Recovering from Detachment: Best Practices for HEK-293T Cells

HEK-293T cells, also referred to as Human Embryonic Kidney 293T cells, are a modified version of HEK-293 cells. They've been modified by introducing a temperature-sensitive gene from the SV40 T-antigen. These cells are extensively used in viral packaging because they're easy to manipulate genetically. Additionally, they are commonly used for research purposes to express foreign genes.

Figure 1. Image of Normal Growth of 293T Cells

These cells don't stick tightly to surfaces, so when you receive them at room temperature, it's common for many of them to detach from the sides of the culture container. When you look at them under a microscope, finding adherent cells can be tricky. Instead, you might see clumps of cells with the naked eye, as shown in Figure 2. This is completely normal.

Figure 2 Upon receipt, 293T cells may exhibit clustering.

To get things back on track, here's a step-by-step guide:

1.Upon receiving the cells, stabilize them in the incubator for 2 to 4 hours before proceeding with further handling. It is not recommended to leave them in the incubator overnight.

2.Transfer all the culture medium from the culture flask into a sterile centrifuge tube. Centrifuge at 1200 rpm for 3 minutes to collect the cells.

3.Remove the supernatant and resuspend the cells in PBS (Cat.No.: PB180327). Resuspend the cells by gently shaking the centrifuge tube; avoid using a pipette to resuspend the cells. Collect the cells into a centrifuge tube and centrifuge again at 1200 rpm for 3 minutes.

4.Remove the supernatant and resuspend the cells in approximately 1 mL of 0.25% trypsin (Cat.No.: PB180225). Mix gently by shaking the centrifuge tube manually; do not pipette the cells. Place the tube in the incubator for digestion for 2 minutes.

5.After 2 minutes of digestion, add 5 mL of complete growth medium (Cat.No.: CM-0005) to stop the digestion. Gently pipette the cell suspension to disperse cell clusters. Centrifuge at 1200 rpm for 3 minutes.

6.Remove the supernatant and add approximately 6 mL of the corresponding complete growth medium to the cells. Determine whether to passage at a ratio of 1:2 or 1:3 based on cell density (due to potential cell damage during transport, it is advisable to slightly increase the seeding density for the first passage).

7.Under a microscope, observe whether the cells have dispersed into single cells. If there are still large cell clusters present, repeat the digestion process once more.

8.On the following day, monitor the cell adhesion status. If there is excessive cell accumulation, causing cell stacking, passage the cells into new flasks after 24 hours. If the density is normal, continue growth without changing the medium. After 48 hours of adhesion, change the medium to remove non-adherent cells.

Here are some easy-to-follow guidelines:

• Treat the cells gently since they don't stick tightly to surfaces.
• Consider wrapping the culture flask or dish while you're working with them.
• Take your time when rinsing with PBS to prevent cells from getting washed away.
• On the second day after passaging the cells, you might notice a bit of clustering. Give them an extra day, and they should spread out. It's best not to change the medium on the second day to avoid losing cells.