Tissue equivalent test systems

Abstract: The present invention provides methods of, apparatus for, and kits for determining the interaction of tissue and at least one agent by use of at least one tissue equivalent. Tissue equivalent includes, but is not limited to, equivalents of epithelial tissue, connective tissue, cartilage, bone, blood, organs, glands and blood vessels, which are composed of living cells and extracellular matrix molecules, principally collagen. Agent includes, but is not limited to, various substances such as chemicals, cosmetics, pharmaceuticals, stimuli, e.g., light or physical injury; and tissue protective agents.

Description: BACKGROUND OF THE INVENTION Many tests have been devised to determine the interaction of various agents and tissues of humans and animals. The effects of chemicals found in the environment, both on man and animals, is of widespread concern. The effects of new drugs, both veterinarian and human, are routinely tested in accordance with federal regulations. Chemical companies, petroleum and paint companies, pharmaceutical companies, and cosmetic companies use test systems to assay the reaction of skin to the substances they use and produce. In addition, biomedical laboratories in pharmaceutical companies, hospitals, and universities use test systems for the study of disease mechanisms and for the evaluation of treatment procedures. At present, systems for determining the interaction of tissues and agents include (i) experimental animals (mainly rodents and rabbits), (ii) monolayer cultures of human cells, (iii) tissue slices or organs from cadavers, and (iv) mathematical models developed to simulate biological responses. Each of these test systems has its advantages and its shortcomings. Experimental animals (excluding human subjects) have been widely used. Because the cells and tissues of these animals are different from those of humans the use of experimental animals to determine the effect of various agents on man is limited. Furthermore, experimental animals are expensive to maintain, and there are ethical considerations associated with the use of animals for such purposes. Cultures of cells are highly reproducible, inexpensive, well standardized test systems, but they do not mimic the state of cells and tissues in the organism. As a consequence, the biosynthetic activities and physiologic functions expressed by cells grown in monolayer cultures are markedly different from those in the organism, thus yielding misleading test findings. Tissue slices from cadavers can provide both the complexity as well as the normal biosynthetic output and cell properties needed for a test system capable of mimicking human responses; however, they are moribund. Some cells are alive, others are dying, and many are already dead. This limits their usefulness since, for example, in a toxicity assay, it may be difficult to distinguish between the effects of the test substance and the natural degenerative changes occurring in the cadaver. Mathematical models are useful when responses are well understood and predictable and when the full range of variables is defined, but they are not appropriate for testing new substances. From the point of view of human health protection, the ultimate test organism is of course the human; however, human testing is subject to stringent limitations. Animals are widely used in testing because they can be dissected and probed invasively, and because they can be used for substances known to be toxic to humans; however, as mentioned previously their responses do not necessarily reflect human responses. The skin, a very important tissue, is the principal barrier between the organism's internal milieu and the chemical and physical world without. It is thus subject to the ravages of the environment. It is exposed to agents, such as, chemicals and antigenic substances, in the workplace, in the home, and in the atmosphere generally. Medicaments are applied to the skin both for the treatment of systemic conditions by topical therapy, as well as for the management of wounds and numerous disorders that afflict the skin itself. The skin is treated cosmetically to improve its appearance and sometimes its health. Today there is broad concern with the necessity of establishing safe practices to protect the individual against the effects of intrusive and injurious substances that come into contact with the skin and to evaluate the effects of cosmetic and remedial emollients that are applied to it. Thus, it is not surprising that many tests have been devised to determine the interaction of various substances and human skin. Skin testing on humans is limited primarily to tests of a "benign" character dealing with sensitization. For example, when human subjects are used to evaluate the effect of test substances on the skin, the skin responses monitored are usually erythema and edema. These are gross manifestations of complex processes that have well defined immunochemical, biochemical, and physiological counterparts at the cellular level. To analyze for such effects requires invasive procedures that are frequently inappropriate. Although excised cadaver skin has been used for skin testing, it is not readily available and it rapidly becomes moribund. As it degenerates, the skin loses its capacity to respond normally, that is, to emit signals or to metabolize foreign substances. Thus, it becomes impossible to distinguish between effects due to the substance being tested and those due to autolysis and deterioration of the organ in vitro. See, e.g., Bronaugh and Stewart, J. Pharm. Sci. 74: 64-67 (1985) and Franz, J. Invest. Dermatol.: 190-195 (1975). The hirsute skin of experimental animals differs fundamentally from the skin of humans in its morphology, its physical properties, and its reactions to allergenic and other stimuli. For example, the rates of percutaneous absorption of animal skin differ considerably from those of human skin. Although animals will continue to be used to determine LD50 values and the responses to toxic substances of internal organ systems, for many other toxicity studies alternatives to animal testing are being sought, both for ethical reasons as well as for the development of more effective tests (See, e.g., Alternatives to Animal Use in Research, Testing, and Education. Office of Technology Assessment. Washington, DC (1985)). Although cell cultures have many uses as test systems, it has been rigorously shown that the cells grown in monolayer cultures exhibit neither the same biosynthetic repertoire nor the same permeability properties as cells in the organism, nor are they organized or differentiated in the same manner as cells in a tissue or organ. In EP-A-0 171 896 there is disclosed a method and apparatus for assaying the sensitivity of biopsied cells to therapeutic agents wherein cells are cultured in a vessel which is a cell culturing apparatus, and then the cells are contacted with a fluorogenic substrate. There is disclosed a membrane which divides the cell culturing vessel into separate chambers and which allows nutrients to perfuse across the membrane to feed the cells in culture. In WO-A-84/003047 there is disclosed a method and apparatus for assaying the sensitivity of biopsied cells to therapeutic agents wherein cells are cultured in a culturing apparatus which consists of an artificial organ, and then the cells are contacted with a fluorogenic substrate. The document discloses