Cat. No.: 17100 Type I
17101 Type II
17102 Type III
17103 Hepatocyte Qualified
17104 Type IV
Sizes: See catalog.
Custom pack sizes available upon request.
Storage: 2 to 8°C （-5 to -20°C after reconstitution）
Avoid moisture and exposure to light.
Avoid inhalation and skin contact.
This product is intended for cell or tissue disaggregation only.
Background
Collagenase （from clostridium histolyticum） is a protease with a specificity for the bond between a
neutral amino acid （x） and glycine in the sequence Pro-X-Glyc-Pro. This sequence is found in high
frequency in collagen and is unique among proteases in its ability to degrade the triplehelical native
collagen fibrils commonly found in connective tissue.
The collagenase most commonly used for tissue dissociation is a crude preparation containing
clostripiopeptidase A and a number of other proteases, polysaccharidases and lipases. This crude
enzyme is ideally suited for tissue dissociation since it contains the enzyme required to attack
native collagen and reticular fibers, in addition to the enzymes which hydrolyze the other proteins,
polysaccharides and lipids in the extracellular matrix of connective and epithelial tissues.
Crude collagenase does exhibit lot-to-lot variability and may produce occasional toxicity. Invitrogen
attempted to minimize these difficulties by tissue-typing their crude collagenase lots based upon a
correlation between various enzyme levels in each preparation and effectiveness in dissociating
certain tissues.
Specifications
Potency: One unit liberates 1 μM of L-leucine equivalents from collagen in 5 hours at +37°C,
pH 7.5.
Types: Particular enzymatic activities of crude collagenases have correlated with the
tissues from which the cells were obtained （or with the uses to which the cells are
put）, and as a result of the correlation’s, several formal types have been established.
Type III Selected because of low proteolytic activity. （casein as substrate）
Type IV Selected because of low tryptic activity. （BAEE as substrate）
These selected types have been found to give better performance in preparation of cells from
the various tissues as tabulated below. It should be noted, however, that while the results
have been greatly improved following this classification, there is still some lot-to-lot variation,
and efforts continue in attempting to gain even better control over crude collagenase.
TYPE TISSUES OR CELLS
I Fat cells, Adrenal, Liver
II Heart, Bone, Thyroid, Cartilage, Liver
III Mammary
IV Islet （insulin receptor sites）
Inhibitors: Metal chelating agents such as cysteine, EDTA or o-phenanthroline but not
DFP. It is also inhibited by a2-macroglobulin, a large plasma glycoprotein.
Instructions for use
A. Preparing stock and working solution
Dissolve the non-sterile, lyophilized enzyme in HBSS （Cat. No. 14025）. Filter sterilize the
solution with a cell culture approved filtration unit. Crude collagenase is most often used
in concentrations from 0.1 to 0.5% （W/V） or 50 to 100 U/mL. Once reconstituted use
immediay or store frozen. Thaw in refrigerator immediay prior to use.
B. Dissociation of tissue
• Tissue is minced with a sterile scalpel or scissors.
• Wash the tissue several times in HBSS.
• The tissue fragments are soaked at +37°C. Increased efficiency is obtained using a
rocker platform and supplementing the digest with 3 mM CaCl2.
C. Organ perfusion
• Digest is prewarmed to +37°C and perfused at a rate preoptimized for the particular
organ. Addition of 3 mM CaCl2 increases the efficiency of dissociation.
• Dispersed cells and tissue fragments are separated from larger pieces by passing the
mixture through a sterile stainless steel or nylon mesh. Fresh collagenase solution
can be added to the fragments if further disaggregation is required.
• Wash several times to eliminate debris and enzyme solution. A density separation
step （Nycodenz） will give a cleaner suspension.
• Resuspend the pellet in the culture medium and incubate under predetermined
conditions.
For further information on this or other GIBCOTM products, contact Technical Services at the
following:
United States TECH-LINE SM : 1 800 955 6288
Canada TECH-LINE: 1 800 757 8257
Outside the U.S. and Canada, refer to the GIBCO products catalogue for the TECH-LINE in your
region.
You may also contact your Invitrogen Sales Representative or our World Wide Web site at
www.invitrogen.com.
For research use only.
CAUTION: Not intended for human or animal
diagnostic or therapeutic uses.

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