Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of co-culturing cells
Reexamination Certificate
2001-01-31
2002-11-05
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of co-culturing cells
C435S347000, C435S383000, C435S357000, C435S372000, C435S004000
Reexamination Certificate
active
06475790
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an in vitro assay of angiogenesis and in particular a multicellular in vitro assay of angiogenesis.
BACKGROUND OF THE INVENTION
Most populations of differentiated cells in vertebrates are subject to turnover through cell death and renewal. Some fully differentiated cells such as hepatocytes in the liver and endothelial cells lining the blood vessels simply divide to produce daughter cells of the same differentiated type. The proliferation rate of such cells is controlled to maintain the total number of cells. Thus if a large part of the liver is destroyed then the remaining hepatocytes increase their division rate in order to restore the loss.
Endothelial cells form a single cell layer that lines all blood vessels and regulates exchanges between the blood stream and the surrounding tissues. New blood vessels develop from the walls of existing small vessels by the outgrowth of these endothelial cells which have the capacity to form hollow capillary tubes even when isolated in culture. In vivo, damaged tissues and some tumors attract a blood supply by secreting factors that stimulate nearby endothelial cells to construct new capillary sprouts. Tumors that fail to attract a blood supply are severely limited in their growth.
The process whereby new vessels originate as capillaries, which sprout from existing small vessels, is called angiogenesis. It can therefore be seen that angiogenesis plays a major role in normal tissue development and repair and in the progression of some pathological conditions.
Once the vascular system is fully developed, endothelial cells of blood vessels normally remain quiescent with no new vessel formation. If disease or injury occurs, the formation of new blood vessels can proceed normally, as in natural wound healing, or be insufficient, as in chronic dermal ulcers, or there is deregulation of growth and an abnormal increase in vessel density ensues as in tumorogenesis, diabetic retinopathy, psoriasis and inflammation. Inhibition of inappropriate angiogenesis or enhancement of angiogenesis in non-healing wounds is therefore an extremely important target for drug discovery programs. However, research in this area leading to new drug development has been hindered by the lack of in vitro models of angiogenesis.
Angiogenesis is an extremely complex process involving a wide range of growth factors, extracellular matrix molecules, enzymes and various cell types. Such a complexity of relationships has resulted in major difficulties in developing an in vitro assay which models the entire in vivo process. Angiogenesis can be subdivided into three phases: proliferation, migration and differentiation. Assays exist which model each of these phases separately. Simple in vitro tests measure changes in proliferation of a range of cell types and assess migration over basement membrane proteins. Current in vitro assay systems, which depend on provision of a protein matrix, effectively measure the ability of endothelial cells to differentiate. Assay systems measuring differentiation involve the formation of cord-like structures by endothelial cells. All such systems depend on supplying the cells with exogenous basement membrane proteins on which the cells migrate to form tubules. Cell migration occurs over relatively short time periods of 2-16 hours to give a three dimensional structure. In addition to the basement membrane proteins, many of the systems require the provision of growth factors to produce acceptable tubule formation. The time scale over which tubules are formed provides an excellent test for inhibition of differentiation but is not so useful when testing for enhancement.
The assay systems described above come closest to modeling angiogenesis but none of them combine all three of the stages required for angiogenesis.
SUMMARY OF THE INVENTION
The object of the present invention is to obviate or mitigate the aforesaid disadvantages of current methodologies by providing an in vitro assay of angiogenesis which encompasses the three stages of angiogenesis and which can be used to examine both stimulation and inhibition of angiogenesis.
One aspect of the present invention relates to a multicellular in vitro assay for modeling the combined stages of angiogenesis, namely, the proliferation, migration and differentiation stages of cell development. The assay provides for a dual culture comprising endothelial cells together with another cell-type which will interact with the endothelial cells to display the combined stages of angiogenesis in vitro. The multicellular in vitro assay for modeling the combined stages of angiogenesis comprises:
(i) providing a culture of endothelial cells;
(ii) adding to the culture of endothelial cells another cell-type exhibiting interaction therewith; and
(iii) growing a dual culture of endothelial cells and another cell-type to display the combined stages of angiogenesis in vitro.
When the other cell type exhibiting interaction with endothelial cells is a fibroblast, the resulting dual culture of fibroblasts and endothelial cells requires no additional growth factors other than those present in standard culture medium. It is postulated that the interaction of these cell types is dependent on cell signaling mechanisms therebetween. The non-reliance on additional growth factors is remarkable and unanticipated considering past research on the subject. In this aspect the invention can be characterized as a multicellular in vitro assay for modeling the combined stages of angiogenesis comprising the steps of (a) setting up growth containers suitable for sustaining dual cell cultures and having a suitable culture medium for sustaining at least growth of endothelial cells therein; (b) seeding a dual culture of human fibroblasts and human endothelial cells into the growth containers; (c) incubating the dual culture, without the provision of any exogenous growth factors, from an initial proliferation phase until a confluent monolayer is produced. The growth medium is replaced at regular intervals, such as every 48 hours, to achieve optimal development.
In a related aspect, the present invention relates to a method of screening agents for their ability to promote or inhibit angiogenesis. The method comprises co-culturing endothelial cells with another cell-type, for example, interstitial cells such as fibroblasts, which will interact to display the combined stages of angiogenesis, and introducing the agent to be screened in controlled amounts to the cultures and monitoring angiogenesis in the cultures. The screening method may be readily automated and angiogenesis may be monitored by known automated counting techniques, image analysis or by spectrographic methods. The multicellular in vitro assay for screening agents that alter angiogenesis comprises: (a) cultivating in a plurality of test containers a plurality of co-cultures of endothelial cells together with another cell-type exhibiting interaction therewith to display the combined stages of angiogenesis; (b) adding an agent to at least one of the cultures; (c) allowing the plurality of cultures to grow; and (d) comparing the culture containing the agent to another of the plurality of cultures not containing the agent.
In yet another aspect, the invention relates to an assay kit including a vessel provided with culture medium appropriate for sustaining fibroblasts and endothelial cells, and seeded with said cells as a dual culture, wherein the cells are preferably Human Adult Dermal Fibroblasts and Human Umbilical Vein Endothelial Cells (HUVEC) respectively. The kit is useful in a multicellular in vitro assay for modeling the combined stages of angiogenesis. It comprises: (a) a vessel; (b) a culture medium appropriate for sustaining fibroblasts and endothelial cells within the vessel; and (c) a dual culture of fibroblasts and endothelial cells within the vessel.
In one embodiment the assay comprises the steps of (a) setting up growth containers suitable for sustaining dual cell cultures and having a suitable culture medium for sustaining at
Bell Graham T.
Bloor Stephen
Grant Eileen T.
Biocure Limited
Caputa Anthony C.
Heslin Rothenberg Farley & & Mesiti P.C.
Nickol Gary B.
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