membranes which divide the cell culturing device into chambers and allow nutrients to perfuse across the membrane to feed the cells in culture. Thus, alternatives to animal testing and cell culture test systems are being sought. Equivalents of tissue that reproduce in vitro many of the physical and biological characteristics of natural tissues would be useful for the study of the tissue cell biology, physiology and pathology. SUMMARY OF THE INVENTION The present invention provides methods and apparatus for determining the response or lack of response of tissue and at least one agent by use of at least one fully formed living skin tissue equivalent. Tissue/agent response or lack of response which may be determined in accordance with the present invention, include but are not limited to, the passage of the agent into or through the tissue equivalent; the production or release of one or more substances by the tissue equivalent; and a change in permeability, proliferation, differentiation, or configuration of cells of the tissue equivalent. In some embodiments the response of the tissue and the agent serves to protect the tissue. The agents tested include, but are not limited to, various stimuli, e.g., light, physical injury, and various substances, e.g., chemicals, cosmetics, pharmaceuticals and tissue protective agents. The composition and configuration of the tissue equivalent will be selected in light of the nature of the response to be studied and limitations of the assay procedure used to determine the interaction. A tissue equivalent may be cast in any desired configuration. The present invention comprises a method of determining the response or lack of response of tissue to at least one test agent by use of at least one fully formed living skin tissue equivalent comprising living cells and extracellular matrix molecules, principally collagen, contracted by a contractile agent, wherein the response of the skin tissue equivalent to the test agent comprises the passage of the agent into or through the skin tissue equivalent, or the response of the skin tissue equivalent to the test agent comprises the production or release of one or more substances by the skin tissue equivalent, the method comprising the steps of: (a) contacting the test agent with the skin tissue equivalent, wherein the skin tissue equivalent is adjacent to a liquid phase; and (b) determining the response or lack of response of the skin tissue equivalent and the agent by analyzing at least one of (I) the skin tissue equivalent, (ii) an intracellular fluid of the skin tissue equivalent, or (iii) the liquid phase. For example, the present invention comprises a method wherein the skin tissue equivalent has an epidermal and dermal layer, the dermal layer of the skin tissue equivalent being adjacent to the liquid phase; in some embodiments of this method the test agent is contacted with the epidermal layer of the skin tissue equivalent. In other methods provided by the present invention, at least one fully formed living tubular skin tissue equivalent is used to determine the interaction of tissue and at lease one agent, the method comprising: a. contacting the agent with the lumen or abluminal surface of the tubular tissue equivalent, such contact being effected by providing a liquid phase adjacent to the lumen or the abluminal surface of the tubular tissue equivalent, and introducing the agent into the liquid phase; and b. determining the interaction of the tubular tissue equivalent and the agent by analyzing at least one of (i) the tubular tissue equivalent, (ii) the intracellular fluid of the tubular tissue equivalent, or (iii) the liquid phase. The present invention also provides an apparatus for determining the response or lack of response of tissue to at least one test agent by use of at least one fully formed skin tissue equivalent, the apparatus comprising a fully formed living skin tissue equivalent comprising living cells and extracellular matrix molecules, principally collagen, contracted by a contractile agent, and a container for the skin tissue equivalent, the container comprising: (I) means for positioning the tissue equivalent in the container, whereby the tissue equivalent defines at least one region in the container; ii) at least one port; and iii) means for closing the container. The apparatus of the invention may include a liquid phase. The tissue equivalent included in apparatus of the present invention is preferably positioned in the container so that it defines at least two regions in the container. In some embodiments the tissue equivalent defines an upper and a lower region in the container. In yet other embodiments wherein the tissue equivalent is a tubular tissue equivalent, the tubular tissue equivalent is positioned so that it defines an inner and an outer region in the container. In some embodiments, the container is further provided with one or more liquid phases. Various means for positioning a tissue equivalent in a container are taught by the present invention. In some embodiments of the present invention, the means for positioning a tissue equivalent in the container is disposed in the container and comprises a permeable member. In yet other embodiments of apparatus according to the present invention, the tissue equivalent is a tubular tissue equivalent, and the means for positioning the tubular tissue equivalent in the container comprises (i) means for attaching the tubular tissue equivalent to the means for positioning the tubular tissue equivalent in the container, (ii) means for limiting the longitudinal contraction of the tubular tissue equivalent, and (iii) means for allowing selected materials to pass between the tubular tissue equivalent and at least one liquid phase. The means for positioning the tissue equivalent may also serve as a support member for the tissue equivalent. Furthermore, in some embodiments, the tissue equivalent may be cast on the means for positioning the tissue equivalent if desired. In preferred embodiments, the tissue equivalent is positioned so that it defines at least two regions. The apparatus may be further provided with one or more liquid phases. In preferred embodiments of the present invention, the apparatus is provided with two or more individual containers. In other embodiments the containers are interconnected so that the liquid phase is common, to each tissue equivalent. One or more reagents for use in determining the interaction of the tissue equivalent and the agent are optionally included in the instant invention. