Sex steroid exert a multitude of effects with humans. They regulate reproductive functions, bone metabolism and neural functions etc. They have also implications to many diseases like cancers (mammary, prostate, uterine), osteoporosis, uterine functional disturbances and postmenopausal symptoms. Steroid action is an important target for drug development.

There are numerous naturally occurring and synthetic chemical compounds that mimic the functions of steroid hormones and which could be hormonally active (e.g. endocrine disrupting compounds).

This is why models that mimic hormonal functions in humans are important for drug development and toxicity testing. The tests can also be used to study  reproductive toxicity.

Endometrial model

Aim. Together with hormonal signals, stromal-epithelial interactions are fundamental to normal endometrial growth and function.  The results from monocultures of uterine epithelial cells, both rodent and human, imply that estradiol may not have a direct mitogenic effect on these cells.  That stromal cells are not only involved in epithelial mitogenesis but also modulate the functional differentiation of epithelial cells has been shown in a number of studies. Reciprocal signals from uterine epithelium to the underlying stroma further emphasize the complex nature of  regulatory interactions in endometrial tissue.   In view of the above it is evident that any culture model intended to mimic normal endometrial responses should include both the epithelial and stromal  compartments of the tissue.

Principle. We have established an organotypic model in which epithelial cells of the normal human endometrium are cultivated as glandular organoids within Matrigel matrix in co-culture with stromal cells seeded on plastic.

Picture 1. Human in vitro endometrial model

Status. In the culture model the organoids maintained their glandular morphology and epithelial phenotype throughout the experimental period as indicated by their in vivo-like expression of cytokeratin 18 and E-cadherin. We have shown that estradiol and progestin exerts in vivo-like effects on epithelial cell proliferation as well as on the regulation estrogen receptor and progesterone receptor expression in this system. Results with selective estrogen receptor modulators (SERM) tamoxifen and raloxifen indicate that tamoxifen is a partial agonist and raloxifen an antagonist to the action of estradiol in the culture model.

The present organotypic model provides a novel in vitro system in which to study the effects of steroids in the normal human endometrium both in terms of cell proliferation and gene expression. The culture system holds promise as a useful method to tissue profile novel steroid compounds.

Mammary gland model

Aim. Ovarian steroid hormones are known to have crucial roles in development and maintenance of normal function of human mammary gland. Estrogen and progestins have further been implicated in breast carsinogenesis. In order to study the possible effects of steroids and steroid like molecules on normal human resting mammry gland, an in vivo culture method was developed based on normal human mammary gland tissue. A large number of studies have shown the importance of stromal-epithelial interaction for the proliferation and differentiation of epithelial cells. Here we have selected a tissue explant culture method in order to preserve the tissuearchitecture.

Principle.We use an absorbable gelatine sponge as a tissue support during the culture. Small pieces of of mammary gland tissue is placed on the top of the sponge and cultured for over one month.

Status. Human mammary gland tissue cultured in this system maintain an excellent morphology and expression of structural protein makers (keratins, vimentin, alpha-actin) and steroid receptors (estrogen receptor, progesterone receptor, androgen receptor) for minimum one month in culture. Regulation of the expression of estrogen recptor, progesterone receptor and androgen receptor

Prostate model

Aim. To facilitate an understanding of the genesis of prostatic disease and to aid the development of new preventive and therapeutic regimens, in vitro models allowing experimentation on normal and pathologic prostate tissue are needed. Since the organogenesis and the differentiated function of the prostate tissue are highly dependent on androgens, maintenance of the androgen signaling mechanism is a prerequisite of any culture model intended to mimic normal prostatic responses in vitro. It has been shown that monocultures of primary prostatic epithelial cells derived from normal or BPH tissues tend to share the features of transit amplifying cells and the challenge remains to create culture conditions that preserve the differentiated phenotype of the secretory epithelium intact. The use of primary epithelial cell cultures as models of normal prostate biology is hampered by a rapid loss of functional androgen receptor in epithelial monocultures, accompanied by a loss of androgen-sensitive cell growth and gene expression.

It is well established that stromal-epithelial interactions are critical for mediating the effects of androgens on the epithelial cells within prostate glands. There is also evidence that stromal components may modulate the initiation and progression of cancer. Therefore, both the epithelial and stromal compartments of the tissue should be included when aiming to model the normal function or pathobiology of the prostate in vitro. A novel tissue slice culture model was developed for maintaining the differentiated morphology and protein expression of normal human prostate in vitro.

Principle. The model is based on aseptic preparation of precision-cut slices of normal or benign prostate hypertrophy tissue and their cultivation in dishes designed to support an optimal gas exchange in static culture. Precision-cut tissue slices represent an in vitro model in which the functionally important features of the tissue of origin, multicellularity together with cell-cell and cell-matrix interactions, are well preserved. We have also introduced novel mixture of serum-free media that is able to preserve excellent tissue viability up to a minimum of 7 days.

Status. Differentiated prostatic secretory cells express androgen receptor (AR) together with a number of androgen-regulated proteins including prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP). An analysis of cytokeratins, AR, PSA and PAP was used to characterize the culture method. The glandular cytoarchitechture was well preserved up to minimum 7 days. Androgen supplementation improved the preservation of the columnal or cuboidal secretory cell phenotype. Glandular cells and stromal cells maintained their capacity to express AR. PSA and PAP were shown to be expressed through out the culture period. The expression pattern and regulation of their expression by steroids in the culture model is in agreement with the normal mammary gland in vivo. The proliferation of the epithelial cell in the culture system is under estrogen regulation.

The present culture system provides an in vitro setting in which to study normal human prostate biology and pathobiology and may help to obviate problems related to the use of established cancer cell lines.