Collagen Attachment Protocols, Solubility, and Stability

By: George Sitterley, BioFiles 2008, 3.8, 5.

Collagen Type I Protocol

Collagen Type I (Cat. Nos. C1809, C7661, C9791, C8919*)

Optimal conditions for attachment must be determined for each cell line and application.

  1. Add collagen to 0.1 M acetic acid to obtain 0.1% (w/v) collagen solution. Allow to stir at room temperature 1-3 hours until dissolved. Collagen solution (Cat. No. C8919) should be diluted 10-fold to obtain a working concentration of 0.01%.
  2. We recommend transferring the collagen solution to a glass bottle with a screw cap and carefully layering chloroform at the bottom. The amount of chloroform to use should be approximately 10% of the volume of collagen solution. DO NOT SHAKE OR STIR. Allow to sit overnight in the cold. Aseptically remove the top layer containing your collagen solution. We do not recommend sterilizing the collagen solution by membrane filtration. We have found substantial protein loss by this method.
  3. Coat dishes with 6-10 ug/cm2. Allow the protein to bind for several hours at room temperature, 37°C, or overnight at 2-8°C.
  4. Remove excess fluid from the coated surface, and allow it to dry overnight. If the collagen solution is not sterile, the dried, coated surface can be sterilized by overnight exposure to UV light in a sterile tissue culture hood.
  5. Rinse with sterile tissue culture grade water or a balanced salt solution before introducing cells and medium.

*Note: Steps 1 and 2 are not necessary for the Collagen Solution, (Cat. No. C8919).

Collagen Type II and IV Protocol

Collagen, Type II and IV (Cat. Nos. C9301, C0543, C5533 )

Optimal conditions for attachment must be determined for each cell line and application.

  1. Collagen Types II and IV may be reconstituted to concentration of 0.5-2.0 mg/mL in 0.25% acetic acid. Dissolve for several hours at 2-8°C, occasionally swirling.
  2. Coating of tissue culture plastic dishes may be performed by air-drying the above protein solution, or by preincubating the same solution overnight at 2-8°C (or several hours at 37°C) without air-drying.
  3. Dried coated dishes can be sterilized by exposure to UV light in a sterile culture hood or by rinsing with 70% ethanol. Alternatively, the collagen solution may be sterilized by dialysis in a 0.25% acetic acid and 0.5% chloroform solution.

Ask a Scientist!

Cat. No. C7661 (Collagen from rat tail)

Q1) How can I sterilize collagen solutions?

A1) We recommend transferring the collagen solution to a glass bottle with a screw cap and carefully layering chloroform at the bottom. The amount of chloroform to use should be ~10% of the volume of collagen solution. DO NOT SHAKE OR STIR. Allow to stand overnight at 2-8°C. The chlorine gas will move up through the collagen solution and sterilize the solution. Aseptically remove the top layer containing the collagen solution to a new bottle. We do not recommend sterilizing the collagen solution by membrane filtration. We have found substantial protein loss by this method.

Q2) Why am I seeing some insolubles in my collagen solution?

A2) The solubility specifications for this product is clear to hazy colorless solution with a few insolubles at 1 mg/mL in water with 2 μL acetic acid (or 0.1 N acetic acid). The insolubles can be removed by settling or centrifugation.

Cat. No. C8919 (Collagen from calf skin solution)

Q1) Why does the vial of collagen solution look milky or contain particulates?

A1) Cat. No. C8919, Collagen Solution from calf skin, is prepared by allowing irradiated sterile collagen strands to mix with sterile filtered 0.1N acetic acid over several hours. The collagen dissolves to an opalescent solution that is aseptically dispensed while stirring. No further filtration is necessary. Due to cross-linkage and sequence variability, as well as structural changes that can occur during isolation and purification, Collagen Type I (Bornstein and Traub) is a heterogeneous mixture rather than a pure compound. This variability, along with slight differences in the raw material, lends itself to shifts in the opacity of the solution from lot to lot. This product is tested for sterility prior to release for sale to our customers. A milky or particulate appearance is not indicative of microbial contamination and does not affect product use.

Q2) The product gelled while refrigerated. Can it still be used?

A2) This can occur but does not harm the product. When warmed to room temperature, the product will liquify. Please make sure it is well mixed prior to dilution for coating cultureware.

Q3) Can this be used to make 3-D collagen gels?

A3) This product is NOT intended to produce 3-D (thick) gels since this solution will not gel (presumably due to gamma-irradiation of the powder during the production process). We recommend using (Cat. No. C7661), Collagen from rat tail or (Cat. No. C4243), Ultra Pure Collagen from Bovine.

Q4) How long can collagen coated plates be stored?

A4) We make no recommendations about storage of plates after coating. Since several factors such as humidity may impact on longevity of coated plates, customers should make their own determination as to whether plates can be stored and under which conditions. We offer Sigma Screen collagen coated plates (Cat. No. S3315 ) that indicates the following: For optimal performance, the unopened product should be stored in a dry place at 2-8°C. The product may be stored at room temperature for up to three months. The product should not be exposed to temperatures above 50°C.

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