Collagenase V (Synonyms: Collagenase; collagen hydrolase)

This protocol only provides a guideline, and should be modified according to your specific needs.
1. Solution Preparation:
(1) Working Solution: Typically prepare the working solution in HBSS balanced salt solution (containing Ca²⁺ and Mg²⁺) at a concentration of 1-2mg/mL. Sterilize by filtration through a 0.22µm filter membrane. For tissue and cell dispersion, a working concentration of 0.5-2.5mg/mL is recommended. For cartilage digestion, a working concentration of 1-2mg/mL is recommended.
 
Note: Unused solution should be aliquoted and stored at -20°C in the dark. Thaw on ice before use and avoid repeated freeze-thaw cycles.
 
2. Tissue Separation Using Collagenase V:
(1) Cut the tissue into 3-4mm³ pieces using sterile scalpels or scissors.
(2) Wash the tissue pieces several times with HBSS containing Ca²⁺ and Mg²⁺.
(3) Add enough HBSS containing Ca²⁺ and Mg²⁺ to immerse the tissue pieces, and add Collagenase V to the desired working concentration.
(4) Incubate at 37°C for 4-18h. Using a horizontal shaker and supplementing digestion with 3mM CaCl₂ can improve digestion efficiency.
(5) The dispersed cells can be collected using a mesh sieve. Undigested tissue can be further digested with fresh Collagenase V working solution at 37°C.
(6) Wash the collected cells with HBSS without Collagenase.
(7) Resuspend the cells in cell culture medium and seed onto culture dishes with appropriate culture medium.
 
3. Protocol for obtaining pancreatic islet cells using Collagenase V^[1] (from the literature, for reference only)
(1) Euthanize the mouse. Cut the upper abdominal cavity and inject 4mL of a mixed solution of Collagenase V (2mg/mL) and VI (0.5mg/mL) into the common bile duct to expand the pancreas.
(2) Transfer the pancreas to a 37°C water bath or incubator and digest for 5min.
(3) Transfer the digested pancreatic tissue to a 50mL centrifuge tube, add an appropriate amount of HBSS buffer, and gently blow the tissue to disperse it into a cell suspension. Slowly add the cell suspension to the upper layer of the Histopaque density gradient solution (1.10g/mL). Centrifuge at 600g for 20min to separate the pancreatic islet cell layer.
(4) Collect the pancreatic islet cell layer, transfer the cell suspension to a new centrifuge tube, add Hank's solution containing 10% FBS, centrifuge at 300g for 2min, and discard the supernatant. Repeat the wash once.
(5) Resuspend the islet cell pellet in a small amount of HBSS and transfer it to a 100mm culture dish. Under a stereomicroscope, manually select the islet cell clusters using a capillary glass tube with an inner diameter of 500μm.
(6) Transfer the islet cell clusters to a new centrifuge tube, add Hank's solution containing 10% FBS, centrifuge at 300g for 2min, and discard the supernatant. Resuspend the cell pellet in RPMI 1640 culture medium (containing 10% FBS), transfer it to a culture dish or culture bottle, and culture it in a 37°C, 5% CO₂ incubator for subsequent experiments.
 
Note: The above experimental protocol provides guidance for tissue separation using Collagenase V. Adjustments can be made according to other literature and specific experimental requirements.

References:
[1]Tripathi D, Cheekatla S S, Paidipally P, et al. c-Jun N-terminal kinase 1 defective CD4+ CD25+ FoxP3+ cells prolong islet allograft survival in diabetic mice[J]. Scientific Reports, 2018, 8(1): 3310.