Collagenase


1. Introduction to Tissue Dissociation Using Collagenases

Collagenase Activity Comments and Example Application**
Collagenase (col G & H) (CDU/mgdw) Caseinase (neutral protease) (u/mgdw) Clostripain (u/mgdw) Trypsin-like (u/mgdw)
Partially Purified for Standard Dissociation Applications, available in 22 µm Filtered and Non-Filtered Formats
Type 1* ≥125 ≥200 ≤4.0 ≤0.5 Balanced activities: Adipose, Adrenal, Epithelial, Liver, Lung
Type 2* ≥125 ≥200 ≥3.5 ≥0.1 Higher proteolytic activities: Bone, Heart, Liver, Thymus
Type 3* ≥100 ≥50 ≤3.0 ≤0.3 Lower proteolytic activities: Mammary
Type 4* ≥160 ≥100 ≤3.0 ≤0.1 Lower tryptic activity: Pancreatic Islets
Type 5* ≥450 ≥450 ≤3.0 ≤0.3 Higher collagenase and caseinase activities
Type 6 ≥400 ≥1000 ≤4.0 ≤0.5 Higher activity with caseinase to collagenase ratio ~2:1, designate
Type 7 ≥1000 ≥2000 ≤8.0 ≤0.5 Contains collagenase and caseinase activities 4× higher than Type 1 & 2
Collagenase/Elastase (CLSH*) ≥125 ≥200 ≤4.0 ≤0.5 Mix of Collagenase Type 1 and Elastase (ESL); Component of the Hepatocyte Isolation System (HIS) Kit
Animal Free for e.g. Bioprocessing, Stem Cell Research, and Clinical Workflows, ensuring highest safety by excluding animal-derived components.
Type A* ≥150 ≥150 ≤8.0 ≥0.1 Balanced activities: Stem Cell & Tissue Bioprocessing
Type B* ≥300 ≥300 ≤5.0 ≤0.5 Higher activities: Stem Cell & Tissue Bioprocessing
Type C* ≥200 ≥150 ≤3.0 ≤0.1 Lower protease activities: Stem Cell & Tissue Bioprocessing
Type D ≥600 ≥600 ≤5.0 ≤0.5 High collagenase and Caseinase activity: Stem Cell & Bioprocessing
STEMxyme® Animal Free Blends
STZ1* ≥250 ≥1000 ≤5.0 ≤0.5 Mix of Animal Free Collagenase Type B & Animal Free Neutral Protease/Dispase® (NPRO)
STZ2* ≥250 ≥2000 ≤5.0 ≤0.5 Same as STZ1, but with higher Caseinase activity
Chromatographically Purified for Collagen Studies and Controlled Addition of Individual Enzymes
CLSPA / CLSPANK*** ≥500 ≤50 ≤2.0 ≤0.25 High Collagenase activity and minimal secondary proteolytic activities: Collagen studies, tissue digestion with other proteases. CLSPANK is a component of the Neonatal Cardiomyocyte Isolation System (NCIS) Kit
CLSAFP (Purified & Animal Free) ≥1500 ≤50 ≤2.0 ≤0.25 Low protease: Collagen studies, tissue digestion with other proteases

* 0.22 µ Filtered version available. ** Correlations between type and effectiveness with different tissues have been good, but not perfect, and may be dependent partly on parameters of use and objectives as well as lot-to-lot variations. For more information see the Collagenase Sampling Program information. *** 0.22 µ Filtered, Chromatographically purified, ≥1,500 units CLSPA Collagenase activity per vial; Component of the Neonatal Cardiomyocyte Isolation System (NCIS) Kit

The Role of Enzymatic Dissociation in Cell Recovery

Enzymatic tissue dissociation is a fundamental technique that enables the recovery of viable, functional cells from solid tissue specimens. It is routinely applied across a wide range of research workflows, including primary cell culture, organoid generation, regenerative medicine research, and single-cell analyses such as flow cytometry and sequencing.

Collagenases derived from Clostridium histolyticum are among the most widely used enzymes for this purpose. These enzymes degrade collagen, a major component of the extracellular matrix (ECM), and thereby facilitate the release of intact, functional cells.

Why Use Collagenases?

