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Protocol no. 27

HUMAN SKIN FIBROBLAST/COLLAGEN LATTICE CYTOTOXICITY TEST

Skin fibroblasts are incorporated into 3-D collagen lattices containing the test compounds. An inhibition of lattice contraction indicates a possible toxic effect which is verified by trypan blue exclusion for cell viability.

CONTACT

Dr. Graham Priestley Department of Dermatology Level 4, Phase 1 The Royal Infirmary Edinburgh, EH3 9YW UK

RATIONALE

The incorporation of skin fibroblasts into a 3-D lattice of collagen fibres constitutes a more realistic model of the living dermis than that presented by monolayer cultures of the same cells. The slower proliferation and metabolism of such cells appear to mimic more closely the events occurring in living skin. One of the main characteristics of fibroblasts is their ability to contract a collagen lattice by reorganising collagen more compactly and expelling fluid by a process which resembles wound contraction in vivo. This model, therefore, offers a useful model for both toxicological and pharmacological studies.

BASIC PROCEDURE

Human skin fibroblasts are cultured in collagen-supplemented DMEM in 6-well plates to form lattices. Varying concentrations of test compounds are incorporated into the lattices which are incubated at 37°C for up to 5 days. Lattice diameter is determined daily using graph paper. Inhibition of the contraction of the collagen lattice gives an indication of the deleterious effect of the compound under test.

CRITICAL ASSESSMENT

Human monolayer cultures of fibroblasts offer advantages over the situation in vivo because they eliminate variables in testing with the result that a number of drug therapies may be compared in identical sister cultures of physiologically normal cells. However, there are vast contrasts between the crowded 2-D array of rapidly proliferating fibroblasts and the sparsely populated living dermis. To a certain extent this can be overcome by the culture of skin fibroblasts in a matrix of collagen producing a lattice which provides a more realistic geometric model of the skin.

TEST STATUS

Developed in-house

CHEMICALS TESTED

Arotinoid (Ro-15-0778) Betamethasone valerate Chlorhexidine acetate Clobetasone butyrate Clobetasol propionate Cycloheximide Dexamethasone Dibutyryl cAMP D-penicillamine Etretin (Ro 13-1670) Griseofulvin Hydrocortisone Minoxidil P-1894B (Streptomyces albogriseolus anthraquinone glycoside) Phenytoin Prostaglandin E2 13-cis retinoic acid all-trans retinoic acid, Sodium salicylate Vitamin C Vitamin E ORGANISATIONS USING THE SYSTEM None at present

REFERENCES

  1. Adams, L.W. & Priestley, G.C. (1986) Contraction of collagen lattices by skin fibroblasts from dystrophic recessive epidermolysis bullosa and other dermatoses. J. Invest. Dermatol., 87, 544-546. Adams, L.W. & Priestley, G.C. (1988) Contraction of collagen lattices by skin fibroblasts: drug-induced changes. Arch. Dermatol. Res., 280, 114-118.
  2. Diegelman, R.F. & Peterkofsky, B. (1972) Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs. Proc. Nat. Acad. Sci., 69, 892.
  3. Elsdale, T. & Bard, J. (1972) Collagen substrata for studies of cell behaviour. J. Cell Biol., 54, 626-639.
  4. Priestley, G.C. & Adams, L.W. (1983) Hyperactivity of fibroblasts cultured from psoriatic skin. I. Faster proliferation and effect of serum withdrawal. Br. J. Dermatol., 109, 149-156.
  5. Priestley, G.C. (1987) Toxicity of the anthraquinone glycoside P-1894B for human skin fibroblasts. British Journal Dermatology, 117, 67-72.
  6. Priestley, G.C. (1986) Skin fibroblast monolayers: A suitable case for treatment? In: Skin Models. (eds. Marks, R. & Plewig, G.) Springer-Verlag, Berlin-Heidelberg, pp297-304. IP-27 © March 1990