Klapperich Lab:Collagen Solution

Overview

Protocol for making 0.05% w/v collagen dispersion in acetic acid.

Materials

Reagents

• 5 mL of 0.05M acetic acid
• 2.5 mg of collagen (from human placenta)

Equipment

• Homogenizer
• 50 ml beaker
• Ice bath
• Ultrasonicator

Procedure

Making 0.05 M solution of acetic acid

Note: This solution has a shelf life of one week.

1. Fill sterilized bottle with 5 mL of Millipore water.
2. Add 14.3 µL acetic acid.

0.5% w/v Dispersion of Collagen in Acetic Acid

Note: Keep collagen mixture at below 20°C.

1. Add 2.5 mg of collagen to a 10 ml sterilized beaker. (0.05 % w/v)
2. Add 5 ml of 0.05 M acetic acid to the beaker.
3. Mix solution with homogenizer at full speed for approximately 5 minutes, or until no large fibrils or chunks are apparent. Make sure that tip has been washed thoroughly (ethanol, rinse with Millipore water)
4. Place beaker in ice bath.
5. Set the sonicater controller to Amplitude 100% (dial), 20 min, 5 sec on, 10 sec off.
6. Place sonicater horn in collar, making sure tip of sonicater does not touch the bottom of the beaker. Press START/STOP on sonicater controller.

Notes

• The mixture may be stored in a capped bottle at 4°C for about 4 months.
• Re-blend at 4°C at an amplitude of 60 with the pulser set to 5 sec for 15 minutes to use.

References

Relevant papers and books

1. Pieper JS, Oosterhof A, Dijkstra PJ, Veerkamp JH, and van Kuppevelt TH. Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate. Biomaterials. 1999 May;20(9):847-58. DOI:10.1016/s0142-9612(98)00240-3 |

Contact

• Jessica
• Eben