Other Cell Applications
The application specific pages describe cell isolation protocols for common applications. VitaCyte shares these methods because the critical steps are described in detail and have been used successfully by multiple laboratories. VitaCyte does not have recommended protocols for many other cell types. However, we can recommend a process for using available information in the literature along with current VitaCyte products to help identify a formulation that will work for the target cell type.
Replicating most cell isolation protocols for other cells can be challenging for several reasons.
Critical steps in the cell isolation method are performed differently.
The tacit or unwritten knowledge of the cell isolator cannot be explicitly written or shared with others in a documented manner.
Lot to lot differences in the collagenase products used by different laboratories.
Modifications are made to reported methods, but those improvements are not recorded or published.
If your goal is to adopt a new cell isolation procedure or find an alternative to your current method, the best place to begin is to search journals or online websites that publish life science protocols. You can also contact your university library to get access to other journals that focus on publishing life science protocols.
Collagenase-Protease Mixtures for Cell Isolation
Selection of the collagenase-protease mixture is crucial in enzyme-mediated cell isolation as it affects cell viability, yields, and function of the cells isolated from tissue. The most common approach is to pre-qualify available “off the shelf” traditional collagenase product by evaluating several lots. The goal is to select the lot of collagenase (i.e., the “good lot”) that most effectively isolates the cells of interest. Once identified, the end-user buys a sufficient amount of the good lot to last a year or more.
VitaCyte’s purified-defined enzymes offer significant advantages over the traditional crude and enriched enzymes as summarized in the table below.
| Traditional Collagenase | VitaCyte Purified-Defined Collagenase | |
|---|---|---|
| Lot Consistency | Minimal: Each lot unique | Maximal: Use of purified enzymes ensures consistency |
| Collagenase Purity | 4-8% (w/w) for crude 15-25% (w/w) for enriched | > 95% (w/w) |
| Need to Pre-Qualify New Collagenase Lots | Necessary to optimize isolation procedure | No need after initial optimization step performed |
| Replication of Results in Other Labs | ? less likely if good lots hard to find | No problem, enzyme composition defined |
| Additional Modification of Enzyme Formulation | Not possible | Always an option |
The difference in lot consistency reflects the lower purity of traditional collagenase products and the potential for other biochemical components in the product to influence cell viability or function. Therefore, it is understandable that most end-users pre-qualify new lots of traditional collagenase. If purified collagenase and protease enzymes are used in their place, then the pre-qualification procedures need to be performed only once. Moreover, once the biochemical composition of the enzyme solution is known, then further adjustments can be made if required.
Product UseVitaCyte Rational Design Enzyme Formulation Method
VitaCyte’s rational design method for formulating enzyme mixtures applies principles from a hypothetical model for enzyme mediated cell isolation described elsewhere. This model simplifies 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.
PD Collagenase 100
Cat # 011-3010
PD Collagenase 800
Cat # 011-3020
Four Key Assumptions of the Enzyme-Mediated Cell Isolation Model
ASSUMPTION ONE
Excess purified collagenase (with minimal protease contamination) will not have an adverse effect on cell viability or function
ASSUMPTION TWO
Collagenase’s collagen degradation activity (CDA) must be in excess to ensure loosening of the extracellular matrix
ASSUMPTION THREE
If CDA in excess, then neutral protease activity is critical to the speed of tissue digestion, and yield of functional viable cells
ASSUMPTION FOUR
Neutral protease activity required for successful cell isolation is equivalent or less than the neutral protease activity found in Worthington Type 1 collagenase
Method Overview
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.
For specific details consult the PD Collagenase 100/800 package insert.
Estimate the collagenase mass in the collagenase product you currently or are planning to use in the cell isolation procedure.
Calculate the amount of collagenase 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 current cell isolation procedure. The table below uses the example of preparing 10 mg collagenase solution in 100 mL (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
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 when using a modified formulation for rodent hepatocyte isolation. This modification had no impact on cell yield or function. Similar modifications have been made for isolation of human adipose derived stromal vascular fraction cells.
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.
Determine if the VitaCyte’s products provides comparable results. You may find 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 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 the protein 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 |
If you have any questions on using enzymes for cell isolation or recovery, contact VitaCyte Technical Support.