Culturing Cells Using a CO2 Incubator

Cell culture is a technology that simulates the in vivo environment in vitro to cultivate, grow, and propagate cells. By culturing diseased cells, researchers can explore the mechanisms of disease occurrence and development. In the drug development process, cell culture technology can be used for drug screening and efficacy evaluation, shortening the drug development cycle and reducing research and development costs. Researchers can also deeply observe life activities of cells such as growth, division, and differentiation by culturing different types of cells, and analyze complex signal transduction pathways and gene regulatory mechanisms within cells. As an important tool to maintain an appropriate cell growth environment, ensure experimental reproducibility, reduce contamination risks, and support industrial production, the CO2 incubator plays an irreplaceable role in cell culture. This article describes how to use the FOUR E's Scientific CO2 Incubator ICO170 to culture HEK-293T cells.

1. Prepare the culture environment

After confirming the incubator is properly installed, disinfect internal components such as the partition plates and water trays, then initiate dry heat sterilization at 180°C. After sterilization is complete, install the HEPA filter. Set the culture conditions to 37°C, 5% carbon dioxide concentration, and 95% humidity. Operate the incubator without load for at least 4 hours. Once the conditions are stable, the culture environment is ready.

Figure 1：CO2 Incubator ICO170 uses ATCC 9372 strain to verify sterilization capability

2. Preparation of culture consumables and reagents

Prepare consumables such as cell culture flasks, petri dishes, pipettes, and centrifuge tubes according to experimental requirements. Ensure all are strictly sterilized to maintain aseptic conditions. Additionally, prepare appropriate cell culture medium based on cell type and experimental needs. Culture media generally include essential nutrients for cell growth, serum, and antibiotics. For example, the common RPMI-1640 medium can be supplemented with 10% fetal bovine serum, and a suitable amount of penicillin-streptomycin double-antibody solution can be added to prevent microbial contamination. After preparation, filter-sterilize the medium by passing it through a 0.22μm filter membrane into a sterile container for later use.

3. Cell preparation

For cryopreserved cells: After thawing, thoroughly disinfect the cryotube with alcohol before opening it in a laminar flow hood to transfer the cell suspension. Transfer the cell suspension to a centrifuge tube containing preheated medium, centrifuge at 1000 RPM for 5 minutes, discard the supernatant, and resuspend the cells in fresh medium.

For primary cell culture from tissue samples: Ensure aseptic conditions during tissue collection. Immediately rinse the harvested tissue with PBS buffer containing double antibiotics to remove blood and impurities. Cut the tissue into small pieces (about soybean-sized), and proceed with subsequent processing using either the tissue block culture method or enzyme digestion method.

For purchased cultured cells in flasks: Disinfect the flask first, then place it in the incubator to stabilize for 2-4 hours. Aspirate the original medium from the flask and add fresh medium for culture.

4. Cell Seeding
Before Seeding, determine the cell concentration in the cell suspension. Take a portion of the mixed cell suspension, stain it with trypan blue, and then count the cells using a hemocytometer or cell counter. Before using the hemocytometer, inspect it under a microscope to ensure it is clean. Place a coverslip over the counting chamber, mix the stained sample thoroughly by pipetting, and then add 10 μL of the sample to the edge of the coverslip. Allow the medium to fill the counting chamber by capillary action, then remove excess liquid with filter paper before counting. Avoid creating air bubbles. When using a cell counter, follow the manufacturer’s instructions and mix the sample well before loading. These methods can also be used to determine cell viability.

Once the cell concentration is determined, Seeding can be performed in a laminar flow hood. Inside the hood, pre-add a certain amount of preheated medium to the culture flask or petri dish. Then, use a pipette or pipettor to aspirate an appropriate amount of cell suspension and slowly add it to the flask or dish. Top up the culture flask or dish with medium and gently shake to evenly distribute the cells. The Seeding information for different culture flasks is shown in the following table:

Table 1: Medium Volume and Cell Seeding Quantity for Common Cell Culture Consumables

5. Incubation in Cultivation Incubator

Gently place the culture flasks or petri dishes into the incubator, taking care not to over-stack them to ensure smooth gas and heat circulation. When closing the incubator door, ensure a tight seal to prevent gas leakage that could alter the internal environment. Check the control panel parameters of the incubator at least 2-3 times daily, and record data on temperature, carbon dioxide concentration, and humidity. Meanwhile, observe the cell growth status under a microscope, including cell morphology, density, and adherence, and check for signs of contamination, such as turbid medium or the presence of filamentous or granular substances in the medium.

6. Cell Passaging

When cells grow to 80%-90% confluence in culture flasks or petri dishes, passaging is required to prevent nutrient deficiency and metabolic waste accumulation from affecting cell status due to overgrowth. In a laminar flow hood, carefully aspirate the old culture medium from the flask or dish using a pipette, taking care not to touch the cell layer. Add an appropriate amount of PBS buffer, gently shake the culture vessel to rinse residual medium and metabolites from the cell surface, then discard the PBS buffer. Next, add an appropriate amount of trypsin-EDTA digestion solution to the culture vessel to cover the cell layer, and incubate at 37°C for 1-3 minutes. Alternatively, observe under a microscope and immediately add culture medium containing serum to terminate digestion when cells round up and begin to detach from the flask wall—the proteins in serum neutralize trypsin activity. Gently pipette the cells to fully detach them from the flask wall and form a uniform cell suspension, then transfer the suspension to a centrifuge tube and centrifuge at 1000 RPM for 5 minutes. Discard the supernatant, resuspend the cells in fresh culture medium, and pipette gently to mix evenly. According to cell growth characteristics and experimental needs, aliquot the resuspended cell suspension into new culture flasks or petri dishes at an appropriate ratio, add sufficient culture medium, shake gently to distribute cells evenly, and return to the incubator for continuous culture.

Figure 2: 293T cells cultured in a CO2 Incubator ICO170 for passaging

7. Cell Cryopreservation

First, prepare the cell cryopreservation solution, then collect cells from the culture flask and adjust the cell concentration to 1×10⁶ to 5×10⁶ cells/mL using the cryopreservation solution. Rapidly aliquot the cell suspension into cryotubes.

If using a programmed cooling container, transfer the aliquoted cryotubes into the container and directly place them at -80°C overnight.

If no programmed cooling container is available, cool the cells in the following order: room temperature → 4°C for 20 minutes → -20°C for 30 minutes → -80°C overnight.

During transfer, maintain thermal insulation to avoid temperature differences or thawing of cryotubes. After removing from -80°C, quickly transfer the cryotubes to a liquid nitrogen tank for long-term storage.

Figure 3: 293T cells cultured in a CO2 Incubator ICO170 for cell cryopreservation

8. Cell Transfection

Exogenous nucleic acids are integrated into cells through physical, chemical, or biological-mediated pathways, enabling cells to acquire new phenotypes. For short-term rapid experiments aimed at quickly obtaining gene expression products, observing the interference effect or overexpression of target genes, transient transfection can be used. For long-term experiments such as pharmacological research, gene editing, or gene function exploration, stable transfection is more appropriate.

Key considerations during experiments:

• Ensure cell density meets the expected requirements based on the transfection method used.

• Avoid using cells that have undergone excessive passaging for transfection.

• Use high-quality nucleic acids for transfection.

• Adopt effective transfection conditions.

• Strictly follow aseptic techniques throughout the transfection process.

Figure 4: 293T cells cultured in a CO2 Incubator ICO170 for cell transfection