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Cell Subculture

Subculture (or passage) refers to the transfer of cells from one culture vessel to another culture vessel. Subculture usually (not always) involves the subdivision of proliferating cells that enables the propagation of a cell line.

Procedure

Take a T25 flask as an example

  • Passaging of suspension cells
  1. Collect cells with medium and briefly centrifuge (150–300 ×g, 3–5 min) to pellet cells.
  2. Remove the medium and gently resuspend cells with a balanced salt solution. Briefly centrifuge (150–300 ×g, 3–5 min) to pellet cells.
  3. Remove salt solution, resuspend cells in fresh medium and count cells.
  4. Plate cells onto a new dish at the desired cell density.
  • Passaging of adherent cells
  1. Remove medium.
  2. Wash cells with calcium- and magnesium-free balanced salt solution (phosphate buffered saline, Hanks’ balanced salt solution, etc.)
  3. Add detaching agent. Incubate at 37°C until cells are fully detached from the dish (2–20 min depending on the cell line).
  4. Resuspend cells in fresh medium, pipette thoroughly to obtain single cell suspension. Count cells.
  5. Place cells onto a new dish at the desired cell density
  • Passaging of semi-adherent semi-suspended cells
  1. Pour out the culture medium in the bottle, add sterile PBS and rinse once, and then add 2mL digestive fluid. Let it stand in the incubator for 3 minutes.
  2. Observe the digested cells under an inverted microscope. If the cells become round and are no longer connected to each other into pieces, immediately add an equal proportion of fresh culture medium, blow, make the cell suspension and centrifuge.
  3. Discard the supernatant, resuspend with 5ml culture medium, passage in a ratio of 1:3 (depending on the number of cells), and send back to the carbon dioxide incubator for further culture.

Basic subculturing procedure.Fig. 1 Basic subculturing procedure.

Features
  1. Passage of cells allows the expansion of cultured cells (formation of cell lines), cell cloning, easy to preserve, but may lose some special cellular and differentiation characteristics.
  2. The greatest benefit of cell line formation from passage cells is that it provides a large amount of durable experimental material, which is convenient for experiments.
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STEMart provides you with a variety of cell culture equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for cell subculture, please feel free to contact us.

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