Collagen Attachment Protocols, Solubility, and Stability
Collagen for Cell Culture
Collagen is the most abundant mammalian protein and is the main component of connective tissues and the extracellular matrix (ECM). In two- and three-dimensional cell culture, researchers often use collagen protein to coat culture dishes.
Typically, collagen is first synthesized as individual glycine-rich pre-procollagen monomeric strands from the ribosomes in the rough endoplasmic reticulum (RER). After transport to the lumen of the RER signal peptides are cleaved to yield procollagen. Lysine and proline are hydroxylated and glycosylated in the lumen and a helix composed of three peptides is formed. It is then transported to the Golgi and excreted from the cell by exocytosis. Procollagen's propetides are then removed to yield tropocollagen which may join to form fibrils. The "collagen type" designations of Bornstein and Traub is determined by the peptide composition of the triple helix.
Collagen is found as several protein types, the most common of which is Type I. Collagen Type I is in found in bone, tendon, ligament, blood vessels, intervertebral disks, and Type III, the second most abundant collagen type, often co-localizes with Type I in skin and blood vessels. Collagen Type II is a major protein component of cartilage, Type IV is a primary protein component of the basal lamina, and Type V is associated with collagen fibril organization and epithelial cell cycle inhibition.
Important points to remember before start
- Ensure the cells are healthy and in adequate numbers.
- Collagen solutions may be sterilized by dialysis in a 0.25% acetic acid and 0.5% chloroform solution.
- We do not recommend sterilizing the collagen solution by membrane filtration. We have found substantial protein loss by this method.
- Coating of tissue culture plastic dishes may be performed by air-drying. Dried coated dishes can be sterilized by exposure to UV light in a sterile culture hood or by rinsing with 70% ethanol.
- Surface coverage concentration may differ with the cells being cultured. Optimal conditions for attachment must be determined for each cell line and application.
- Do not over-dry culture plates/coverslips post coating as this affects the attachment of cells.
- All the steps in the protocols need to be performed in sterile conditions.
Collagen Type I coating protocol for culture ware
- Add collagen to acetic acid to obtain 0.1% (w/v) collagen solution. Stir at room temperature for 1-3 hours until dissolved. Collagen solution should be diluted 10-fold to obtain a working concentration of 0.01%.
- 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. Let stand overnight in the cold. Aseptically remove the top layer containing your collagen solution.
- Coat dishes and allow the protein to bind for several hours at room temperature, 37 °C, or overnight at 2-8 °C.
- 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.
- Rinse with sterile tissue culture grade water or a balanced salt solution before introducing cells and medium.
Figure 1. Collagen Type I protein structure.
Collagen Type II and IV coating protocol for culture ware
- Collagen Types II and IV may be reconstituted to a concentration of 0.5-2.0 mg/mL in acetic acid. Dissolve for several hours at 2-8 °C, occasionally swirling.
- Coat the tissue culture plastic dishes by air-drying. Alternatively, preincubate the coated culture ware overnight at 2-8 °C (or several hours at 37 °C).
- Dried coated dishes can be sterilized by exposure to UV light in a sterile culture hood or by rinsing with 70% ethanol.
Figure 2.A. Collagen Type II protein structure, predominantly three strands. B. Collagen Type IV protein structure, predominantly two strands. C. Collagen Type IV protein structure, predominantly one strand.
Collagen Type IV
Other Collagens: Type II, Type III, Type I & III, Type I & IV, and Type V
Why am I seeing some insoluble particles in my collagen solution?
The solubility specification for some collagen products, like C7661, 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.
The product gelled while refrigerated. Can it still be used?
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 culture ware.
Can all collagen products be used to make 3-D collagen gels?
No, not all collagen products can form gels or be used for 3D applications. Suitability depends on the collagen type, concentration, and specific formulation. Check product specifications in the tables above to ensure it meets the requirements for gel formation and 3D applications.
How long can collagen-coated plates be stored?
We make no recommendations about the storage of plates after coating. Because several factors, such as humidity, may impact on the longevity of coated plates, customers should make their own determination as to whether plates can be stored and under which conditions. The product should not be exposed to temperatures above 50 °C.
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