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a cross-section through the center of one apparatus according to the present invention. Fig. 1B is a diagrammatical view of another apparatus of the present invention. Fig. 2 is a cross-section through the center of a container according to the present invention. Fig. 3 is an isometric view of a means for positioning a tubular tissue equivalent together with a cover means both in accordance with the present invention. DETAILED DESCRIPTION OF THE INVENTION The tissue/agent responses determined in accordance with the present invention include the myriad of responses normal tissues are subject to. These responses include, but are not limited to, the rate and extent of penetration of agents into or through the tissue, changes in tissue permeability, the release of one or more substances by a tissue into its intracellular tissue fluids, the effect on tissue metabolism or cell proliferation or differentiation, and reorganozation of the cells of the tissue. In some embodiments of the present invention, the response determined comprises the effect of the tissue on the agent, e.g., where a tissue breaks down an agent. In other embodiments, the agent is a nutrient or precursor and the response is the synthesis or production of a substance. In yet other embodiments, the response determined may be the protection conferred on a tissue by one or more agents. In yet other embodiments of the present invention, more than one agent is used to determine the agent/tissue response or lack of response, e.g., the response of a tissue and a first agent may be determined by use of a second agent. For example, a tissue equivalent is contacted with a first agent. A second agent is subsequently contacted with the tissue equivalent, and the first agent prevents or reduces the response of the tissue equivalent and the second agent. A fully formed skin tissue equivalent as used herein comprises living cells and extracellular matrix molecules, principally collagen, contracted by a contractile agent. See, for example, U.S. Patent No. 4,604,346. Tissue equivalents for use in accordance with the present invention may optionally be provided with components not typically found in normal tissue. Tissue equivalents for use in the present invention are populated with cells that can remain alive for long periods and can be produced in quantity with the assurance that all units fabricated will be essentially uniform. Cells in the tissue equivalents used in accordance with the present invention resemble those of normal tissue in their structural arrangement, in their biosynthetic output, and in their permeability. It should be understood that tissue equivalents for use in the present invention need not be human but may be those of any animal as desired. In some embodiments of the present invention it is desirable to provide tissue equivalents with protective means, e.g., by disposing a removable means for protecting the tissue equivalent on exposed surfaces thereof. A thin, flexible film, e.g., of a plastic, is acceptable for such applications. Human skin tissue equivalents used in the practice of the present invention permit the growth of normal human epidermal cells that differentiate fully producing a normal stratum corneum and a complete basal lamina which have not, to date, been obtained by routine culture methods. Such skin tissue equivalents have been extensively used as a permanent skin replacement in animal experiments and recently in initial human trials in France; the morphological appearance of such skin tissue equivalent is normal, its constituent cells persist after grafting as shown by genetic marking, and its functional performance has been demonstrated. See, e.g., Science, 21: 1052-1054 (1981); J. Invest. Dermatol. 81: 2s-10s (1983). Skin tissue eqivalent fabricated in vitro bears a close resemblance to natural skin. It consists of a multilayered epidermis with well developed basal cells joined to the dermal layer by a fully structured basal lamina. The dermal layer is a collagen matrix in which dermal fibroblasts are distributed. Cells in the three-dimensional collagen matrix achieve a state of differentiation in many respects similar to that which prevails in vivo . For example, fibroblasts are synthetically active and enrich the matrix in vitro with collagen, as well as with a number of other molecular species, and exhibit permeability properties typical of a cell in vivo . See, e.g., Collagen Rel. Res. 4: 351-364 (1984). The effects of steroids on the capacity of human and rat fibroblasts to contract tissue equivalent lattices has been evaluated ( J. Invest. Dermatol. 82: 341-344, 1984). A skin tissue equivalent model has been used to fabricate tissues with psoriatic and normal cells for the study of the disease psoriasis ( Science, 230: 669-672, 1985). Recently it has been shown that skin tissue equivalents can be pigmented by inclusion of melanocytes that donate pigment to keratinocytes and that the process is speeded up in vitro by UV radiation ( J. Invest. Dermatol. 87: 642-647, 1986). The configuration of apparatus according to the present invention will depend upon the tissue equivalent used as well as the nature of the interaction to be determined. Tissue equivalents for use in the present invention are generally cast as a flat sheet, a hollow tube or a network of hollow tubes. However, they can be cast in any desired shape. For example, in some embodiments of the present invention, it is desirable to change the natural geometry of the tissue equivalent. For example, skin tissue equivalent may be cast as a cylinder rather than as a sheet and the layers of blood vessel tissue equivalent may be cast in the reverse of the order of natural blood vessels. The present invention provides apparatus for determining the interaction of tissue and at least one agent by use of at least one tissue equivalent, the apparatus comprising a container for the tissue equivalent, the container comprising (i) means for positioning the tissue equivalent in the container, whereby the tissue equivalent defines at least one region in the container; (ii) at least one port; and (iii) means for closing the container. In preferred embodiments of the present invention the tissue equivalent defines at least two regions in the container. Some embodiments of the instant invention further comprise cover means for closing the container. In some such embodiments the cover means is removably sealable to the container. In some embodiments the tissue equivalent defines an upper and lower region in the container, the lower region further comprising a liquid wherein one surface of the tissue equivalent is adjacent to the liquid. In some such embodiments of the present invention, the upper region is further provided with a second liquid phase. The present invention also includes within its scope apparatus comprising a tubular tissue equivalent, wherein the tubular tissue equivalent defines an inner and outer region in the container, the inner region comprising a first liquid phase and the outer region comprising a second liquid phase. Referring to the drawings, Fig. 1A illustrates one embodiment of an apparatus according to the present invention for determining the interaction of skin and one or more agents by use of skin tissue equivalents, the apparatus comprising a container 10, the container providing means for positioning the skin tissue equivalent 60, a lower chamber 20 and an upper chamber 30, the chambers being sealably connected by means of an upper gasket means 40, a lower gasket means 41 and connecting means 50, 51. This embodiment is provided with a skin tissue equivalent 60, removably positioned between the lower chamber 20 and the upper chamber 30. The skin tissue equivalent 60 comprises two layers, layer 62 comprising an epidermal layer, layer 64 comprising a dermal layer. In this embodiment the container 10 is provided with cover means 70 which is placed over the upper chamber 30, and the lower chamber is provided with ports 80, 90. In the embodiment show in Fig. 1A, the container is provided with sealably connected lower and upper chambers 20 and 30 to provide means for postioning the skin tissue equivalent 60 in the container 10. Other means may be used for positioning a tissue equivalent in the container 10 such as positioning the