Collagen, Type I , from rat tail

The surface coating of cell culture vessels: Recommended concentration: 1-5 μg/cm²

Take a coating concentration of 2 ug/cm2 as an example:

Dilute the collagen to 0.012mg/ml with sterile 0.006mol/L (0.36g/L) acetic acid. Add the volumes as indicated in Table (I) to the corresponding culture vessels:

Make sure the collagen solution covers the entire surface of the container, then open the lid and let it dry overnight on the ultra-clean bench. Alternatively, it can be left at room temperature for 1 hour, washed with PBS 3-4 times, and then used directly.

The sealed containers can be preserved for at least 3 months or more at a temperature ranging from 4 to 25 degrees Celsius.

2. Preparation of three-dimensional collagen

Type I mouse tail collagen can form a three-dimensional gel with a certain strength when the concentration is above 1mg/ml and the pH is around 7. It is recommended that the gel-forming concentration be 1-2mg/ml. Hygromycin collagen dissolves in 0.006mol/L acetic acid. During the gel formation process, 0.06X volume of 0.1mol/L NaOH needs to be added to neutralize.

Required solutions (all need to be sterile and pre-cooled): 10x PBS (can contain 10 mg/L phenol red for pH indication) or 10x culture medium, 0.1 mol/L NaOH, 0.1 mol/L acetic acid (usually not used), double-distilled water

 A. Preparation of three-dimensional collagen without cells (for preparing 1 ml, 1 mg/ml three-dimensional collagen): Add 200 ul of type I rat tail collagen (5 mg/ml) to a centrifuge tube placed in an ice bath, then add 690 ul of H₂O. Next, add 12 ul of 0.1 mol/L NaOH (if 12 ul of 0.1 mol/L NaOH is added to the collagen solution instead, local collagen coagulation will occur due to the inability of NaOH to be thoroughly mixed quickly), and mix immediately. Then add 100 ul of 10xPBS or 10x culture medium, mix well, and immediately add to the culture dish (after mixing, the pH is about 7, if phenol red is not added to the PBS or culture medium, pH test with pH paper should be conducted before initial use). Place the culture dish at room temperature (around 25 degrees) for 20 minutes until the collagen solidifies, then transfer it to the incubator. If 10xPBS is used in the preparation, an appropriate volume of cell culture medium should be added for pre-equilibration before use.

B. Preparation of three-dimensional collagen containing cells (for preparing 1 ml, 1 mg/ml three-dimensional collagen): Prepare the cells suspended in the culture medium and place them in an ice bath. Add 200 ul of type I rat tail collagen (5 mg/ml) to 12 ul of 0.1 mol/L NaOH (if instead, 12 ul of 0.1 mol/L NaOH is added to the collagen solution, local collagen coagulation will occur due to the inability of NaOH to be thoroughly mixed quickly), and mix immediately. Then add 23 ul of 10x PBS or 10x culture medium, and mix (after mixing, the pH is around 7, if no phenol red is added to the PBS or culture medium, it is necessary to test the pH with pH paper when using for the first time). Add 760 ul of cell suspension, mix, and immediately add to the culture dish. Place the culture dish at room temperature for 20 minutes until it gels, then add an appropriate volume of cell culture medium and transfer to the incubator for cultivation.

Note: Type I collagen from mouse tail can rapidly gel at room temperature in a neutral pH environment. During the operation, it is necessary to maintain a low temperature as much as possible.