Collagen Gelation Diagnosis

Reasons Your Collagen Might Not be Gelling

1. The pH is not neutral
   • Double check to make sure the pH is 7.0-7.4. 
   • Even if using a neutralization solution, the pH may be off due to mixing issues or collagen "hold-up" in pipette tips, slightly altering the collagen:neutralization solution ratio.
2. Incorrect levels of salts
   • Ensure the salts of the neutralization collagen solution are isotonic (~300 mOsmo). We accomplish this by adding 1 part of 10X PBS or Media in most collagen hydrogel preparations.
3.  Temperature
   • Collagen gels best at 37C. Ensure the temperature does not exceed 38.5C, as the collagen will begin to denature.
4. Collagen Concentration
   • Low collagen concentration solutions will have a hard time forming a gel. We recommend keeping the collagen concentration >1.5 mg/ml for best results.
5. Mechanical agitation
   • Vortexing the collagen solution can have negative impacts on the collagen and will negatively affect the ability of the solution to form a hydrogel. Do not vortex.
   • Do not agitate the collagen solution during incubation and gelation. This will interfere with the fibrillogenesis process. 
6. Material was frozen or left at room temperature
   • If the collagen solution was frozen or left at room temperature for a long period of time, it may have begun to denature, which would affect its ability to gel.
7. Very small volumes of collagen were used in a non-confined space
   • We have found that using microliters of collagen on top of glass coverslips tend to not gel very well. 
   • We recommend ensuring the collagen is within a confined space (ie. wells or walls)
8. Neutralization Solution came out of solution
   • If using a collagen product that comes with a neutralization solution, ensure that the neutralization solution is still fully in solution. Getting it too cold can cause it to come out of solution. If this happens, simply warm it back up.
9. Weights vs Volume
   • Due to pipette hold-up volume, dispensing 1 mL of collagen (3 mg/ml) using a pipettor actually only dispenses about .89 grams. For the most accurate results, we recommend usings weights for viscous materials, such as collagen. The neutralization solutions, buffers and medias can use volume, as their viscosity is close to water. 
10. Expired material
    • Collagen tends to be stable when stored properly, but after a few years, the "gel strength" slowly declines. Expired collagen may still form hydrogels, but it will be significantly softer than a fresh batch of collagen.
11. Heat transfer
    • Warming collagen to 37C for polymerization in a test tube, in a water batch will be much more efficient than gelling collagen in an enclosed 6-well plate, with a lid, in a dry incubator. Look for ways to improve heat transfer if quicker polymerization is desired.
12.  Mixing
    • Ensure proper mixing to create a homogeneous solution (without vortexing) for best results.
13. Lyophilized Collagen
    • Atelocollagen that has been lyophilized (such as lyophilized PureCol, catalog #5006), tends to form weaker hydrogels, or not polymerize at all.
14. Insoluble Collagen
    • Advanced BioMatrix offers two insoluble collagen products, #5162 and #5164. These collagen powders are extremely robust and will not fully go into solution.