Collagenases are enzyme preparations composed of multiple enzymes working together. They exhibit collagenolytic and proteolytic activity, enabling efficient matrix degradation while preserving cell membrane integrity. Their unique enzymatic profiles allow tailored application depending on tissue type and target cell population.

  • Broad applicability across soft and connective tissues.
  • The combination of different dissociating enzyme activities within collagenase preparations improves tissue dissociation efficiency.
  • Customizable enzyme selection enhances viability and yield of specific cell types.

Key Questions for Selecting the Right Collagenase Preparation

Commercially available collagenase “types” refer to partially purified enzyme preparations derived from Clostridium histolyticum. These are not pure collagenase enzymes, but rather complex blends containing:

  • two collagenase isoforms (called Collagenase Type I and Collagenase Type II)
  • the sulfhydryl protease
  • Clostripain
  • a Trypsin-like Protease
  • an Aminopeptidase

This enzymatic diversity contributes to effective degradation of extracellular matrix components and facilitates tissue dissociation across a broad range of sample types.

Not necessarily. Commercial collagenase types (e.g., Type 1, Type 2, …) describe enzyme mixtures, but their exact composition can vary between manufacturers. A “Type 1” from one supplier may differ in enzyme balance and activity compared to another brand’s Type 1. It’s important to review product details carefully and test which preparation works best for your specific tissue and application.

For more information on enzyme activities across the different types, see 2. Collagenase Types Overview, including the table detailing their composition and activity profiles.

The relative activity of each enzyme component influences:

  • Digestion kinetics
  • Cell yield and viability
  • Preservation of membrane antigens and receptors

Different tissues and cell types vary in matrix density and sensitivity to proteolysis. Choosing the right preparation ensures a balance between efficient dissociation and minimal functional damage to target cells.

Key considerations include:

  • Tissue characteristics: fibrotic vs. soft tissue
  • Target cell population: parenchymal, stromal, endothelial
  • Downstream application: cell culture, FACS, RNA extraction
  • Marker sensitivity: receptor preservation is critical for immunophenotyping

Tip: Explore our Tissue Dissociation Guide for examples of published protocols and practical tips on choosing the right enzymes for your application.

Collagenase Type 2 is formulated to contain higher levels of protease activity, especially clostripain, which enhances its ability to break down tough extracellular matrix components. Because of this, it is especially well suited for isolating primary cells from tissues like bone, heart, liver, thyroid, and salivary glands. This makes it a versatile and popular choice for many tissue dissociation protocols.

Collagen Type 2 offers a good compromise between digestion strength and antigen preservation, especially in dense, well-perfused tissues.

Each product is supplied as a standardized lyophilized powder, tested and defined for:

  • Collagenase activity
  • Caseinase (non-specific proteolytic) activity
  • Clostripain activity and
  • Trypsin-like activity.

2. Collagenase Types Overview

Collagenase preparations from Clostridium histolyticum are available as lyophilized powders in various purification grades and enzyme compositions. They differ in their relative levels of collagenolytic activity—primarily Collagenase Types I (gene ColG) and II (gene ColH)—as well as secondary proteolytic activities, including caseinase (a non-specific protease) and clostripain, a trypsin-like enzyme. This enzymatic diversity allows for tailored selection depending on tissue type, cell sensitivity, and downstream applications.

The non-animal-free collagenase preparations, labeled Types 1 through 7, differ in their specific enzyme compositions and activity profiles. Animal-free collagenase variants, designated Types A through D, offer similar functional diversity while being produced without any animal-derived components.

Preparation Formats

All products are supplied as lyophilized powders and are available in two main preparation formats:

  • Standard (non-filtered) Preparations
    The base form, without filtration through a 0.22 µm mesh, is suitable for many standard protocols in tissue dissociation and cell isolation.
  • 0.22 µm Filtered Preparations
    Collagenase Types 1 – 5 are also available in a 0.22 µm sterile-filtered format, offering enhanced purity and consistency. These preparations are:

    • free from microbial contamination
    • ready-to-use after reconstitution
    • optimized for sensitive cell isolation and culture workflows
  • Animal-Free Collagenases
    For applications where the exclusion of animal-derived components is critical, animal-free collagenase types are produced using entirely animal-free culture media and processes. They offer enzymatic profiles similar to traditional types (e.g. Types 1 and 2) and are suitable for bioprocessing, stem cell research, or clinical workflows requiring the highest safety standards.

