title: In vitro systems for the study of T cell development: fetal thymus organ culture and OP9-DL1 cell coculture.
abstract: Most T cell development occurs within the thymus and includes a series of selection processes that are, in large part, still poorly understood. Studies of T cell development have been greatly advanced by the description of multiple phenotypic subsets of T cells and their maturational relationships. This unit describes a system for observing and modulating T cell development in vitro via the culture of entire mouse fetal thymic lobes. Methods are included for the isolation of fetal thymi and culture to allow for normal T cell development on either transwell plates or Gelfoam sponges. A method for depleting hematopoietic cells from thymic lobes using 2'-deoxyguanosine and subsequent reconstitution with precursor cells is also described. This protocol is valuable for the study of tolerance and T cell selection. A support protocol describing methods of altering and monitoring T cell development are outlined. In addition, methods for culturing fetal thymic lobes under high oxygen submersion conditions and for the preparation of reaggregate thymus organ cultures are provided. Finally, a simple and practical method that allows for the thymus-independent generation of T cells from defined sources of stem/progenitor cells by OP9-DL1 coculture is described.
authors.affiliation: Immunex Corporation, Seattle, Washington, USA.
authors.forename: Fred
authors.initials: F
authors.lastname: Ramsdell
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Zúñiga-Pflücker
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Takahama
mesh_terms: D017667:Adipocytes / Q000166:cytology; D000818:Animals; D002454:Cell Differentiation; D018920:Coculture Techniques / Q000379:methods*; D005333:Fetus; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000502:physiology; D038041:Lymphopoiesis; D051379:Mice; D009924:Organ Culture Techniques / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology*; D013950:Thymus Gland / Q000166:cytology / Q000196:embryology* / Q000502:physiology