KR20040070343A - In vitro micro-organ, and uses in related thereto - Google Patents

Abstract

The present invention describes micro-organ cultures comprising isolated cell populations with specific characteristics. The main feature of the micro-organ cultures of the present invention is the preservation of organ microstructures, which facilitates cell-cell interactions and cell-epileptic interactions similar to those found in source organs, as well as the ability to remain in culture for relatively long periods of time. It includes. The micro-organ culture of the present invention can be used for the delivery of gene products to test receptors, the identification of cell proliferative agents and cell differentiators, and the identification and isolation of progenitor and stem cells. In addition, the micro-organ cultures of the present invention can be used in methods for identifying inhibitors for cell proliferation, cell differentiation and viral infectivity. In another embodiment, the micro-organ culture of the invention can be used for transplantation.

Description

In vitro micro-organs and related uses {IN VITRO MICRO-ORGAN, AND USES IN RELATED THERETO}

Eukaryotic cell cultures were first obtained in the early 1950s. Since then, various transformed and primitive cells have been cultured using various media and regulatory supplements such as growth factors and hormones and the like, and nonregulated supplements such as serum and other body extracts. For example, fibroblasts obtained from the skin of an animal can be cultured in a customary manner through various cell generations as cells that are nucleomorphically diploid, or indefinitely in a customary manner as an established cell line. However, epithelial cells have different morphological properties and proliferation than fibroblasts, making them more difficult to culture. Moreover, when epithelial cells and fibroblasts are propagated in the same culture, epithelial cells generally overgrow compared to fibroblasts.

Two-dimensional cell proliferation is a convenient way to prepare, observe, and study cells in culture by providing high cell proliferation rates, but the characteristic cell-cell and cell-epileptic interactions of whole tissues in vivo are unknown. .

For these functional and morphological interaction studies, several researchers have described collagen gels (Douglas et al., (1980) In Vitro 16: 306-312; Yang et al., (1979) Proc. Natl. Acad. Sci. 76: 3401; Yang et al. (1980) Proc. Natl. Acad. Sci. 77: 2088-2092; Yang et al., (1981) Cancer Res. 41: 1021-1027); Cellulose sponge alone (Leighton et al., (1951) J. Natl. Cancer Inst. 12: 545-561) or collagen coated (Leighton et al., (1968) Cancer Res. 28: 286-296); Three-dimensional substrates such as gelatin sponge, gelfoam type (Sorour et al., (1975) J. Neurosurg. 43: 742-749) were used.

Various culture conditions were used to proliferate epithelial cells in a competent cloned manner. For example, the epithelial cells, specifically dermal epithelial cells (keratocytes), may be treated with a lethal layer of irradiated fibroblasts (Rheinhardt et al. (1975) Cell 6: 331-343) and semisynthetic collagen matrix (US Patent No. 5,282,859; European Patent Application No. 0361957). In some cases, biological extracts such as pituitary extracts and serum and growth supplements such as epidermal growth factor and insulin were added to the medium used for the proliferation of such cells (Boisseau et al. (1992) J. Dermatol. Sci 3 (2): 111-120; US Pat. No. 5,292,655).

Numerous attempts have been made to propagate skin in vitro. Such attempts generally involve separating keratinocytes in the epithelium from fibroblasts and adipocytes in the dermis. After isolation, keratinocytes are generally proliferated in a way to form a stratified epithelium. However, epithelium prepared in this way lacks hair follicles and sweat glands. Moreover, the cell culture does not preserve the natural relationship between the epithelium and the dermis. In addition, the method of proliferating keratinocytes on non-proliferative fibroblasts (Rheinwald wt al. (1975) Cell 6: 331-343) or on the skin matrix of collagen and fibroblasts, either synthetic or derived from a source other than the dermis. Culture of methods for providing keratinocytes (Sugihara et al. (1991) Cell. Dev. Biol. 27: 142-146; Parenteau et al. (1991) J. Cell Biochem. 45 (3): 245-251) Methods were also performed. In some cases, however, keratinocyte separation was not performed and the entire organ was cultured. Attempts to cultivate organs in vitro only resulted from culturing organs in serum-containing medium (Li et al. (1991) Proc. Natl. Acad. Sci. 88 (5): 108-112).