tissue equivalent in the container by means of a permeable support member disposed in the container, wherein the tissue equivalent attaches to or is cast on the permeable support member. One such embodiment of the present invention is shown in Fig. 1B. Elements similar to those in other described embodiments are indicated by the same numeral. This embodiment comprises a container 10 defining a holder for a skin tissue equivalent 60, container 10 having disposed therein a permeable support member 65, the support member 65 providing means for positioning the skin tissue equivalent 60 in the container 10. This embodiment further comprises a skin tissue equivalent 60, further defining a lower chamber 20 and an upper chamber 30. In this embodiment, the container 10 is provided with a cover means for closing the container 70, which is placed over the upper chamber 30, and the lower chamber is provided with ports 80, 90. Figs. 2 and 3 show yet another apparatus according to the present invention. Elements similar to those in other described embodiments are indicated by the same numeral. The apparatus comprises a cylindrical container 10 for a tubular tissue equivalent, the container having two ports, 81, 82, at the bottom, a hollow mandrel 100, the upper end of the mandrel being provided with a port 71, the lower end of the mandrel being open, and a cover means 70 for closing the container 10, the cover means being sealably connectable to the mandrel 100. The inner surface 12 of the container 10 comprises an inert, non-wettable material. The container 10 is threaded to accept the cover 70 for removably sealing the container 10. The cover means 70 is provided with a port 72. The port 72 may also serve as a vent during filling or a separate vent 73 may be provided. The container 10 is further provided, at the bottom thereof, with a means (not shown) for sealing the mandrel 100 to the base of the container. In some embodiments, the bottom of the mandrel 100 is provided with an inlet or outlet means which is provided with means for sealing the bottom of the container. The mandrel 100 shown in Fig. 3 comprises a number of regions: region 105 comprising a means for sealably covering the container; regions 101 and 108 comprising means for the cells of the tubular tissue equivalent to adhere to; regions 103 and 107 comprising means for limiting the longitudinal contraction of the tubular tissue equivalent; region 104 comprising a liquid permeable means for allowing selected materials to pass through the tubular tissue equivalent and, in some embodiments for supporting the tissue equivalent; and region 109 comprising means (not shown) for removably sealing the mandrel 100 to the base 11 of the container 10 (e.g., groove for O-ring or screw threads). The container 10 and cover means 70 may be made of any desired material which does not react with or have an undesirable effect on the components of the assay, including the tissue equivalent. In some embodiments, it is desirable that the container 10 be made so that the tissue equivalent is visible through the container, e.g., through the walls of the container or through a window in the container. Preferred materials for the container include glass, polycarbonate, polystyrene, TEFLON® and stainless steel. In yet other embodiments, the inside 12 of the container 10 comprises an inert, non-wettable surface such as TEFLON®, polycarbonate or stainless steel or the inside 12 is coated to make it non-wettable. The container may be of any shape and volume which will accomodate the size and shape of the desired tissue equivalent. The dimensions of the container will again depend upon the size and shape of the desired tissue equivalent and the desired assay volumes. For example, a container having an outer diameter of about 25mm and a volume of about 5ml is useful in practicing the present invention. In some embodiments of the present invention, multiple containers will be provided in a base or holder. In embodiments wherein a permeable support member 65 is provided for positioning the tissue equivalent 60 in the container 10 the permeable support member 65 preferably comprises a membrane or a mesh. Preferred materials include polypropylene, nylon, and polycarbonate. The gasket means 40, 41 may be made of any material which is inert to the conditions of the assay and the tissue equivalent being used, as well as provides a good seal between the upper and lower chambers 20, 30. Preferred materials include silicone and TEFLON®. In the embodiment shown in Figure 1, the upper and lower chambers 20, 30 are attached by screw means 50, 51. It will be appreciated by those of ordinary skill in the art that any suitable connecting means may be used to sealably attach the upper and lower chambers. The apparatus shown in Figs. 1A and 1B are provided with ports so that a liquid phase may be passed into and out of the lower chamber 20. The desired number of ports for the lower chamber will depend upon the assay being conducted. In the embodiment shown in Fig. 3, the cover means 70 for the container 10 is provided with a valve 71, a port 72 and a vent 73. In some embodiments, a vent is not provided, and a port 72 may serve both as a port and as a vent. The port may be used, e.g., for epidermalizing the tissue equivalent or applying test agents. It must be understood that although the apparatus shown in the Figures are provided with cover means 70 for closing the container 10, alternative means for closing the container 10 are acceptable. In alternative embodiments, access to the inside of the container or a tissue equivalent positioned therein is achieved by means of ports or other openings or passages appropriately disposed on the container. These openings or passages will be provided with valve means or other means for closure. In yet other embodiments the means for closing the container may be sealed or fused to the container, e.g., as by heat sealing. In such embodiments access to the container is achieved by removing the sealed or fused means for closing the container. In the embodiment shown in Figs. 2 and 3, the mandrel 100 provides a means for positioning the tissue equivalent in the container 10. In some embodiments of the present invention, the tissue equivalent is cast on the mandrel 100 after the mandrel 100 has been disposed in the container 10. As has been described above, the mandrel 100 is comprised of a number of regions. Preferred materials for regions 105 and 109 are rigid, nonporous and inert, and include plastics such as polycarbonate or polystyrene, TEFLON®, glass or stainless steel or combinations thereof. Regions 101 and 108 comprise means for the cells of the tissue equivalent to adhere to and in some embodiments also provide a seal between the tissue equivalent and the mandrel 100. Preferred materials for regions 101 and 108 include glow discharge treated plastic, collagen or fibronectin-coated glass or plastic. Regions 103 and 107 provide means for limiting the longitudinal contraction of the tissue equivalent. Regions 103 and 107 preferably are highly textured. Texture can be provided, e.g., by providing the region with holes or with numerous fine projections. Preferred materials include, but are not limited to, VELCRO® textured stainless steel, e.g., wire cloth, textured plastics, textured TEFLON® and polyurethane foam. Preferred materials for region 104 include materials which are permeable to small molecules such