rattailtendon collagen type I was prepared by Birkedal-Hansen method through acetic acid extraction, sodium chloride precipitation, disodium hydrogen phosphate precipitation and other steps. Our company's rat tail collagen can be used in coated cell culture vessels to culture some cells that are not easy to stick to the wall in ordinary cell culture vessels. It can also be used to prepare three-dimensional glue to simulate the real growth environment, so that cells grow in a three-dimensional environment.

1, the adherence and growth of PC-12 cells were examined in cell culture dishes coated with Solebo rat collagen I.

2, three-dimensional glue with a certain strength can be formed when the concentration is above 1mg/ml and the pH is about 7. It was found that NIH-3T3 cells grew normally in the three-dimensional glue and PC-12 cells grew normally on the surface of the three-dimensional glue.

Usage:

1, the recommended concentration of the surface package of cell culture utensils: 1-5ug/cm2

Taking the coating concentration of 2 ug/cm2 as an example, collagen was diluted to 0.012mg/ml with sterile 0.006mol/L(0.36g/L) acetic acid.

Ensure that the collagen solution is spread all over the surface of the utensil, and leave the lid on a super clean table overnight to dry. It can also be placed at room temperature for 1 hour, washed with PBS 3-4 times and then used directly. The well-wrapped utensils can be stored for at least 3 months at 4-25℃.

2. Preparation of three-dimensional collagen

Rat tail collagen type I can form a three-dimensional gel with a certain strength when the concentration is above 1mg/mL and the pH is about 7. It is recommended to form a gel concentration of 1-2mg/mL. Collagen is dissolved in 0.006mol/L acetic acid, and 0.06× volume of 0.1mol/L NaOH is added to neutralize it during the gelation process.

Required solution (all sterile and pre-cooled) : 10×PBS (can contain 10 mg/L phenol red for pH indication) or 10× culture solution, 0.1mol/L NaOH, 0.1mol/L acetic acid (usually not used), double steaming water

A. Preparation of cell-free 3D collagen (as an example of preparing 1mL, 1mg/mL 3D glue) : 200μL rat tail collagen Type I (5mg/mL) was added to a centrifuge tube placed in an ice bath and 690μL H2O was added. Then add 12μL 0.1mol/L NaOH to the collagen solution (if you add 12μL 0.1mol/L NaOH to the collagen solution in reverse, local collagen condensation will occur because NaOH cannot be mixed quickly), mix immediately. Then add 100μL 10×PBS or 10× culture medium, mix and immediately add to the culture vessel (the pH after mixing is about 7, if the PBS or culture medium is not added with phenol red, the pH test paper should be used for the first time). Place the culture vessel at room temperature (about 25 degrees) for 20 minutes until the glue sets, and transfer it to the incubator. If 10×PBS is used in the formulation, an appropriate volume of cell culture medium should be added for pre-balancing before use.

B. Preparation of three-dimensional collagen containing cells (for example, preparation of 1 mL, 1mg/mL three-dimensional glue) : Prepare the cells suspended in a culture solution and place them in an ice bath. Add 200μL rat tail collagen type I (5mg/mL) to 12μL 0.1mol/L NaOH (if 12μL 0.1mol/L NaOH is added to the collagen solution in reverse, local collagen condensation will occur because NaOH cannot be mixed quickly), mix immediately. Then add 23μL 10×PBS or 10× culture medium and mix well (the pH after mixing is about 7, if no phenol red is added to PBS or culture medium, it is necessary to use pH test paper for the first time). Add 760μL cell suspension, mix well and immediately add to culture vessel. Place the culture vessel at room temperature for 20 minutes until the glue solidifies, add the appropriate volume of cell culture solution, and transfer to the incubator for culture.

Note:

Rat tail collagen type I can quickly form a gel when pH is neutral at room temperature, and should be kept as low as possible during operation.

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