Collagen Attachment Protocols, Solubility, and Stability

Important points to remember before start

1. Ensure the cells are healthy and in adequate numbers.
2. Collagen solutions may be sterilized by dialysis in a 0.25% acetic acid and 0.5% chloroform solution.
3. We do not recommend sterilizing the collagen solution by membrane filtration. We have found substantial protein loss by this method.
4. 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.
5. Surface coverage concentration may differ with the cells being cultured. Optimal conditions for attachment must be determined for each cell line and application. Recommended concentrations are listed in Table 1.
6. Do not over-dry culture plates/coverslips post coating as this affects the attachment of cells.
7. All the steps in the protocols need to be performed in sterile conditions.

Collagen Type I coating protocol for culture ware

1. Add collagen to acetic acid (refer to Table 1) 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%.
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. Let stand overnight in the cold. Aseptically remove the top layer containing your collagen solution.
3. Coat dishes with recommended concentrations in Table 1. 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.
5. 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.
6. Rinse with sterile tissue culture grade water or a balanced salt solution before introducing cells and medium.

Collagen Type II and IV coating protocol for culture ware

1. Collagen Types II and IV may be reconstituted to a concentration of 0.5-2.0 mg/mL in acetic acid (refer to Table 1). Dissolve for several hours at 2-8 °C, occasionally swirling.
2. 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).
3. Dried coated dishes can be sterilized by exposure to UV light in a sterile culture hood or by rinsing with 70% ethanol.

Alternatively, we offer ready-to-use, collagen-coated 96-well strips (Table 1).

Table 1. Collagens for Cell Attachment

Product No.	Description	Coating Concentration	Dilute in
11179179001	Collagen from rat tail tendon	5 μg/cm2	Sterile 0.2% acetic acid
08-110	ECL Cell Attachment Matrix	5 μg/cm2	Sterile basal medium (without serum)
C0543	Collagen from Engelbreth-Holm-Swarm murine sarcoma basement membrane	6‑10 μg/cm2	0.25% Acetic acid
C2124	Collagen solution from bovine skin	6‑10 μg/cm2	Sterile PBS
C2249	Collagen Solution from human fibroblasts	6‑10 μg/cm2	Sterile PBS
C3867	Collagen, Type I solution from rat tail	6‑10 μg/cm2	Sterile water
C4243	Collagen solution from bovine skin	6‑10 μg/cm2	Sterile PBS
C5533	Collagen from human placenta	6‑10 μg/cm2	0.25% Acetic acid
C6745	Collagen Type IV from human cell culture	6‑10 μg/cm2	Sterile HBSS
C7661	Collagen from rat tail	6‑10 μg/cm2	0.1 M acetic acid
C8919	Collagen from calf skin	6‑10 μg/cm2	Sterile water
C9301	Collagen from chicken sternal cartilage	6‑10 μg/cm2	0.25% Acetic acid
C9791	Collagen from calf skin	6‑10 μg/cm2	0.1 M acetic acid
234184-M	Collagen, Type II, Bovine Joint Cartilage	0.2 - 2 µg/cm2	500 mM acetic acid
08-115	Collagen Type I, rat tail	10 µg/cm2	Sterile PBS
122-20	Rat Tail Collagen Coating Solution (20 ML)		Ready-to-use
ECM104	Millicoat Human Collagen Type I Coated Strips (96-Wells)		Ready-to-use
ECM105	Millicoat Human Collagen Type IV Coated Strips (96-Wells)		Ready-to-use

Collagen Gels

Products that are compatible for the preparation of collagen gels are in listed Table 2.

Table 2. Collagens for Gel Preparation

Product No.	Description
11179179001	Collagen from rat tail tendon
234138	Collagen, Type I, Human Skin
234154	Collagen, Type IV, Human Placenta
CC050	Human Collagen Type I
CC052	Human Collagen Type II
CC054	Human Collagen Type III
CC076	Human Collagen Type IV
CC077	Human Collagen Type V
CC078	Collagen Type III, bovine purified protein
CC083	Bovine Collagen Type IV
CC086	Bovine Collagen Type VI
5006	PureCol™
5074	PureCol™ EZ Gel solution
5168	Bovine Collagen
234149	Collagen, Type I, Human Placenta

Frequently Asked Questions (FAQs)

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

Due to cross-linkage and sequence variability, as well as structural changes that can occur during isolation and purification, Collagen Type I (C8919) 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. Products listed as sterile is tested for sterility prior to release for sale to our customers. No further filtration of such products is necessary. A milky or particulate appearance is not indicative of microbial contamination and does not affect product use.

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.

What is the best solvent to dilute collagen?

The solvents that are compatible to dilute collagens are provided in Table 1.

What is the optimum concentration of collagen for coating?

What is the optimum concentration of collagen for coating?

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?

The collagen products that can be used to make gels are listed in Table 2.

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.