as nutrients, but are of limited permeability to larger molecules such as collagen and especially to collagen fibrils. Such liquid permeable means include strong but inert materials such as nylon or polypropylene. Pore sizes of about 0.2 to 5µm are useful; pores of 0.5 to 3µm are preferred. In some embodiments support means is provided for the membrane. Preferred materials include a framework or a screen of a rigid and inert material, such as stainless steel, plastics including polycarbonate or polystyrene, TEFLON®. Apparatus according to the present invention may be provided with means for controlling the flow of the liquid phase(s) into and out of the apparatus. Means for controlling the flow of a liquid are well known to those skilled in the art. For example, means for sampling, circulating, exchanging or feeding the liquid phase(s) adjacent to the tissue equivalent may be provided. In the embodiment shown in Fig. 1A, the liquid phase in chamber 20 may be moved through the apparatus by attaching ports 80 and 90 to any appropriate circulatory means known to those skilled in the art. In some embodiments of the present invention, the response or lack of response of skin and at least one agent is determined by the use of at least one fully formed living skin tissue equivalent, the skin tissue equivalent having an epidermal and a dermal layer, the method comprising the steps of: (a) contacting the agent with the epidermal layer of the skin tissue equivalent, wherein the dermal layer of the skin tissue equivalent is adjacent to a liquid phase; and (b) determining the effect of the agent on the skin tissue equivalent by analyzing at least one of (i) the skin tissue equivalent, (ii) an intracellular fluid of the skin tissue equivalent, or (iii) the liquid phase. Apparatus and methods based on human skin tissue equivalent are provided by the present invention for use in determining the response or lack of response of skin and various agents, including, but not limited to, the measurement of: 1) the rate and extent of penetration of substances into and through the skin (Skin Absorption Test); 2) the interaction of agents reflected by changes in cell permeability (Skin Cell Permeability Test); 3) the responses of skin cells to agents that provoke or promote the release of various regulatory or signaling molecules into the intracellular tissue fluids (Skin Chemical Response Test); and 4) the responses of skin cells together with specialized immune cells to substances that are true allergens (Skin Immunoreactivity Test). Such apparatus and methods provide means for quantifying the response of human skin tissue equivalent to agents. The results of such tests should reflect the response of natural human skin more closely than a corresponding test conducted with animal or cadaver skin. In some methods according to the present invention, the epidermis is exposed to a gaseous atmosphere, while the dermis is bathed by a sterile tissue culture fluid freely exchangeable with the tissue fluids of the skin tissue equivalent. The skin tissue equivalent is positioned between a gaseous phase (e.g., air) and a fluid phase (e.g., culture medium) (See, e.g., Fig. 1). In such embodiments, the dermal layer of the skin tissue equivalent is adjacent to, typically in contact with, the fluid phase that serves as a nutrient medium simulating the "milieu interieur" while the cornified epidermal layer contacts the gaseous phase simulating the environment. The skin tissue equivalent provides a fluid-tight seal between the two phases; the test substance is applied to its epidermal surface in an appropriate vehicle. The skin tissue equivalent itself, the tissue fluids thereof and the culture medium are each available for analysis. The composition of skin tissue equivalents for use in tests according to the present invention will vary depending upon the nature of the test to be conducted. The present invention provides apparatus and methods for use in skin absorption tests which permit a determination of the amount of an agent that traverses the skin during a predetermined period of time. With the exception of the initial phase of absorption, appendageal absorption has generally been found to play a minor role. Thus standard skin tissue equivalents, although lacking appendageal openings such as hair follicles and sweat glands, are expected to be suitable for most steady-state absorption studies. However, in some embodiments of the present invention, it may be desirable to fabricate skin tissue equivalents with a number of cell-lined pores that provide a physiologic ratio of appendageal to non-appendageal percutaneous absorption. Long term testing using skin tissue equivalents is possible because skin tissue equivalents can be maintained in a viable state for many months or even longer. Skin absorption tests according to the present invention may include measuring the amount of an agent in the liquid phase adjacent to the dermal component of the skin equivalent, or directly in the skin tissue equivalent itself or intracellular fluids thereof since lipid soluble substances, for example, tend to become entrapped in skin. Agents used in such tests are capable of being detected in some way, e.g., labeled with radioactivity, or are measured by direct assay, using methodologies known to those skilled in the art. Some methods according to the present invention measure the kinetics of skin penetration by the agent(s) under test. In some such embodiments, an apparatus according to the present invention is provided with multiple containers in a holder therefor, mounted into a jig and coupled to a reservoir for feeding each sample skin tissue equivalent periodically or continuously with, e.g., a nutrient solution to bathe the dermis. The pumped or gravity-feed output allows for fraction collecting so that flow through as a function of time under conditions simulating blood flow is measurable. The data is used to calculate the permeability constant or other relevant parameters, such as, the time for initial permeation or the percentage absorbed of the agent. Such methods also provide an optional calibration step that uses tritiated water before and after testing penetration so that the effects of the agent on the barrier properties of the skin tissue equivalent are determined. The present invention also provides apparatus and methods for use in skin cell permeability tests to determine changes in cell permeability brought about by the interaction of skin and agents such as chemicals, cosmetics or drugs. The release by cells of strictly intracellular proteins may serve as an indicator of cell damage due to the agent under test. Dose response data is derived by testing multiple skin tissue equivalents using apparatus and methods in accordance with the present invention, each skin tissue equivalent being positioned in an individual container which is provided with its own fluid phase, the contents of which are available for assay. The release of a cytoplasmic protein such as LDH is measured, for example, chromogenically. Release of at least one cytoplasmic and one lysosomal enzyme is measured by use of appropriate assay techniques well known to those skilled in the art. For example, various protein-binding methodologies including radioimmunoassay (RIA), enzyme immunoassay (EIA), enzyme-linked immunoassay (ELISA), and fluorescent immunoassay are useful. The skin cell permeability test of the present invention