Storage Condition

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

Components

Description

Collagenase I is purified from Clostridium histolyticum. It specifically cleaves at Pro-X-Gly-Pro motifs, abundant in collagen. Unlike most proteases, it degrades the native triple-helical collagen fibrils of connective tissue.

Type I collagenase with ≥125 U/mg solid, suitable for tissue and cell dissociation in epithelium, liver, lung, adipose tissue, adrenal gland, cartilage, and more.

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 Storage Solution

• Dissolve 100 mg Collagenase in 1 ml buffer (e.g., HBSS with Ca²⁺/Mg²⁺) → 100 mg/ml stock.
• Sterile filter through 0.22 μm membrane; aliquot and store at −20℃ protected from light.
• Thaw on ice; avoid repeated freeze-thaw cycles.

Recommended working concentration: 0.5–2.5 mg/ml for tissue dissociation; 1–2 mg/ml for cartilage digestion.

2) Tissue Dissociation

1. Mince tissue into 3–4 mm pieces; wash with buffer.
2. Submerge tissue in buffer containing collagenase.
3. Incubate at 37℃ for 4–18 h (rocker platform + 3 mM CaCl₂ improves efficiency).
4. Disperse cells via mesh; re-incubate residual tissue if needed.
5. Wash cells by centrifugation in buffer without enzyme.
6. Resuspend in culture medium; determine viability and seed into vessels.

3) Organ Perfusion

1. Add collagenase to prewarmed buffer (+3 mM CaCl₂ for efficiency).
2. Perfuse organ at optimized flow rate.
3. Filter perfusate to remove large debris; re-incubate residual tissue if needed.
4. Wash cells by centrifugation in buffer without enzyme.
5. Resuspend in medium; determine viability; seed into culture vessels.