FAQ: Collagen

How do I prepare a 3D gel using the ibidi Collagen Type I, Rat Tail solution?

The Collagen Type I forms a firm and clear gel when brought to neutral pH. This gelation process works at room temperature or 37 °C. We provide detailed 3D collagen gel protocols in the Instructions of our Collagen Type I Rat Tail and in our Application Note "Fabrication of Collagen I gels" (AN 26) (PDF).

My collagen 3D gel looks very inhomogeneous. What is the reason for that?

Generally, an inhomogeneous appearance of the collagen gel is caused by starting polymerization too early. This can happen either due to insufficient cooling or incomplete mixing of the collagen solution. We recommend cooling the reagents you are using and keeping them on ice while preparing the 3D gel solution. We further recommend mixing the viscous collagen solution thoroughly before gel preparation.

My cells need pH 6.0 in the 3D collagen matrix. Can the gel be adjusted to a pH lower (or higher) than the 7.2–7.4 that is recommended in the instructions?

Most cell types grow best at neutral pH, which is around 7.2–7.4. However, if the experimental conditions require an environment with slightly basic or acidic pH, this can be achieved by increasing or reducing the amount of NaOH in the gel. For details, please follow the General 3D Gel Protocol (PDF), which is included in the Collagen I, rat tail Instructions.

Does the temperature play a role in the initiation of collagen polymerization?

No. Unlike other ECM materials such as Matrigel, the collagen polymerization is initialized when the pH is raised to neutral. A temperature change to room temperature or even higher cannot initiate the polymerization process. After pH neutralization, the temperature plays a significant role, since the polymerization time highly depends on the temperature. The colder the mix, the slower the polymerization.

Why does the 10 mg/ml solution of the ibidi Collagen Type I, Rat Tail have such a high viscosity?

Collagen is a fibrous molecule that naturally exhibits a very high viscosity, especially at higher concentrations. Therefore, we recommend handling the collagen solution with special pipetting equipment that is suitable for highly viscous solutions, such as Eppendorf Visco Tips or Gilson Microman E.

Why is the viscosity of the 10 mg/ml solution of the ibidi Collagen Type I, Rat Tail different to other collagens on the market?

The concentration, the pH, the animal origin, and the level of natural crosslinking influence the viscosity of the collagen solution. ibidi's mild manufacturing process preserves a very high level of nativity leading to a higher viscosity at the same protein concentration. For handling the viscous solution, we recommend pipettes for highly viscous solutions, such as Eppendorf Visco Tips or Gilson Microman E. For lowering the viscosity and concentration, we recommend a dilution step in 17.5 mM acetic acid (∼ 0.1%).

Can the viscosity of the ibidi Collagen Type I, Rat Tail solution be different from lot to lot?

Yes. The concentration and the pH highly influence the viscosity of the collagen solution. Both values can vary slightly and are given in the CoA of each lot.

Can I pipet the collagen gel after polymerization?

No. Defined pipetting is not possible after polymerization. Any mechanical force will destroy the structure and the mechanical properties of the collagen gel.

Why does my gel become turbid after polymerization?

The macroscopic appearance of non-polymerized collagen in solution is clear to slightly opaque, depending on the concentration. As soon as the small collagen fibrils polymerize to larger fibers, a gel with a macroscopically turbid appearance is created. The forming collagen fibers are the size of a wavelength of visible light, which makes the gel become visible due to light scattering. In the phase contrast microscope, the polymerized fibers are also visible, while the fibrils in solution are not.