Collagenase Tissue Digestion/Dissocation Trouble Shooting and References

BioFiles 2006, 1.2, 6.

Tissue Digestion—Dissociation by Collagenase


Problem Cause Soultion Ref.
Digestion is Poor
 
 
 
Inactive enzymes
 
Inadequate Ca++
Insufficient enzymes
Store collagenase cold and dry.
Store collagenase solution in frozen aliquots
Include 5 mM Ca++ in collagenase solution
Use more collagenase. Try Sigma Blends with more protease activity.
1 
 
2 
 
Released Cells are Trapped
 
 
 
 
DNA released from broken cells
 
Non-protein gums
 
 
Reduce agitation
Add DNase**
Flush tissue well before digesting it***
Use no Mg++ in digestion solution
Add hyaluronidase with enzymes**
2 
3 
2 
2 
4
Cells are Killed
 
 
 
 
 
Excess protease
 
pH changes
 
Too little oxygen
 
Reduce exposure to proteases*
Add albumin or heated serum
Use buffers (e.g., HEPES) instead of HCO-3.
Check and re-adjust pH often
Aerate digestion solution (air or O2)
Digest faster by using more enzymes
 
 
2 
 
5 
 
Cell Yield is Low
 
 
Enzyme balance
 
Adhesion factors
Use more collagenase*
Include elastase with collagenase**
Perfuse tissue fi rst to remove Ca++***
 
6 
2,8
Many Cells are Damaged
 
 
Excessive protease
 
Physical damage
Use less protease*
Add albumin or heated serum
Handle tissue and cells very gently
7 
 
2,8
New Lot Doesn’t Work
 
Lot variation
 
Use fractionated Sigma blend collagenases
(Sigma Catalog Nos. C7926, C8051 or C8176)*
 
 

* The separately prepared collagenase and protease enzymes in the “Sigma Blend” products (Cat. No. C7926, C8051 or C8176) give reproducible control of how much of each is used.

** DNase will be inactivated by the shear of excessive stirring, and added enzymes may be digested by the neutral protease present in the collagenase.

*** Use EGTA (or EDTA) to remove Ca2+ and flush away microorganisms, then wash tissue with buffer to remove the chelating agent. Do not add EGTA or EDTA to the enzyme solutions.

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Factors that Affect Tissue Digestion-Dissociation by Collagenase

Based on our own R and D and from discussions with customers it is clear that the way a particular tissue is dissected and prepared has a significant effect on the speed and efficiency of any tissue digestion-dissociation with collagenase. Differences in the ages of the tissue donors can also be a major source of variation over time. Make sure that calcium ions are present in the digestion buffers at 5 mM. Chelating agents EGTA and EDTA can severely inhibit collagenase activity by removing calcium ions required for enzyme stability and activity. β-mercaptoethanol,9 cysteine9 and 8-hydroxyquinoline-5-sulfonate9 are other inhibiting substances. A new lot of collagenase with higher specific activity could cause excessive cell death at an established concentration. In that case use less collagenase and/or add BSA or serum (up to 0.5% and 5–10% respectively) to stabilize the cells during digestion.

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Materials

     

References

  1. Fain, J.N., Meth. Enzymol., 35, 555 (1975)
  2. Seglen, P.O., Methods in Cell Biology, 13, 29 (1976)
  3. Buitrago, A., et al., Biochem. Biophys. Res. Commun., 79, 823 (1977)
  4. Berry, M.N., and Friend, D.S., J. Cell Biol., 43, 506 (1969)
  5. Bellemann, P., et al., Anal. Biochem., 81, 408 (1977)
  6. Ives, H.E., et al., J. Expt. Med., 148, 1400 (1978)
  7. Fain, J.N. and Loken, S.C., J. Biol. Chem., 244, 3500 (1969)
  8. Berry, M.N., et al., Isolated Hepatocytes; Preparation, Properties and Applications. Elsevier. 1991
  9. Seifter, S., et al., J. Biol. Chem., 234, 285 (1959)

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