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Protocol no. 28

HUMAN OESOPHAGEAL CULTURE

This procedure describes a method for establishing short-term explant cultures of oesophageal mucosa. Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth.

CONTACT

Dr. C. Mothersill Radiobiology Research Group Nuclear Energy Board Geological Survey Office Beggars Bush Dublin 4 Ireland

RATIONALE

When mucosa explants are plated into culture there is an initial migration of cells outward, followed by a period of mitotic activity resulting in a pronounced outgrowth. Such explant cultures can be exposed to test compounds and radiation, after the initial period of migration has occurred, and the effects of these can then be quantified as an inhibition on the rate of cell growth. As the cells grow outward they form a definite area surrounding the tissue initially plated which, after fixing and staining, can readily be quantified by visual examination.

BASIC PROCEDURE

Dissect mucosa samples from the oesophagus, place the pieces of tissue in growth medium and cut into small sections. Incubate in a trypsin/collagenase mixture for a short period then select suitable tissue samples and culture in growth medium until required for testing. After » 2 days add test chemicals to the cultures and, after the required exposure period, assess toxicity by fixing, staining and measuring the area of outgrowth of the epithelial cells.

CRITICAL ASSESSMENT

Primary cultures of epithelial cells are very difficult to achieve, require a high degree of technical expertise, and can be very time-consuming to maintain. Although a number of primary "normal" cell lines have been established from adenomatous tissue it is quite common for these to become contaminated by fibroblasts, and thus no longer be considered "normal". A further complication with regard to maintaining oesophageal mucosa cells in culture is their lack of clonogenicity. The epithelial nature of the outgrowth was confirmed in representative samples of cultures using a low molecular weight general cytokeratin antibody with indirect peroxidase development. The presence of stromal or endothelial elements can be monitored using anti-vimentin and anti-human endothelium. This method, however, provides a relatively simple, fast, easy to perform technique for establishing short-term cultures of relatively pure populations of mucosal epithelial cells. Should contamination with fibroblasts occur, these cells are readily discernable after staining and can be allowed for in calculation of cell growth. Unfortunately, the major drawback of this technique is that the cultures are only viable for a limited period of » 2 weeks (but up to 4 weeks is possible). The viability of the culture can be extended by changing half the medium weekly. In addition, each new culture is dependent upon a fresh supply of human tissue, which may not always be readily available. It should also be noted, however, that the technique is readily adapted to healthy and diseased tissue, thus enabling the differential response to compounds to be examined in both tissue types. Cell outgrowth is measured directly by quantifying the area of cells around the explant. This may provide a more reliable measure of cell growth and survival than other methods currently used e.g. growth curve extrapolation in monolayer cell culture systems. The possibility that the outgrowth of cells from the original explant is simply due to a process of cell migration has been considered. This is largely true in the initial stages of culture, however the contribution of migrating cells to overall outgrowth at later stages is insignificant. This has been confirmed through experiments designed to measure the incorporation of tritiated thymidine (coupled with autoradiographic analysis) and examination of cultures by electron microscopy, which have illustrated that the cells present in the area of outgrowth have a high rate of mitotic activity and are actively dividing (see Mothersill et al., 1988). Cell proliferation in cultures can be studied using the monoclonal antibody, Ki 67 (DAKO) and an indirect peroxidase immunocytochemical development technique. The effects of irradiation can also be studied using this cell system either in the presence or absence of the test compounds. Irradiation is carried out 2 days after the explant is established and/or 12 hours after exposure to the test compound. Preliminary results using this culture system have shown that a reduction in the outgrowth of cells from the explant occurs after radiation treatment. The dose-response relationship that is obtained is within normal mammalian limits and tissue specific differences can be detected.

TEST STATUS

In-house and interlaboratory validation.

CHEMICALS TESTED

Benzo[a]pyrene Nitrosamines Glycolysis inhibitors

REFERENCES

  1. Mothersill, C., Cusack, A., MacDonnell, M., Hennessy, T.P. & Seymour, C.B. Differential response of normal and tumour oesophageal explant cultures to radiation. Acta Oncologica 27, 275-280
  2. Mothersill, C., Cusack, A. & Seymour, C.B. (1989) Enhanced proliferation of cells from human tissue explants following irradiation in the presence of environmental carcinogens. Radiat. Environ. Biophys., 28, 203-212.

IP-28 © November 1990