Single-Cell Cloning of Hybridoma Cells by Growth in Soft Agar

Single-cell cloning during hybridoma production ensures that cells that produce the antibody of interest are truly monoclonal and that the secretion of this antibody can be stably maintained. Cloning of hybridoma cells in semisolid medium is one of the most commonly used methods for producing single-cell clones. The technique is easy, but, because it is performed in two stages, it does take longer than other methods. Not all cells will grow in soft agar, and there may be a bias on the type of colony that appears. However, most of the commonly used myeloma fusion partners have relatively good cloning efficiencies in soft agar, and, consequently, so do most hybridomas. Even though every attempt is made to ensure that the cells are in a single-cell suspension before plating, there is no way to guarantee that the colonies do not arise from two cells that were stuck together. Therefore, single-cell cloning in soft agar should be repeated at least twice before the cells are considered clonal.