Scientists commonly study the development of lymphocytes in two ways, adoptive transfer of hematopoietic stem cells or progenitor cells into recipient mice and fetal thymic organ culture (FTOC). Both strategies, especially the first one, are still widely used. However there are some limitations of these two methods such as being time consuming, resulting in limited cell yield, and challenges in the technology. During the last decade, OP9 stromal cells co-culture system has been modified to support lymphocyte development in vitro. This alternative way offers researchers a simple, efficient approach to support lymphoid progenitors to develop in vitro. A more important advantage of this system is that a lot of factors involved in lymphocyte development, such as cytokines and Notch signaling pathway, can be manipulated in order to delineate the mechanisms more clearly. This protocol is based on the experience of supporting T cell development by OP9-DL1 stromal cells. As a matter of fact, this system can be used to facilitate other lymphocytes development in vitro, such as B cells and NK cells, depending on the type of stromal cells and different combinations of cytokines.

Stromal cell line, OP9, was derived from the bone marrow of op/op mice that are deficient for macrophage-colony stimulating factor (M-CSF). In order to investigate the role of Notch signaling on the differentiation of T cells in vitro, intensive studies have been recently done through the co-culture system including lymphoid progenitor cells and modified OP9 stromal cells that express Notch ligands such as Delta-like 1, Delta-like 4, or control vector. These derived OP9 stromal cells lines are termed as OP9-DL1, OP9-DL4, and OP9-V or OP9-vector, respectively.