Human Tissue Cell Culture
Introduction
Materials
- Cell Culture Hood (Sterile Environment)
- Personal Protective Equipment (PPE)
- Cell Culture Flasks (or plates)
- Cell Culture Medium
- Trypsin-EDTA Solution
- Phosphate-Buffered Saline (PBS)
- 37°C CO2 Incubator
- Pipettes and Pipette Tips
- Microscope
- Conical Tubes
Procedure
Subculturing Cells
- Preparation:
a. Ensure that your cell culture hood is clean and the UV light is turned on for sterilization.
b. Put on the appropriate PPE, including gloves, lab coat, safety goggles or face shield, and a mask.
c. Warm the required amount of cell culture medium to 37°C in a water bath or incubator.
- Cell Detachment (only for adherent and hybrid cells):
a. Remove the existing culture medium from the cell culture flask or dish.
CRITICALIf cell type is HYBRID transfer the medium which contains suspension cells into a conical tube.
b. Carefully rinse the cells with 1x PBS to remove any traces of serum.
c. Add an appropriate volume of trypsin-EDTA solution (typically 1-2mL per 25 cm² flask or dish) and incubate at 37°C for 2 - 3 minutes minutes until the cells detach. Check under the microscope.
d. If cell type is HYBRID: Go and centrifuge cells at 1,000rpm for 5 minutes.
- Neutralize the Trypsin (only for adherent and hybrid cells):
a. Add 2-times the volume of complete culture medium containing serum to neutralize the trypsin activity.
b. Transfer the volume into a conical tube.
c. If cell type is HYBRID: Transfer detached cells into the same conical tube that was centrifuged in step 2bp.
- Cell Counting (if needed for experiment):
a. Take a small aliquot of the cell suspension and mix it with trypan blue (1:1 ratio). Count live cells using a hemocytometer or an automated cell counter.
- For more info use protocol: Counting cells with Hemocytometer (manual)
b. Calculate the desired cell density for your experiment (e.g., cells per cm² or mL).
- Cell Seeding (aka. subculturing):
a. Plate the cells in a fresh culture flask or plate, in the case of an experiment use the calculated cell density.
b. Gently rock the dish or plate to distribute the cells evenly.
- Incubation:
a. Place the culture dish or plate in a 37°C CO2 incubator.
- CRITICALMonitor and Maintain:
a. Regularly check the cell cultures for confluence and change the culture medium as needed.
Thawing Cells (Seeding Cells)
- Small Surface (25 cm2 – 75 cm2)
1) If you are going to thaw one vial in a small surface you MUST dilute the DMSO first. This is done by quickly thawing the vial in a 37ºC clean water bath, and the cells suspension is transferred to a 15mL test tube with 10mL of medium.
2) Centrifuge the new suspension at 1500 rpm for 5 minutes.
3) Remove the medium and swirl the cells to break the pellet.
4) Add an appropriate amount of medium and transfer the cells to a bottle.
- Big Surface (150 cm2)
1) Quickly thaw the vial in a 37ºC clean water bath, and the cells suspension is transferred directly to a 150 cm2 mL bottle with 20mL of medium.
2) Change the medium after 24 hours.
This protocol outlines the procedures for growing and maintaining cells (adherent, suspension or hybrid) in a controlled environment (in vitro). Adherent cell culture is essential for various research and biotechnological applications. This guide covers the key steps for seeding (plating) and splitting (subculturing) cell cultures, ensuring proper technique and aseptic conditions to maintain cell health and purity. Adherence to these procedures is crucial for achieving reliable experimental results.
Author:
Osvaldo D. Rivera-Gonzalez, PhD
Rivera Omics Group
Revision: Sep/01/2023