These formulation options provide flexibility in adapting enzymatic tissue dissociation to the specific requirements of research or bioprocessing applications.

To support selection for specific tissue types and workflows, the table below outlines the various collagenase types, their enzymatic activity ranges, and typical applications in cell isolation and tissue dissociation.

3. Product Formats & Handling

Worthington collagenase products are supplied as lyophilized powders in a range of package sizes to suit different scales of research and production needs.

  • The smallest standard package size is typically 50 mg respectively 100 mg.

  • Larger quantities are commonly available as 5 × 50 mg, 1 g, and 5 g packs.

  • Chromatographically purified collagenases are offered in activity-based packages, typically containing 4 Kunitz Units (ku) or 10 ku.

  • For larger scale applications, all collagenase types are also available in bulk quantities.

These packaging options provide flexibility for both small-scale experiments and larger bioprocessing workflows, ensuring users can select the format best suited to their requirements.

Storage and Reconstitution:
Lyophilized collagenase should be stored tightly sealed at 2–8 °C and protected from moisture. Reconstituted solutions should be freshly prepared and used promptly or aliquoted and frozen to maintain enzymatic activity.

4. Application

Collagenase is typically used at concentrations ranging from 0.05% to 0.5% (w/v) in balanced salt solutions such as Hank’s, Earle’s, or similar buffers. Achieving optimal results requires carefully matching the collagenase type and its associated proteolytic activities to the specific tissue being dissociated. Worthington’s range of partially purified collagenase types offers tailored mixtures of enzymatic activities, recommended for particular tissue types based on extensive evaluation and experience from numerous researchers.

Among the available options, Collagenase Types 1, 2, and 4 are the most commonly used and are suitable for dissociating a wide range of tissues in both research and bioprocessing applications.

Beyond using collagenase alone, many tissue dissociation protocols include additional enzymes to enhance efficiency and protect cell viability. Typical combinations pair a collagenase suited for the specific tissue with one or more complementary enzymes:

  • Collagenase + Elastase: particularly useful for tissues with significant elastic fibers, such as lung, liver or skin

  • Collagenase + Hyaluronidase: helps break down extracellular matrix components like hyaluronic acid, aiding dissociation of connective tissues

  • Collagenase + DNase I: reduces viscosity caused by free DNA released from lysed cells during dissociation, improving cell yield and handling

For researchers seeking detailed guidance, Worthington offers the Tissue Dissociation Guide, which can be obtained through us. This comprehensive resource consolidates information on standard laboratory procedures, provides a logical framework for developing tissue-specific cell isolation protocols, and includes extensive references covering a wide variety of tissues.

5. Support for Selecting the Right Collagenase

Choosing the optimal collagenase for your tissue type and experimental setup can significantly impact your results. We’re here to help you navigate this choice with personalized guidance and practical services designed to reduce variability and ensure confidence in your workflows.

Personal Consultation
Our team is available to discuss your specific application, tissue type, and experimental requirements. We’re happy to help you narrow down suitable collagenase types and activity profiles before you proceed with testing. Contact us.

Collagenase Sampling Program
Once you have a recommendation—or if you’d like to evaluate options yourself—we offer a Collagenase Test Program to help you select the best-performing lot for your assays:

  • Request 100 mg samples of up to two different lots for testing in your own assay systems.

  • A 4-week evaluation period is provided to assess performance.

  • We’ll reserve a minimum of 5 grams per lot under your name during this testing phase.

  • Once you identify the best-performing lot, simply specify it when placing your order to ensure continuity in your experiments.

Lot Matching Service
Switching collagenase lots can sometimes introduce variability. To mitigate this risk, we offer a Lot Matching Service:

  • Provide the lot number of a collagenase that has worked well in your application.

  • We will identify the closest available match based on enzymatic activity, including secondary proteolytic activities, to help maintain consistency in your protocols.

These services are part of our commitment to supporting your specific research needs and ensuring reliable results. Don’t hesitate to reach out for a consultation or to initiate a sampling request.