is especially useful for discriminating between irritant and corrosive agents since, in general, the former are expected to give negative results, and the latter, positive results. The present invention also provides apparatus and methods for use in skin chemical response tests for the measurement of physiological responses of skin cells to agents that induce edema and erythema or inflammation. The test is designed to quantitate complex tissue reactions resulting from multiple chemical triggers. Chemicals released from cells into the tissue fluids in response to the agent(s) under consideration are assayed. These include, for example, prostaglandin E2, prostacyclin and other signaling factors of arachidonic acid derivation as well as Interleukin I that can amplify the response by stimulating fibroblasts of the dermis to secrete prostacyclin. Multiple skin equivalent samples provide data for dose response curves. The degree of release is quantitated using techniques known to those skilled in the art. By testing known contact irritants, release of one or more of these mediators is correlated with the classical, though difficult to quantitate, responses of erythema and edema. While the keratinocytes of the skin tissue equivalents for use in methods provided by the present invention are expected to be the principal emitters of prostaglandin E2, in some embodiments capillary endothelial cells are optionally included in the dermis of the skin tissue equivalent to provide a response source for prostacyclin. Although both factors have relatively short half lives, they can be measured by assaying their degradation products using methods known to those skilled in the art. Apparatus, and methods for use in skin immunoreactivity tests are also provided by the present invention to measure responses to various agents which emanate from immune cells incorporated into the skin tissue equivalent, for example, an agent capable of causing contact sensitivity by forming covalent bonds with proteins. The immunogen will often be a complex of self-protein acting as "carrier" and the contact sensitizer acting as hapten. In some embodiments of skin immunoreactivity tests according to the present invention, the macrophage-like Langerhans cells (LC) of normal skin which process imunogenic complexes for presentation to other immune cells are incorporated in the epidermis of the skin tissue equivalent to provide the basis for the first step in the immune reaction chain involving the skin. Measurement of the migration of activated LC out of the epidermis of the skin tissue equivalent and of the protein-hapten complex are suitable assays of allergenicity. The movement of cells may be followed by immunofluorescence or other methods known to those skilled in the art and may be correlated with allergenicity by using substances of known allergenicity. In other embodiments, macrophages are optionally included in the skin tissue equivalent to provide another source of the assayable lymphokine, IL-1, secreted by the cells in response to substances that initiate humoral immune reactions. In yet other embodiments, subsets of sensitized T cells that will respond to particular classes of immunogens, together with mast cells that release strong signals such as histamine, are optionally incorporated in the skin tissue equivalent to provide immune signals, easily assayed because of their degree of amplification. Mediators are collected from the culture fluid in contact with the dermis or from the tissues themselves for assay. With the skin irritation test and skin chemical reaction test, the user will be able to discriminate between irritants and allergens. The apparatus and methods of the present invention may be used for determining the interaction of tissue with at least one agent by use of one or more tissue equivalents. The present invention may comprise the following in combination: (a) an apparatus for determining the interaction of tissue and at least one agent by use of at least one tissue equivalent, the apparatus comprising a container for the tisuse equivalent, the container comprising: (i) means for positioning the tissue equivalent in the container, whereby the tissue equivalent defines at least one region in the container; (ii) at least one port; and (iii) means for closing the container; and (b) a tissue equivalent. In preferred embodiments, the tissue equivalent defines at least two regions. In some embodiments according to the present invention, the tissue equivalent defines an upper and a lower region in the container. In yet other embodiments wherein the tissue equivalent is a tubular tissue equivalent, the tubular tissue equivalent defines an inner and outer region in the container. In some such embodiments, the container is provided one or more liquid phases. In preferred embodiments according to the present invention, the apparatus is provided with two or more individual containers. In some such embodiments, the apparatus may be provided with means for hooking to a manifold in order to sample or perfuse individual containers. In yet other embodiments, the containers are interconnected so that the liquid phase adjacent to the tissue equivalents is common to all the tissue equivalent samples. In such cases the tissue equivalent in each container is accessible for contact with the agent(s) and the liquid phase in each container is accessible for analysis. The apparatus may optionally comprise one or more agents as well as one or more of the reagents necessary for determining the interaction of the agent and the tissue equivalent. Various assay techniques known to those skilled in the art are used to determine the interaction of the tissue equivalent and the agent and, include, but are not limited to, histological analysis, mass spectrometry, magnetic resonance imaging, ultrasonic imaging, radioactive tracer methodologies, radioimmunoassay, enzyme immunoassay, enzyme-linked immunoassay, and fluorescent immunoassay. The present invention provides apparatus wherein a second liquid phase is disposed between the skin tissue equivalent and the means for closing the container. In such embodiments, it may be desirable to remove the second liquid phase before contacting the agent and the skin tissue equivalent. In yet other embodiments the apparatus is provided with two or more individual containers or two or more interconnected containers. In some embodiments of the present invention, it may be desirable to determine the response or lack of response of an agent with different types of tissue. For example, the response of liver tissue and an agent which has penetrated the skin could be determined by first passing the agent through a skin tissue equivalent in accordance with the present invention, collecting the agent or its breakdown products, and then contacting the collected agent or breakdown products, with a liver tissue equivalent. The invention will be further understood with reference to the following examples, which are purely exemplary in nature, and are not meant to be utilized to limit the scope of the invention. EXAMPLE 1 -- SKIN ABSORPTION TEST Skin tissue equivalent(s) cast as a sheet used to test percutaneous absorption of benzoic acid. 1) A dermal equivalent is cast with human dermal fibroblasts using the method of Patent No. 4,485,096. A suitable size for casting is a total volume of 15 ml. in a Petri dish 100 mm in diameter. 