For reference, the table below shows the mg of collagenase in the enzyme solutions used to isolate the listed specific cells noted in the table. A reference is provided to show the method used for cell isolation.
| Tissue | Species | Cell isolated | mg/ml collagenase enzyme solution | Reference |
|---|---|---|---|---|
| Adipose parametrial fat | Rat | ADSVF | 3.0 | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684382/ |
| Heart | Adult rat or mouse | Cardiomyocytes | 0.06 for mouse 1.6 mg for rat | https://pubmed.ncbi.nlm.nih.gov/35813022/ |
| Venous | Adult humans umbilical tissue | HUVECs | 0.07 | https://pubmed.ncbi.nlm.nih.gov/18978951/ |
| Kidney | Adult mice | Renal tubular epithelial cells | 0.25 | https://pubmed.ncbi.nlm.nih.gov/29985358/ |
| Skeletal muscle | Rats | Satellite cells | 0.15 | https://pubmed.ncbi.nlm.nih.gov/23542587/ |
Calculate the RCD used in the enzyme solution prepared for your cell isolation. From this value, calculate the amounts of PD Collagenase 100, PD 800, and a 50:50 mixture of the two products required to prepare an enzyme solution containing the same mass of collagenase as found in your current lot of collagenase product.
Prepare three enzyme solutions containing PD Collagenase products, each containing the same collagenase mass that you use in your RCD for cell isolation. The table below uses the example of preparing 10 mg RCD solution in 100 mL enzyme solution (see pack insert for additional details).
Enzymes Solution 1 Solution 2 Solution 3
Total mg collagenase in enzyme solution 10 10 10
Total mg BP Protease in the enzyme solution 3.27 0.72 0.41
ug BP Protease per mL enzyme solution 32.7 7.2 4.1
Neutral protease activity (NPA U/mL) 4578 1008 574
In the example presented above, the neutral protease activity found in Solution 1 is similar to the protease activity in Worthington Type 1 Collagenase. This activity is further reduced in solutions 2 and 3 enabling the end user to assess the effect of neutral protease activity on cell isolation.
The need to titer the neutral protease activity in the rational design method is based on feedback from users who found that this activity could be reduced further, likely resulting in an increase in cell viability and less damage to cell membrane markers. For example, customers who purchased VitaCyte’s enzymes for human hepatocyte isolation found that the neutral protease activity could be reduced about 4-fold when using a modified formulation for rodent hepatocyte isolation. This modification had no impact on cell yield, viability, or function. The neutral protease activity was also successfully reduced for isolation of adipose stromal vascular fraction cells.
Determine if the VitaCyte’s products provides comparable results. You may observe slower digestion times as the neutral protease activity is decreased. This is expected from the hypothetical model of enzyme mediated tissue digestion. You will need to determine if extending the digestion time provides other benefits to recovered cell population.
Reviewing Results
If the results are comparable to those obtained with your current lot of product, congratulations! You now know more about the enzyme formulation required for isolating your cell of interest.
If you do not obtain equivalent results with any of the enzyme mixtures above when compared to your current lot of collagenase, the enzyme formulation may need to be adjusted or supplemented with another protease, Clostripain, as described in the package insert. Clostripain may be needed because it has a complementary enzyme activity when compared to BP Protease. Clostripain is a trypsin-like enzyme that cuts at different regions of proteins than BP Protease.
The effort required to assess the performance of the three PD Collagenase enzyme solutions above will not change the effort you presently take to qualify new lots of traditional crude or enriched collagenase products. The benefits of adopting the PD Collagenase enzyme in place of traditional collagenase are summarized in the table below.
| Lot Qualification | PD Optimization | |
|---|---|---|
| Effort Required | Equivalent | Equivalent |
| Knowledge Gained | None, no knowledge of enzyme composition | Essential: Enzyme composition defined |
| Lot Pre-Qualification | No change, must prequalify new lots | Once defined, no need to prequalify future lots |
| Ability to Modify Formulation | None | Yes |
| Shelf Stability for Product | ? | 4 years |
The method above is not restricted to the PD Collagenase products but can be used for any VitaCyte product. If you have any questions on using enzymes for cell isolation or recovery, contact us.