General

Growth on dog paw pad

Growth on dog paw pad

Growth on dog paw pad explants is an objective method for determining a test chemical's activity, and is useful for screening potential therapeutic agents. The assay is based on the stimulation of cell division in explanted hair follicles and sebaceous glands of the dog. It is more sensitive than the mouse ear test and the rabbit eye test and is particularly useful for agents with low toxicity.

Excisional biopsies of skin or mucosal tissues are placed in a moist chamber and treated with test chemicals. Excisional biopsies (tissue explants) may be placed into individual wells of a multi-well plate or into a flat-bottom 96-well microtiter plate. Growth is allowed to proceed for 2 to 14 days, depending on the compound being tested.

Tissue explants are generally composed of epidermis, dermis and/or sebaceous glands. Growth is determined by a direct method, in which the explants are cultured in an appropriate media containing an appropriate enzyme, e.g., adenosine triphosphatase (ATPase), which is specific to a given tissue type and which promotes the growth of that particular tissue. A more sensitive method is to monitor cell proliferation through the incorporation of 3H-thymidine. Cell proliferation is evaluated by determining incorporation of radioactive thymidine (3H-Tdr) into the cell DNA.

The following method describes the preparation and growth of explants in 96-well microtiter plates.

Excisional biopsies are excised from the dorsal neck of the hairless (HR) mouse, the dog, or the human. In general, the biopsies may be taken from a site which is easily accessible, is unlikely to cause pain to the patient, and contains a large amount of tissue. The biopsies are excised using an 8 mm diameter biopsy punch. The biopsy is placed on a 0.4 micron cellulose membrane filter mounted on a 96 well culture plate, and is held in place with a biopsy clip. The biopsies are then covered with skin equivalent culture medium, consisting of an extract of porcine stratum corneum. The well is incubated at 37.degree. C. for 8 hours to allow the tissue to settle to the bottom of the well, and then the membranes are cut off. The contents of the well, along with the stratum corneum extract, are removed and added to a well of a 96 well tissue culture microtiter plate. The resulting cell suspension in the stratum corneum extract is incubated at 37.degree. C. for 24-48 hours.

The cell suspension is rinsed once with phosphate buffered saline and incubated for an additional 24-48 hours in fresh media containing 10% fetal bovine serum and 2.5% human epidermal growth factor (HEGF). The cells have the appearance of a single layer of epithelial cells with cell-cell junctions at the periphery. An intact biopsy may contain from 100-200 xcexcm of tissue. Because the biopsy is removed prior to cell culture, the biopsy contains a significant amount of material not used in this assay, including fibroblasts, endothelial cells, smooth muscle cells, nerve cells, blood vessels, and keratinocytes, and the amounts of each cell type may vary from biopsy to biopsy. The tissue is also damaged by the time of the biopsy which may have been done a significant length of time before it was processed for cell culture. As a result of this, the cells being tested for potential usefulness in cell based assays or as a cell or tissue source for cell based assays are not a homogeneous culture, but contain a mix of cell types derived from the biopsy.

There are a number of problems with the current approach. When an epithelial tissue such as a skin biopsy is used for cell culture, the presence of contaminating stromal cells will affect the results of any assay being performed using the cultured cells. Additionally, the epithelial cells obtained from the biopsy do not proliferate in the culture medium in a uniform manner. This leads to clumps of cells which may have significant numbers of stromal cells and which are difficult to dissociate into a homogenous single cell population. Furthermore, when a large biopsy is removed from a subject, the skin may be under substantial tension. As a result, the removal of the biopsy may actually stretch the epithelium of the skin, causing a change in the cell morphology of the keratinocytes. This is particularly true of samples taken from the palms and soles of the feet.

The biopsies of the present invention solve these problems by providing a large area of non-skin tissue which may be removed from a patient, and which provides a high yield of epidermal cells suitable for a variety of applications. The large surface area of this tissue also ensures that a large number of the keratinocytes will remain viable, by being away from the site of the injury.

The present invention is based on the finding that epidermal cells, including all cells of the epidermis, can be cultured directly from a large biopsy of non-skin tissue such as the epiglottis.

It is the object of the present invention to provide an improved biopsy tissue which will be less traumatic to the patient than a skin biopsy, yet will provide a greater yield of epithelial cells and other cells from which to derive various applications.

This object and other objects and advantages of the invention will be apparent from the following description.


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