I. What Are Cell Culture Flasks?
Cell culture flasks are a common piece of laboratory equipment used for cell culture. They are manufactured from high-quality polystyrene (PS) material using ultra-precision molds and fully automated production processes. These products are utilized in laboratory cell culture applications; their excellent optical properties facilitate microscopic observation, and their surfaces undergo tissue culture (TC) treatment to enhance cell adhesion.
II. Inoculating Cell Suspensions into Culture Flasks
1. Mixing the Cell Suspension: Prepare the cell suspension at the concentration required by your experiment, and mix it thoroughly within a medium bottle, culture flask, or other suitable container.
2. Using a pipette or Pasteur pipette, slowly and steadily dispense the cell suspension into the cell culture flask, taking care to avoid splashing the liquid or generating foam. (For multi-layer cell culture flasks, particular attention must be paid to ensuring uniform liquid distribution; this can be achieved by tilting or rotating the flask to allow the liquid to flow evenly into each layer.)
3. Gently place the culture flask on a stable work surface, ensuring that the smooth, flat side faces upward and the side featuring the graduation marks faces downward (this corresponds to the flask's stackable design, which facilitates easy handling).
4. Place the cell culture flask into an appropriate incubator, setting the temperature (e.g., 37°C), humidity, and gas concentration (e.g., 5% CO₂) to the required levels.
5. Allow the flask to sit undisturbed for a period of time to permit the cells to grow within the culture vessel.
III. Changing the Culture Medium
1. Removing the Old Medium: When aspirating the culture medium, tilt the flask body to a 45-degree angle, positioning the smooth side of the flask toward yourself. Use a Pasteur pipette or standard pipette to gently aspirate the old medium; perform this action with care to avoid dislodging any adherent cells. If the medium contains a significant number of suspended cells or impurities, you may first wash the cells once or twice with PBS buffer to remove these contaminants.
2. Adding Fresh Medium: After removing the old medium, add an appropriate volume of fresh culture medium to the flask. When adding the liquid, dispense it along the side wall of the flask to avoid directly impacting the adherent cells on the growth surface. Concurrently, the volume of fresh culture medium to be added should be determined based on the cell density and growth rate to ensure that the cells can grow normally.
IV. Cell Collection
1. Removal of Spent Medium: Gently aspirate the spent culture medium from the cell culture flask. Add an appropriate amount of PBS buffer and gently rock the flask to wash away residual medium, ensuring that the cell surfaces are clean.
2. Cell Digestion and Neutralization: Add an appropriate amount of trypsin solution (≥ 3 mL) to digest the cells for 2–3 minutes. Neutralize the trypsin activity by adding serum-containing culture medium.
3. Cell Collection: Use a pipette to transfer the cell suspension into a centrifuge tube (taking care to avoid generating foam). Place the tube in a centrifuge and spin at an appropriate speed (e.g., 800 rpm) for 5 minutes to pellet the cells at the bottom of the tube.
4. Collection of Cell Pellet: Once centrifugation is complete, gently decant the supernatant from the centrifuge tube (taking care not to discard the cell pellet). Collect the cell pellet remaining in the centrifuge tube and proceed with subsequent processing according to experimental requirements.