Use of human tissue explants to study human infectious agents

Abstract

The study of human cell-cell and cell-pathogen interactions that occur in the context of complex tissue cytoarchitecture is critical for deciphering the mechanisms of many normal and pathogenic processes. This protocol describes methods for culturing and infecting explants of human tissues to study the pathogenesis of human infectious agents and their local interactions. The protocol relies on the use of fresh human tissues dissected into small blocks or biopsies that are cultured at the liquid-air interface on collagen rafts. These tissue blocks retain their cytoarchitecture and support productive infection of various pathogens without exogenous stimulation. Experimental details for setting up cultures of human tonsils, lymph nodes and cervicovaginal and rectosigmoid tissues, including protocols for their infection with HIV-1 and other pathogens, are described here. Using this protocol, culture and infections can be set up in 3-6 h and be maintained for 2-3 weeks, depending on the tissue used.

Figure 1

Synopsis of explants culture setup. Six major stages of the protocol to set up human tissue explants to study human infectious agents: (a) Tissue (tonsils, lymph nodes, cervicovaginal, rectosigmoid) is obtained through surgery or from cadavers, and delivered to the laboratory; (b) tissue is dissected into small blocks; (c) blocks are placed at the medium–air interface on collagen rafts in appropriate culture vessels. (Note: the minimum number of wells that constitute an experimental condition is boxed in red on each culture plate and labeled with a ; the number of tissue blocks per well is indicated); (d) tissue is infected with a pathogen of interest (note that the modality and order of infection for cervical tissue is different from those of other tissues); (e) tissue is cultured for 2–3 weeks and samples of medium are collected periodically and analyzed for pathogen components and for various metabolites of interest; (f) at various time points, tissue blocks are collected and analyzed by use of flow cytometry or microscopy.

Figure 2

Selected sequential steps of the preparation of a tonsillar tissue for histoculture. (a) Black arrows point to the parts of the tissue that cannot be used and should be discarded (tonsilolithes, cauterized tissue, bloody tissue and green necrotic tissue). Usable tissue parts are cut into large pieces (Step 10A(iv)) and strips (Step 10A(v)). (b) Tissue blocks before (Step 10A(v)) and after (Step 10A(vii)) being deposited on top of the gelfoam sponge in a six-well plate.

Figure 3

Replication of HIV-1 in human explants of different tissues. Blocks of human tissues were prepared and infected with the R5 HIV-1 strain SF162 and cultured as described in the protocol. The culture media were collected and replaced at different time points following viral infection. The concentration of viral antigen p24 was measured in each sample. (a) The graphs in the blue box represent the primary data measured for each sample; the solid blue curve is the interpolation of these data points. Note: the interpolation curve does not take into account the fact that after a medium change, the concentration starts from almost zero to reach the level measured at the following time point. The dark dotted line reflects this fact. (b) The graphs in the red box represent the plots of the cumulative production of p24 obtained for each datum point by summing up the measurement at this time point with the measurement of all the preceding time points. Note that the scale of the y axis has been changed between a and b to emphasize the difference between cumulative and differential mode of data presentation. To avoid distracting the reader of this technical article with the scientific problem of the different replicative capacity of different HIV variants in different tissues, we present here only one (R5) HIV-1 variant replicating in different tissues and chose experiments in which the efficiency of replication in different tissues were comparable. For the differences in the average replication of HIV variants in different tissues, see refs. ,.