Methods of organ culture

Several general methods exist for the organ culture of lymphoid tissue. The first of these is termed the 'plasma clot' method, and is very similar to the method described in the early 1900s. In the plasma clot method, crude embryo extract is added to a small amount of plasma and allowed to clot. The tissue or organ to be cultured, usually derived from fetal donors, is placed on top of the clot (Figure 1A) and cultured for various time periods to observe temporal changes in morphology and/or cell populations. Plasma clot techniques, like other organ culture methods to be described, allow the sequential analysis of development without the complexities of cell traffic (immigration and emigration), found in vivo. In addition, it frequently allows the maintenance of an organotypic three-dimensional structure with a complex cellular heterogeneity similar to the in situ organ. However, its utility in the study of the interactions between selected lymphoid populations or their precursors and their cellular microen-vironment, and how that interaction may change with time, is limited by the heterogeneity of the initial population.

A similar technique for conducting these cultures without the plasma clot by using a supporting mem-

(A) Plasma clot

(A) Plasma clot

60 mm culture dish

Tissue Plasma clol

^ Media

60 mm culture dish

Tissue Plasma clol

^ Media

'GeHoaim" sponge

0.22 jim pore membrane

'GeHoaim" sponge

Media

(C) Transmembrane

Target tissue

0.45 nm pore membrane

Target tissue

0.45 nm pore membrane

Donor tissue

(D) Hanging drop

(D) Hanging drop

60 mm dish (or a well in an inverted 96-well plate)

Culturo media

60 mm dish (or a well in an inverted 96-well plate)

Figure 1 Approaches to the organ culture of lymphoid tissues. Fetal or adult tissues can be organ cultured in systems that employ: (A) plasma clot cultures; (B) raft cultures; (C) transmembrane cultures; or (0) hanging drop cultures. Raft cultures of fetal materials may be treated with deoxyguanosine or low temperature to first deplete the lymphoid populations, prior to being used as a 'target' tissue for a source of donor cells in the transmembrane system brane raft in liquid culture (Figure IB) is now employed by most investigators. Like the plasma clot technique, the raft systems allow the target tissue to be maintained at a gas:media interface in a relatively well-controlled in vitro environment. In addition, raft techniques provide for better control of soluble factors through the use of defined media and growth factors, if necessary. Clearly, one of the major drawbacks of the plasma clot method is its required use of an undefined embryo extract as a source of growth promotion in the culture medium. Thus raft techniques allow controlled studies on the role ot soluble factors on lymphocyte differentiation and function.

In many studies it would be useful to be able to separate the developing and/or functional lymphopoietic cells from their native stromal cell micro-environment and then selectively recombine them to better understand the nature of their interactions. Further modifications of raft organ culture techniques which employ temperature change or media supplements allow for the preferential depletion and/or enrichment of cell populations, with the potential for a purification of specific cell populations. Using these organ culture purification techniques, elements of the microenvironment have been isolated from the progenitor cell population and the two purified populations recombined under controlled conditions for the study of the influence of the microenvironment on differentiation of the progenitors. Such techniques have been utilized extensively in the study of lymphocyte differentiation in the thymus or B lymphocyte development in a marrow environment, as well as the ability of various tissues to serve as a potential source of lymphopoietic progenitor cells.

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