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Protocol no. 57
HUMAN AND BOVINE LENS EPITHELIAL CULTURE

This procedure describes a method for routinely establishing finite but abundant primary explant cultures of human (and bovine) lens epithelial cells.

CONTACT

Ms S.F. Webb

    Biomedical Research Centre
    School of Biological Sciences
    University of East Anglia
    Norwich NR4 7TJ UK
    Tel: England - 0603 592781
    Fax: England - 0603 259492

RATIONALE

Whilst some research groups have a readily available supply of human donor lens epithelial cells, the material is nevertheless unpredictable. This protocol can be employed as a means of initiating and maintaining human explant lens epithelial cultures in order to provide a homogenous and reliable source of experimental material. It is also suitable for bovine lens cultures, which can in certain cases be used as a back-up source when human material is unavailable. The cultures are initiated from the anterior lens capsule and its attached epithelium. A closed system, initiated and maintained in a flask lessens the risk of fungal infection during the 3-8 weeks required for human tissue to reach confluency. Cells seeded from established cultures into 96-well plates provide thin monolayers which are ideal for toxicity testing. The anterior capsules may be transferred to a new flask in order to initiate a second, and in some cases also a third, primary culture.

BASIC PROCEDURE

Whilst some research groups have a readily available supply of human donor lens epithelial cells, the material is nevertheless unpredictable. This protocol can be employed as a means of initiating and maintaining human explant lens epithelial cultures in order to provide a homogenous and reliable source of experimental material. It is also suitable for bovine lens cultures, which can in certain cases be used as a back-up source when human material is unavailable. The cultures are initiated from the anterior lens capsule and its attached epithelium. A closed system, initiated and maintained in a flask lessens the risk of fungal infection during the 3-8 weeks required for human tissue to reach confluency. Cells seeded from established cultures into 96-well plates provide thin monolayers which are ideal for toxicity testing. The anterior capsules may be transferred to a new flask in order to initiate a second, and in some cases also a third, primary culture.

CRITICAL ASSESSMENT

The cells that make up the mammalian lens consist of two types: lens fibre cells, which form the bulk of the lens, and a monolayer of epithelial cells over the anterior surface of the fibres. Pure cultures of lens cells can be derived from the anterior epithelium. The standard procedures for the establishment of such cultures have, up until now relied on the mechanical or enzymatic disaggregation of the lens tissue in order to obtain a cell suspension for seeding into the culture vessels. However, it was found that human lens epithelium, especially from older donors, proved too fragile to provide sufficient viable cells for culture. A second problem relating to cultures of human lens epithelium is the fact that they generally require 3-8 weeks to reach confluency, as opposed to the 2-5 weeks required by bovine tissue. Therefore, the problem of fungal contamination is greater with human cells. For this reason, it is preferable to use a closed culture system for human cells, although bovine lens epithelium can be grown in an open system. A modification of the technique of Bermbach (1991) avoids the need for disaggregation, or initiating the explant in a Petri dish, and provides a closed system in which both bovine and human lens tissue has been successfully cultured. The establishment of bovine and human lens cultures differs in two main aspects. Firstly, the source of culture cells for bovine culture is from calves of less than two years old; whereas the source of human culture cells varies from foetal lenses of 14 weeks to adults of 90 years old. Secondly, the bovine material is obtained and used within 5 hours of death. This ensures that the cells are still in an optimum condition when culture is initiated, which is reflected in the ease of production of bovine cell lines. In the case of human tissue there can be a delay of up to 3 weeks between the death of a donor and the time at which the material is made available for culture. Keeping the passage number low is an important consideration as the number of senescent cells increases with each passage. The success of the culture is also affected by the age and state of health of the donor at the time of death, as well as the condition of the dissected out lenses when made available for culture. Younger donors produce initially higher yields of proliferating cells and the cells can undergo more passages before senescence occurs. Cells from healthier donors produce better cultures, than those from donors with certain diseases. As a general rule, cells are not used beyond the second passage, except when very young donors (less than 4 years old) are involved. However, the yield from a single donor can be optimised by transferring the anterior capsules to a new flask in order to initiate a second primary culture. Below are some representative culture yields: No. of cells (a) Foetal lenses 14 weeks 3 x 106 (b) 3-4 year old 1 x 106 (c) 25 year old 3 x 105 (d) 60 year old 8 x 104 (e) 80 year old 4 x 104

TEST STATUS

In-house development

REFERENCES

    1. Bermbach, G., Mayer, U. & Gottfried, O.H.N. (1991) Human lens epithelial cells in tissue culture. Experimental Eye Research, 52, 113-119
    2. Chandrasekharam, N.N. & Bhat, S.P. (1989) Human foetal lens epithelial cells in culture: an in vitro model for the study of crystallin expression and lens differentiation. Current Eye Research, 8(3), 285-291
    3. Jacob, T.J.C. (1987) Human lens epithelial cells in culture: a quantitative evaluation of growth rate and proliferative capacity. Experimental Eye Research, 45, 93-104
    4. Lipman, R.D. & Taylor, A. (1987) The in vitro replicative potential and cellular morphology of human lens epithelial cells derived from different donors. Current Eye Research, 6(12), 145-157
    5. Reddan, J.R., McGee, S., Goldenburg, E.M. & Dziedzic, D.C. (1982/1983) Both human and newborn rabbit lens epithelial cells exhibit similar limited growth properties in tissue culture. Current Eye Research, 2(6), 399-405

IP-57 July 1992