Most of the dermis in the in vitro model of the dermis is devoid of hair follicles, sweat glands and sebaceous glands (Coulomb et al. (1992) Pathol. Biol. Paris 40 (2): 139-146 for review of epithelial cell culture). Exceptionally, a gel-supported skin model has been presented in which skin explants with dimensions of 2 × 5 mm 2 and 2.0 mm thickness have maintained viability for several days in the presence of serum-containing medium (Li et al. (1992) Proc. Natl.Acad.Sci. 89: 8764-8768).

Bioreactors and other mammalian cell culture methods, in addition to the drawbacks of cell damage, were very limited due to the lack of ability to combine the cells into tissues similar to the spatial three-dimensional form of the actual tissue present in the original organism. . For this reason, conventional tissue culture methods provide the ability to express cultured tissues in a functionally highly granular or differentiated state, which is considered to be an important factor for mammalian cell differentiation, and the ability of microbial biologically active molecules, which are under investigation and pharmaceutical agents, to be studied. There is a limit on secretion. Unlike microorganisms, cells of higher organisms, such as mammals, themselves form higher-order multicellular tissues. The exact mechanism of such self-assembly is not known, but the examples studied so far indicate that the development of cells into the tissues is characterized by the orientation of the cells relative to each other (homologous or heterologous) or to other adherent substrates and And / or depending on the presence or absence of certain substances (factors) such as hormones, self-secreting factors or peripheral secretion factors. Summarizing this, it is possible to simultaneously achieve a period of significant cell interaction (cell-to-cell or cell-substrate) that is sufficiently long to allow sufficiently low shear stress, sufficient three-dimensional spatial freedom and good modeling of tissue structures in vivo. There is no cultivation method.

Thus, there is a need for an in vitro method of generating and maintaining some organs in culture in which the cells of the culture preserve their original intercellular relationships for a long time. The availability of tissue and organ models in which cell differentiation, cell proliferation, and cell function are similar to those observed in whole organs in vivo can be used to understand how organs remain healthy and thus how abnormal processes can be restored. It will be useful.

1 is a schematic diagram of a micro-organ with dimensions measuring Alep (where x is the thickness of the tissue and a is the width of the tissue).

FIG. 2 is a bar graph showing BrdU labeling after cell incubation for several hours in guinea pig micro-organ cultures.

3 is a histogram measured by BrdU labeling after cell proliferation in human epidermal micro-organ culture for 1 to 8 days.

4A-4D show mouse skin (50 magnification) (FIG. 4A), guinea pig skin (75 magnification) (FIG. 4B), human foreskin (50 magnification) (FIG. 4C) and human foreskin (75 magnification) (FIG. 4D). It is a micrograph showing the immunofluorescence of each of the cloned cells.

5A-5C are cross-sectional views of human epidermal micro-organ explants (75x magnification) showing tissue microstructures on culture days 0 (FIG. 5A), 3 (FIG. 5B) and 6 (FIG. 5C).

Figure 6 (a) is a bar graph showing the effect of epidermal proliferation measured through BrdU incorporation by changing the thickness (x) of the guinea pig micro-organ culture to maintain a constant 4mm.

7A and 7B are micrographs (75 ×) showing immunofluorescence corresponding to proliferative cells present in pancreas derived micro-organ cultures.

8 is a bar graph showing the amount of insulin released from micro-organ cultures of mature pig pancreas.

9 is a bar graph showing the ¹ H-thymidine incorporation rate of proliferative cells present in micro-organ cultures of colon, liver, kidney, duodenum and esophagus on days 3, 4 and 6 of culture.

