Application of organ culture techniques to the culture of lymphoid tissues

Organ culture techniques have been widely applied to examine normal development and the role of microenvironmental elements on hematopoietic differentiation in several different species. Many of these studies have also employed a transmembrane system (Figure 1C) that allows the initial separation and subsequent controlled recombination of purified progenitor and stromal cell populations. For example, since chick and quail cells can be distinguished on the basis of nuclear morphology, L.c Douarin and colleagues employed the primitive thymic rudiment of the chick and the yolk sac, spleen or bone marrow cells of the quail in a transmembrane organ culture system to demonstrate that the thymic anlagen taken prior to colonization can become lymphoid if given a source of lymphopoietic progenitors at an appropriate time. In related studies, several waves of receptivity of the thymus anlagen could be demonstrated. The same group has used similar organ culture systems to investigate the relative lymphopoietic potential of the embryonic bursa.

In the murine system, the use of organ cultures to define the role of the thymic microenvironment has been widespread. Flarly studies of thymic epithelium and the thymic microenvironment utilized monolayer cultures derived from rhvmocyre cell snspen-

sions. Although many investigators reported the isolation of 'epithelial' cells, the techniques were generally unsatisfactory. A major problem in these systems was the presence of excessive numbers of macrophages and fibroblasts within the cultures, often overshadowing the role of the epithelium. In addition, since several epithelial cell populations found within the thymus in vivo may be required for normal lymphocyte development, one runs the risk of deleting an important cell population by preferential growth conditions or subcioning, both of which are common in monolayer systems.

Techniques which use treatment of thymic rudiment organ cultures with deoxyguanosine, low temperature or even a change in oxygen tension have all been shown to selectively enrich for the epithelial cell populations and thus facilitate the study of T cell differentiation, both in vitro and in vivo. Like the unco-Ionized thymic anlagen, purified thymic epithelium may become lymphoid when the appropriate progenitor cells are provided in transmembrane organ cultures where donor tissue expiants are placed below a cell permeable filter upon which the lymph-oid-free thymic expiants are placed. These experiments have generally used bone marrow, fetal liver, or fetal thymus as a stem cell source. In situations where cell suspensions are used for the donor cells, often first highly purified by positive and/or negative selection techniques, the lymphoid-free thymic anlagen can be directly injected with a single putative stem cell or another appropriate source of progenitors. Alternatively, a hanging drop modification of the organ culture system (Figure ID) may be used to facilitate cell interaction. Hanging drop systems also allow the selective continued separation of donor and target cell populations. In an elegant set of experiments utilizing deoxyguanosine-treated thymic rudiment organ cultures, combined with transmembrane cultures, hanging drop cultures or direct injections, Owen's group and others have demonstrated that single lymphopoietic progenitor cells were capable of giving rise to all of the subpopulations of thymocytes found within the developing thymus.

Organ culture-purified murine thymic epithelium of both fetal and young adult origin has been used in in vivo transplantation experiments to examine the ability of the grafts to recapitulate ontogeny and provide functional reconstitution. These studies have shown that although the grafts are colonized, they give rise to a variable number of peripheral T cells of possibly different subpopulation composition. Also in vivo, grafts of organ-cultured thymic epithelium have been used to investigate the role of epithelium on immune recognition and regulation. After grafting deoxyguanosine-treated thymic lobes into allogeneic recipients, Jenkinson and colleagues were able to demonstrate that maturing host lymphoid progenitors were not rendered tolerant to the major histocompatibility complex (MHC) antigens exprès sed by the grafted thymic epithelial organ culture.

More recently, similar organ culture systems have been applied effectively to the study of human materials. Many of these procedures utilize "tissue slices' and their utility in evaluating three-dimensional interactions of cell populations in assays with functional end-points is just emerging. Furthermore, the additional potential to use human feral thymic fragment organ culture models to assess human immunodeficiency virus (HIV) pathogenesis ma\ serve as an important functional model for that criti cal area of research.

Finally, in addition to the more traditional organ culture techniques already described, another system has been used which is often considered to be a method of organ culture. Utilizing a system similar to that described by Dexter and coworkers, Whirlock and Witte developed a monolayer culture svsrem which supported the long-term production of R cells from an initial inoculum of mouse bone marrow cells. Once established, the cultures consist of a dense stromal cell adherent layer associated with a number of loosely adherent developing B cclls. These cultures are capable of maintaining a population of early B cell progenitors in the presence of heterogeneous and poorly defined stromal cell population. VI a m stromal cell lines have been isolated from these cultures, a number of which are capable of inducing early events of B cell differentiation in B cell progenitors. Thus, like more classical organ culture techniques, the Whitlock-Witte culture system ma\ allow for the purification and recombination of the stromal components of lymphoid microenvironments with early lymphoid progenitor cells.

See also: Aging and the immune system; Avian immune system; Bone marrow and hematopoiesis; Bursa of Fabricius; Chimerism, hematopoietic; Cytokines; Deoxyguanosine; Hematopoietic stem cell transplantation; T lymphocyte differentiation; Thymus; Tolerance, peripheral; Transplantation.

How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment