Robert McCarthy
After closely reviewing my earlier blog post’s content, “Are you tired of lot qualification? try this,” I realized that optimizing collagenase-protease enzyme mixtures could become simpler. The revised method continues to be based on the mechanistic model of collagenase-protease-mediated cell isolation summarized in an earlier blog post (“Occam’s razor guides mechanistic model of tissue dissociation”).
The method is now termed the Rational Design Method to Optimize Tissue Dissociation Enzymes (RDMTDE) for cell isolation. The two changes from the earlier method are that Collagenase Gold is used in place of PD Collagenase 100 and an Excel-based enzyme calculator is included to simplify the preparation of the enzyme solutions.
The method works only if you can reference a method or have a method that uses a Clostridium histolyticum collagenase-protease product(s). Knowledge of the collagenase mass used in your enzyme solution is critical because the preparation of the enzyme solution depends on the Reference Collagenase Dose (RCD) value. The RCD should provide sufficient collagenase activity that consistently provides an adequate yield of functional cells. These results indicate that collagenase’s collagen degradation activity must be in excess.
Worthington Collagenase Types 1, 2,3, and 4 contain about 6% collagenase per dry weight. Roche’s Liberase research products, sold by Sigma, contain either 5 or 50 mg of purified collagenase per bottle. For other products, contact VitaCyte technical support for additional information.
The other critical assumption is that the neutral protease activity contained in Worthington Type 1 Collagenase is sufficient to digest any mammalian tissue. This statement is supported by the numerous references listed in Worthington’s Tissue Dissociation Guide for different tissues from many species. The neutral protease activity in PD Collagenase 800 is similar to that in Worthington Type 1 Collagenase.
The PD Collagenase 800 and Collagenase Gold products are used in place of the PD Collagenase 100 and 800 products presented in the earlier blog post for several reasons:
- Preparation of the enzyme solutions is simpler since both products contain a high percentage of purified collagenase (≈ 44%), 8x the amount found in Worthington Type 1 Collagenase, allowing the use of less product to prepare the enzyme solutions.
- The amount of PD Collagenase 800 determines the amount of neutral protease activity whereas the Collagenase Gold is a “collagenase filler” to ensure the targeted dose of collagenase is used in the enzyme solution
The simplest way to teach the method of enzyme preparation is to use an illustration. Let’s assume you are using 3 mg/mL of Worthington Type 1 Collagenase are prepare 100 mL of enzyme solution for your cell isolation method. Your interest in adopting the RDMTDE method is to define the collagenase and neutral protease activities required to isolate the cell population of interest. You also want to reduce neutral protease activity in the enzyme mixture to see if it will improve cell yield, viability, or function.
First, you need to determine the collagenase mass in 3 mg of Worthington Biochemicals’ Corporation Type 1 Collagenase (WBC T1). Multiplying 3 by 0.06 (% collagenase in WBC T1) = 0.18. Since 100 mL of enzyme solution is needed, the total mass of collagenase required to prepare the enzyme solution is 18 mg.
Second, you must determine the amount of neutral protease activity used in the enzyme solution. In the RDMTDE method, the amount of neutral protease activity references the activity found in WBC T1 Collagenase. You decided to use 60, 80 and 120% of this activity.
To determine the amount of each product you require to prepare these enzyme solutions, plug the values into the VitaCyte TDE Excel calculator as shown below.
Up to five different enzyme solutions can be calculated using this spreadsheet. Only three entries are made above, and the following information is presented to prepare the enzyme solutions.
- The amounts of PD Collagenase 800 and Collagenase Gold required to prepare the enzyme solutions
- The amount of purified collagenase in each product
- The total NP U and mg of collagenase in the enzyme solution
- Re-verification of the targeted doses of enzymes expressed as the percentage of neutral protease activity found in WBC T1 Collagenase, the mg/mL of collagenase, and NP U/mL of enzyme solution
The last and final step is to test the effectiveness of these solutions to isolate your cell of interest. Is the purified enzyme solution’s performance comparable to those obtained with your current lot of product? If the results generated from the PD Collagenase enzyme mixtures do not perform to your expectations, an additional neutral protease, clostripain, is likely. Details for using this product as a supplemental enzyme are found in the Clostripain package insert or contact VitaCyte technical support for additional help.
The enzyme calculator can be found below. To download the calculator, you will need to provide your contact information followed by email verification. This request is made because VitaCyte wants to follow up by email with the hope that you will share your experience using this new approach for optimizing enzyme formulation for cell isolation. The plan is to extend this method to using other VitaCyte-purified enzyme products.
If you do take this step and use the RDMTDE method, the three benefits of this approach are:
- Identification of the collagenase and protease activities required for cell isolation
- Assessment of the effect of neutral protease activity on cell viability and function
- Avoid pre-qualification of new collagenase lots since the purified enzyme composition is defined and new lots of purified products are consistently manufactured
TDE Excel Calculator
Fill out the form below to verify your email and then the excel calculator will be sent to you.