A cell suspension refers to a liquid mixture containing dispersed individual cells that are not attached to each other or to any surface. In cell culture, a cell suspension can be created by detaching adherent cells from their growth surface, such as a culture flask or plate, and resuspending them in a suitable culture medium. Suspension cultures can also consist of cells that naturally grow in suspension without the need for detachment. Cell suspensions are used in various applications in biological research, biotechnology, and medicine, including cell counting, cell viability assays, flow cytometry, cell sorting, and the production of recombinant proteins or viral vectors.
To create a cell suspension of adherent cells, such as HEK293 cells, the following steps can be performed:
- Remove culture medium: Aspirate the culture medium from the flask or plate containing the adherent cells.
- Wash the cells: Gently wash the cell monolayer with phosphate-buffered saline (PBS) or another suitable buffer to remove residual culture medium and any dead or detached cells.
- Detach the cells: Add an appropriate volume of cell detachment solution, such as trypsin-EDTA, to the flask or plate. Incubate at 37°C for a few minutes to allow the enzymatic detachment of the cells from the surface. Monitor the detachment under a microscope to avoid over-trypsinization.
- Neutralize trypsin: Add an equal volume of complete culture medium containing serum to the flask or plate to neutralize the trypsin-EDTA solution. Serum contains trypsin inhibitors that will halt the enzymatic activity of trypsin.
- Collect the cells: Gently pipette the cell suspension up and down to ensure that the cells are well dispersed and detached from each other. Transfer the cell suspension to a sterile tube.
- Optionally, centrifuge the cells: If necessary, centrifuge the cell suspension at 200-300 x g for 5 minutes to pellet the cells. Aspirate the supernatant and resuspend the cell pellet in fresh culture medium or buffer, depending on the intended application.
For cells that naturally grow in suspension, such as certain lymphocyte or hybridoma cell lines, no detachment step is required. The cells can be directly collected from the culture medium and used for various applications as needed.