Storage Condition

Transport at room temperature. Store at 4℃ protected from light.

Components

Description

Collagenase products are purified from Clostridium histolyticum. They cleave at the Pro-X-Gly-Pro motif (X = neutral amino acid), which occurs frequently in collagen. Collagenase uniquely degrades native, triple-helical collagen fibrils found in connective tissue.

This product is Type II collagenase (≥125 U/mg solid) suitable for dissociation of tissues and cells in heart, thyroid, salivary gland, liver, bone, cartilage, and related tissues.

Definition of Activity Unit

One unit liberates 1 μmol of L-leucine equivalents from collagen in 5 hours at 37℃, pH 7.5.

Protocol

1) Preparation of Collagenase Storage Solution

• Add 1 ml suitable buffer (e.g., HBSS with Ca²⁺/Mg²⁺) to a 100 mg vial; gently vortex to dissolve → 100 mg/ml stock.
• Sterile-filter through a low-protein-binding 0.22 μm membrane; aliquot and store at −20℃ protected from light.
• Thaw on ice; avoid repeated freeze–thaw cycles.

Typical working concentration: 0.5–2.5 mg/ml for tissue/cell dispersion; 1–2 mg/ml for cartilage digestion (optimize per sample).

2) Tissue Dissociation

1. Mince tissue to 3–4 mm pieces; wash several times with buffer (e.g., HBSS).
2. Add buffer to submerge tissue; add collagenase to desired concentration.
3. Incubate at 37℃ for 4–18 h (rocker platform + 3 mM CaCl₂ improves efficiency).
4. Disperse cells by passing through sterile stainless-steel or nylon mesh; re-incubate remnants if needed.
5. Wash dispersed cells several times by centrifugation in buffer without collagenase.
6. Resuspend in culture medium; assess viability and seed into vessels.

3) Organ Perfusion

1. Add collagenase to prewarmed (37℃) buffer (e.g., HBSS); adding 3 mM CaCl₂ increases dissociation efficiency.
2. Perfuse organ at a pre-optimized flow rate.
3. Filter perfusate to remove large debris; re-incubate remaining tissue in fresh collagenase if needed.
4. Wash cells several times by centrifugation in buffer without collagenase.
5. Resuspend in culture medium; determine viable cell density; seed into culture vessels.