2) The dermal equivalent is seeded with human epidermal cells. Small droplets of a cell suspension, made with cultured epidermal cells or freshly dissociated epidermis, are applied to the surface of the dermal equivalent (See, e.g., Patent No. 4,485,096). Alternatively, pieces (strips, punches, or other shapes) of skin or of skin tissue equivalent are applied to the surface of the dermal equivalent (See, e.g., Patent No. 4,604,346). The seeded dermal equivalent is cultured to allow a complete epidermal layer to form. 3) The lower half of the test chamber is filled with fluid (typically culture medium or buffered salt solution). The completed skin tissue equivalent is transferred to an apparatus in accordance with the present invention such as shown in Figs. 1A or 1B, chamber so that the entire lower chamber and gasket are covered. The upper gasket is placed on upper surface of the skin tissue equivalent. The diameter of the chamber is about 25 mm. The upper half of the test chamber is screwed on tightly enough to make an effective seal without crushing the skin tissue equivalent. 4) Any excess fluid is aspirated from the upper surface of the skin tissue equivalent. Flow of buffered salt solution or other collection fluid is started through the lower chamber and collected in a fraction collector. 5) The agent(s) may be applied, its passage through skin tissue equivalent assayed, and time course and absorption calculated as in J. Pharm. Sci. , 74, 65-67 (1985). The test sample is applied to the upper surface of the skin tissue equivalent. For example, [ 14 C] Benzoic acid in petrolatum as a vehicle (suitable concentrations are 10, 100, and 1,000 ng/mg) with 25 mg of vehicle applied per cm 2 of skin tissue equivalent. 6) Samples of effluent are collected and counted by standard methods known in the art in a liquid scintillation counter. Alternatively, the skin tissue equivalent sample itself may be washed (with buffered saline), removed from the container, solubilized (for example in a solution 0.5% sodium dodecyl sulfate and 4M urea in water), and aliquots counted to determine radioactivity trapped in the skin tissue equivalent. This approach is useful for hydrophobic compounds which may not readily pass from the skin tissue equivalent into the collection fluid. This method is further refined by separating the epidermal and dermal layers using methods known to those skilled in the art, e.g., mild trypsin treatment. EXAMPLE 2 -- SKIN CELL PERMEABILITY TEST 1) Prepare and mount the desired amount of skin tissue equivalents in apparatus in accordance with the present invention as described in example 1, above. 2) Apply the agent(s) to upper (epidermal) surface of the skin tissue equivalent A. As an example, the chemicals used in the FRAME test (Bells, M. & Bridges, J. W., in Acute Toxicity Testing; Alternative Approaches, pp. 63-79, 1984; A. M. Goldberg, ed., M. A. Liebert, Inc., New York), are applied to individual samples of skin tissue equivalent in aqueous solutions at various concentrations. 3) The effluent fluid is collected from the lower chamber and assayed for a cytoplasmic enzyme produced by the skin tissue equivalent in response to the agent(s) (e.g., Lactate Dehydrogenase, EC 1.1.1.27) and a lysosomal enzyme (e.g., β-glucuronidase, EE 3.2.1.31). The enzyme released is assayed by standard methods known to those skilled in the art (See, e.g., Methods of Enzymatic Analysis , H. U. Bergmeyer, E. Verlag Chemie, Weinheim, 1983 III, 118-138 and IV, 246-256 respectively). 4) Dose response curves of enzyme release which is related to cellular damage are prepared from the data. EXAMPLE 3 -- SKIN IMMUNOREACTIVITY TEST 1) A container is used which consists of two chambers defined by a membrane or mesh. See, e.g., Fig. 1B. The membrane or mesh is a disk about 25 mm in diameter and has an average pore size in the range 0.5 to 3.0 µm. The membrane is made of, or coated with, a material to which cells and collagen adhere (e.g., glow discharge treated polystyrene or collagen coated controlled pore size glass). The lower chamber is provided with ports for medium sampling or flow-through. The upper chamber is provided with a cover means, removable for sample application. The walls of the upper chamber do not promote cell adhesion (e.g., polycarbonate, TEFLON®, or siliconized polystyrene). 2) A dermal equivalent is cast, as described in Example 1, above, in the upper chamber. In addition to dermal fibroblasts, the mixture contains cells of the immune system. One or more of the following cell types are included: Langerhans cells, macrophages, T-cells, mast cells and microvascular endothelial cells. Other cell types may be included as desired. A specific formulation includes Langerhans cells, macrophages, and mast cells. 3) The dermal equivalent contracts, constrained by adhesion to the membrane, to form a disk which separates the upper and lower chambers. 4) The dermal equivalent is seeded with epidermal cells (typically 3-10 days after casting). The skin tissue equivalent is cultured to develop a fully keratinized epidermal layer. See, e.g., J. Invest. Dermatology 81:25-105 (1983); 5) An agent (potential allergen) is applied to the epidermal surface. One or more mediators of allergic responses is collected in the lower chamber and assayed by standard means. Examples include interleukin (IL-1) released by Langerhans cells and macrophages, and histamine released by mast cells. EXAMPLE 4 -- SKIN CHEMICAL RESPONSE TEST Irritant Chemical. 1) The mandrel 100 and container 10 shown in Figs. 2 and 3 are assembled. A dermal equivalent is cast through port 72 as described in Example 1 in the volume or space defined by the mandrel 100 and the container 10 as assembled. The volume of dermal equivalent casting mixture is adjusted so that the entire mesh 104 and means for cells to adhere 103 are covered. Thus, for the dimensions given, a volume of about 50-60 ml would be required. The dermal equivalent is allowed to gel and to contract around the mandrel 100. The holes 103, 107, limit longitudinal contraction so that the mesh or membrane portion 104 remains completely covered. If necessary, the lumen of the mandrel 100 can be filled through port 71 simultaneously with the container 10 to prevent flow of the casting mixture into the lumen of the mandrel 100. 2) Then (typically after 3-10 days), the dermal equivalent will be seeded with a suspension of epidermal cells through the port 72. A multiple barelled micropipettor is inserted through port 72 and tiny droplets (typically 1-5 µl) of a cell suspension are applied so that the entire surface of the dermal equivalent is uniformly seeded with epidermal cells. These are grown to form a keratinized epidermal layer. 3) The agent(s) is then applied to the epidermal layer of the formed skin tissue equivalent and the lumen of the mandrel is perfused with the collection fluid. An irritant substance (See, e.g., M. Steinberg et al. (In Animal Models in Dermatology, H. Maibach, ed., Churchill Livingstone, 1975, pp. 1-11)) may be applied in a dressing (e.g., gauze pad). This is occluded by covering with an impermeable material, e.g., plastic film, if desired. The test dressing is removed after a suitable time (e.g., 24 hr). 4) The perfusate is assayed for substances released by the cells in response to the agent(s) (e.g., Prostaglandin E 2 ) using methods known to those skilled in the art. For example, commercially available Prostaglandin E 2 radioimmunoassay kits may be used.

