Cell culturing

Hela cells

Passaging

• Remove and discard culture medium using the vacusafe and pasteur pipette. Rinse the cells with PBS (around 10mL) and remove it. This is because the Trypsin is inactivated by FBS in the media.
• Add 2.0 mL of Trypsin-EDTA solution to flask and put it back in the incubator and wait about 5 minutes. Tap the flask and observe cells under an inverted microscope until cell layer is dispersed.
• Add 8.0 mL of complete growth medium, this will stop the action of trypsin. Pipette up and down (gently) a few times to break the clusters of cells.
• If you are keeping the same flask:
  • Pipette away and discard a certain amount of media. The exact amount depends on the desired density of the passaged cells.
  • Add fresh media to the flask.
• If you are moving to a new flask:
  • Pipette the amount of cells you want in a new flask (less than the total if you want to decrease the density of cells in your flask).
  • Add some media to the new flask (12 - 15 mL in T75 or 5 mL in T25 - total).
• Put your name (if not already present) the date and increase the passage number on the flask and put them back in the incubator.

Muscle cells

In the Bioactuator project, we use C2C12 myoblasts. For more information about C2C12 myoblasts click on the following link: https://en.wikipedia.org/wiki/C2C12#:~:text=C2C12%20is%20an%20immortalized%20mouse%20myoblast%20cell%20line.&text=C2C12%20cells%20are%20used%20to,to%20explore%20mechanistic%20biochemical%20pathways.

We use the following cell culture protocol for the C2C12 cells:

C2C12 cells protocol

I-General information

1.      The base medium for this cell line Dulbecco's Modified Eagle's Medium (DMEM, Sigma D6429) with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin.

2.      The seeding density should be 5.0 X10³viable cells/cm²

3.      IMPORTANT - DO NOT ALLOW CULTURES TO BECOME CONFLUENT.

Cultures must not be allowed to become confluent as this will deplete the myoblastic population in the culture.

Myotube formation is enhanced when the medium is supplemented with 10% horse serum instead of fetal bovine serum.

II-Trypsinizing or passaging the cells

1.      Remove and discard culture medium. Rinse the cells with PBS twice.

2.      Add 2.0 mL of Trypsin-EDTA solution to flask and put it back in the incubator and wait about 5 minutes. Tap the flask and observe cells under an inverted microscope until cell layer is dispersed.

3.      Add 8.0 mL of complete growth medium and dispense cells by gently pipetting.

4.      Tranfer the cell suspension into 5a 0 ml tube.

5.      Centrifuge the cell suspension to make cell pellet (300g, 3 min). Remove the supernatant and re-suspend the cells in 10 ml of culture medium.

6.      Add appropriate aliquots of the cell suspension to new culture vessels.Inoculate at a cell concentration between 1.5 X 10⁵ and 1.0 X 10⁶ viable cells/75 cm² bottle.(Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product.

7.      Incubate cultures at 37°C.

8.      Medium Renewal: Every two to three days (see the medium colour)

Collagen for 3D or coating surfaces

Type I Collagen is a major structural component of the extracellular matrix (ECM). Therefore, this fibrous protein is often used in three-dimensional (3D) collagen gels that simulate the in vivo cell environment better than the traditional 2D systems. Additionally, Collagen I is ideal for coating surfaces, as it can form thin layers for culturing cells.

Type 1 Collagen from Ibidi, Protocols for use. (bought through Inter Instrument https://interinst.no/. The product number is IB 50202 (4x5ml 5 mg/ml), price 3226,- + MVA Feb 2021).