Cell-Seeded Collagen Gel Protocol

By Youssef Farhat, Written 4/17/11, Last updated 5/6/14

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Collagen gels are very commonly used for tissue engineering and cancer research, because collagen is an abundant and important part of the extracellular matrix of humans and other living things.  Given that cells behave differently depending on their environment, it is thought that collagen gel experiments provide more meaningful results than 2D cell culture experiments, because collagen is more similar to the cells’ native environment than culture dishes which are flat, rigid and made of plastic.

Important Notes and Hints

  • The collagen used in this protocol comes in an acidic solution and needs to be neutralized with NaOH and made isotonic with 10X PBS before mixing with cells.  If the pH of the collagen solution is not close to 7.4 or the ion balance incorrect, the cells may die, ruining the experiment.  For this reason, a sample from every collagen solution much be tested to ensure the pH of the solution is about 7.4.  After neutralizing the collagen, it must be kept on ice until the cells have been added in order to prevent it from gelling.
  • As in all cell culture methods, work under a culture hood and keep all materials sterile.  See Tips for Sterile Technique.
  • Before beginning, the amount of collagen needed for the experiment must be determined.  For generalization, “X” is used below to represent this volume of collagen.
    • Out of precaution, factor in about 30% excess materials when determining the desired amount of collagen, because collagen is very viscous and a considerable amount gets lost during pipetting.
  • Consider keeping the 10X PBS and 0.1 M NaOH chilled in the refrigerator (4°C) so that they do not warm the collagen solution upon adding and cause it to gel.


  • Collagen I (PureCol®, Advanced BioMatrix, Cat. # 5005-B)
  • 10x PBS (Invitrogen, Cat. # 70013)
  • 0.1 M NaOH (mix 399 mg NaOH into 100 mL DI H2O and sterile-filter)
  • pH strips (EMD, Cat. # 9588)
  • 50 mL centrifuge tubes
  • 1.5 mL Eppendorf tubes
  • Pipettors, serological pipettes, and pipette tips
  • Essentials for counting cells, such as a microscope, Trypan blue (Gibco, #15250), hemocytometer, and cover slips
  • Oven set to 37°C or cell culture incubator with the water bath removed (atmospheric humidity)
  • Container with ice


Step 1: Neutralize the collagen solution (Estimated time: 20 min)

1)     Recommended: download our collagen gel calculator to simplify calculations.

2)     Keep the collagen on ice. Place all reagents under the hood.

3)     Add X mL of collagen to a 50 mL centrifuge tube.

4)     To every X mL of collagen, add X/8 mL of 10x PBS and mix with gentle pipetting.

  • This step makes the collagen solution isotonic with the cells.

5)     Add X/8 mL of 0.1 M NaOH and mix with gentle pipetting.

6)     Take 1 mL of collagen into a 1.5 mL Eppendorf tube and test the pH with a pH strip.  As mentioned in the introduction, the success of this experiment depends on the collagen solution having a pH around 7.4.

  • If the pH is too high/basic (e.g. >7.6), add 0.1 mL of 10X PBS, add 0.9 mL of collagen I, and repeat step 5.
  • If the pH is too low/acidic (e.g. <7.2), add 0.1 mL of 10X PBS, add, 0.2 mL of 0.1 M NaOH, add 0.7 mL of collagen I, and repeat step 5

7)     Once the pH of the collagen solution is about 7.4, keep it on ice until the cells are ready to mix into it.

Step 2: Suspend cells and gel the collagen solution (Estimated time: 2.5 hour)

1)     Trypsinize and count the cells.

2)     Place the number of cells required for the experiment into an empty 50 mL centrifuge tube.

3)     Spin them down to a pellet and remove the supernatant.

4)     Resuspend the cells in X/19 mL of media

  • I use a ratio of 1 part media to 19 parts collagen.  This is arbitrary and can be modified as needed.

5)     Add X mL of collagen to the cell suspension and mix with gentle pipetting.

6)     Aliquot the appropriate amount of cell-seeded collagen to the desired container (i.e. a 24 well plate) and allow the solution to gel at 37°C, atmospheric humidity.

  • In our experience, gelling in atmospheric humidity (for example, in an oven) is faster than gelling in the humidified cell culture incubator.  The water bath from the incubator can be removed and the door left open for a minute to achieve atmospheric humidity, but be sure not to harm any other cells or experiments if the incubator is being used by others!  Return the water bath when finished.
  • Gelation may take anywhere from 30 minutes to 2 hours depending on the size of the gels (smaller volumes of collagen take less time to gel).

7)     Remove the gels from the oven or incubator and use a sterile pipette tip or spatula to score the edges of the gels, if desired.  This will release them from the sides to which they are adhered and allow the gels to float freely in the media.

8)     Add culture media to the gels and place them in a humidified cell culture incubator.

Citing this Protocol

Farhat, Y. “Protocol for Cell-Seeded Collagen Gels.” The Protocol Place. April 2011. Accessed on mm/dd/yyyy. <http://protocol-place.com>

One Response to Cell-Seeded Collagen Gel Protocol

  1. Thanks… This was a great protocol. I was able to replicate the same with some lyophlized collagen that I resuspended myself. It was a lot less expensive. I liked ecmscience but you can get some at sigma too.

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