Claims: A method of determining the response or lack of response of tissue to at least one test agent by use of at least one fully formed living skin tissue equivalent comprising living cells and extracellular matrix molecules, principally collagen, contracted by a contractile agent, wherein the response of the skin tissue equivalent to the test agent comprises the passage of the agent into or through the skin tissue equivalent, or the response of the skin tissue equivalent to the test agent comprises the production or release of one or more substances by the skin tissue equivalent, the method comprising the steps of: (a) contacting the test agent with the skin tissue equivalent, wherein the skin tissue equivalent is adjacent to a liquid phase; and (b) determining the response or lack of response of the skin tissue equivalent and the agent by analyzing at least one of (I) the skin tissue equivalent, (ii) an intracellular fluid of the skin tissue equivalent, or (iii) the liquid phase. The method of claim 1 wherein the skin tissue equivalent has an epidermal and dermal layer, the dermal layer of the skin tissue equivalent being adjacent to the liquid phase. The method of claim 2 wherein the test agent is contacted with the epidermal layer of the skin tissue equivalent. The method of any preceding claim, wherein the skin tissue equivalent is a fully formed living tubular skin tissue equivalent. The method of claim 4 comprising: contacting the test agent with a lumen or the abluminal surface of the tubular skin tissue equivalent, contact being effected by providing a liquid phase adjacent to the lumen or the abluminal surface of the tubular skin tissue equivalent and introducing the test agent into the liquid phase. The method of claim 5 comprising: contacting the test agent with the tubular skin tissue equivalent, wherein (I) the lumen of the tubular tissue equivalent is adjacent to a first liquid phase, and (ii) the abluminal surface of the tubular tissue equivalent is adjacent to a second liquid phase, contact being effected by introducing at least one test agent to the first or second liquid phase. The method of any preceding claim, wherein the response of the fully formed living tissue equivalent to the test agent has an effect which causes a change in (I) permeability, (ii) proliferation, (iii) differentiation, or (iv) configuration, of cells of the tissue equivalent. The method of any preceding claim, wherein the response of the fully formed living tissue equivalent to the test agent protects the tissue. An apparatus for determining the response or lack of response of tissue to at least one test agent by use of at least one fully formed skin tissue equivalent, the apparatus comprising a fully formed living skin tissue equivalent comprising living cells and extracellular matrix molecules, principally collagen, contracted by a contractile agent, and a container for the skin tissue equivalent, the container comprising: (I) means for positioning the tissue equivalent in the container, whereby the tissue equivalent defines at least one region in the container; ii) at least one port; and iii) means for closing the container. An apparatus for determining the response or lack of response of tissue to at least one test agent by use of at least one fully formed living skin tissue equivalent, the apparatus comprising a fully formed living skin tissue equivalent comprising living cells and extracellular matrix molecules, principally collagen, contracted by a contractile agent, and a container for the skin tissue equivalent, the container comprising: I) a liquid phase; ii) means for positioning the skin tissue equivalent in the container, whereby the skin tissue equivalent defines at least two regions in the container and the skin tissue equivalent is adjacent the liquid phase; iii) at least one port; and iv) means for the closing container. An apparatus according to claim 10, wherein the means for closing the container comprises a cover means, removably sealable to the container, or comprises an opening or a port, the opening or port having a valve or cover means. An apparatus according to claim 10, wherein the skin tissue equivalent defines an upper and a lower region in the container. An apparatus according to claim 12, wherein the lower region further comprises the liquid phase and one surface of the skin tissue equivalent is adjacent to the liquid phase. An apparatus according to claim 13, wherein the upper region further comprises a second liquid phase and one surface of the tissue equivalent is adjacent to the second liquid phase. An apparatus according to claim 10, wherein the container is provided with at least two ports. An apparatus according to claim 10, wherein the means for positioning the skin tissue equivalent in the container is disposed in the container and comprises a permeable member, preferably a selectively permeable membrane or mesh. An apparatus according to claim 16, wherein the skin tissue equivalent is cast on the permeable membrane. An apparatus according to claim 10, comprising at least two containers wherein the containers are interconnected so that there is a liquid phase common to both containers. An apparatus according to claim 10, comprising a fully formed living tubular skin tissue equivalent, wherein the tubular skin tissue equivalent defines an inner and outer region in the container, the inner region comprising a first liquid phase and the outer region comprising a second liquid phase. An apparatus according to claim 19, wherein the means for positioning the tubular skin tissue equivalent in the container comprises valved cannulae, portions of which are disposed inside the container. An apparatus according to claim 19, wherein the means for positioning the tubular skin tissue equivalent in the container comprises (I) means for attaching the tubular skin tissue equivalent, (ii) means for limiting the longitudinal contraction of the tubular skin tissue equivalent, and (iii) means for allowing selected materials to pass between the tubular skin tissue equivalent and at least one of the first or second liquid phase. An apparatus according to claim 19, wherein the tubular skin tissue equivalent is provided with at least one support member. An apparatus according to claim 21, wherein the tubular skin tissue equivalent is cast around the means for positioning the tubular skin tissue equivalent in the container. An apparatus according to claim 19, wherein the apparatus is incorporated into a circulatory loop. An apparatus according to any of claims 9 to 24 further provided with means for changing, sampling, circulating or feeding at least one of a first liquid phase or a second liquid phase, preferably at a predetermined rate or frequency. A method according to any of claims 1 to 8 of determining the response or lack of response of tissue to at least one test agent with the aid of the apparatus of any one of claims 9 to 24. A method according to claim 26 of determining the response or lack of response of tissue to at least one test agent with the aid of the apparatus of claim 19, wherein the second liquid phase is circulated through the fully formed living tubular skin tissue equivalent to simulate the flow of blood. A method according to any preceding method claim, wherein the test agent is selected from the group consisting of: light; physical injury, chemical substances; cosmetic substances; pharmaceutical substances; and skin tissue protective agents.

Inventors: BELL EUGENE, WEINBURG CRISPIN, BELL, EUGENE, WEINBURG, CRISPIN
Applicants: ORGANOGENESIS INC, ORGANOGENESIS INC.
Application year: 1988
Main classification: G01N33
IPC codes: C12Q1, G01N33