10A to 10C are micrographs showing immunofluorescence measurements of active proliferation of hair follicles in micro-organ cultures. Magnifications are 40x (FIG. 10A), 40x (FIG. 10B) and 75x (FIG. 10C).

Figure 11 is a bar graph showing the size distribution of the hairy stem at the beginning and end of microculture.

12 is a bar graph showing mitogenic activity of micro-organ cultures in guinea pig skin culture with 2.5 ng / ml TGF-β.

FIG. 13 is a schematic diagram of a micro-organ explant for treating chronic skin ulcers showing an incomplete fraction of tissue sections for maintaining a structure that can be easily processed in vivo.

14 is a photograph of the surface of the mouse after replacement of a portion of normal skin with micro-organ culture, where healing, generation of new hairs in the graft, and implantation of the graft in normal mouse skin can be observed (10 ×).

FIG. 15 is a graph depicting the luciferase reporter gene expression seen in skin micro-organ cultures of guinea pigs after transfection of a culture with a plasmid encoding the luciferase reporter gene.

FIG. 16 is a graph depicting the luciferase gene expression seen in plasmids encoding the luciferase reporter gene and cation lipid mediated transfection of rat lung and thymus micro-organ cultures.

Figure 17 is a graph showing the activation of resting hair follicles during FGF treatment in the micro-organ culture of the present invention.

18 is a graph showing the expression of the mutant luciferase gene present in the micro-organ explant of the present invention.

Detailed description of the invention

The present invention relates to a three-dimensional organ explant culture system. This culture system can be used for long-term proliferation of in vitro micro-organ explants in an environment similar to that found in whole organs in vivo. The culture system described herein provides proliferation and proper cell maturation to maintain a structure similar to the corresponding organ in vivo.

The micro-organ culture of the present invention provides an in vitro culture system capable of maintaining a tissue or organ section and preserving the function of the section for a long time. Such a culture system provides an in vitro model that allows for easy and accurate testing of cell differentiation, cell proliferation, cell function, and methods of altering such cell characteristics and functions. The resulting culture can be used for a variety of applications, from in vivo transplantation to the selection of cytotoxic and pharmaceutical compounds in vitro, to the production of biologically active molecules in "bioreactors" and to the separation of progenitor cells from tissues. .

For example, certain embodiments of the invention include (i) micro-organ bone marrow culture implants used to replace bone marrow that will be destroyed during chemotherapy; (ii) micro-organ liver transplants for use in enhancing liver function in sclerosis patients; (iii) genetically modified cells propagated in test micro-organ cultures (such as pancreatic micro-organs expressing recombinant genes encoding insulin); (iv) dental prostheses coupled to micro-organ cultures of oral mucosa, but are not limited thereto.

As another non-limiting embodiment, the microorgan culture of the subject can be used in vitro to select various compounds, such as cytotoxic compounds, growth / regulatory factors, pharmaceutical agents, and the like. For this purpose, micro-organ cultures are maintained in vitro and exposed to the compounds to be tested. The activity of the cytotoxic compound can be measured, for example, through the ability to damage or kill cells present in the explant.

This activity can be readily assessed through vivid staining techniques. Growth / regulatory factors can be assessed by analyzing the cell contents of the explant, such as total cell number, and differentiated cell number. This assessment can be performed by standard cytological and / or histological techniques, including the use of immunocytochemical techniques with antibodies that define type specific cellular antigens. The effects of various drugs on normal cells cultured in three-dimensional systems can be assessed. For example, drugs that increase red blood cell formation can be tested with bone marrow micro-organ cultures. Drugs that affect cholesterol metabolism (such as by lowering cholesterol production) can be tested with liver micro-organ cultures. Micro-organ cultures of abnormal tissues can also be used, for example, to facilitate the study of hyperproliferative or neoproliferative diseases. For example, micro-organ explants in which tumor cell proliferation has occurred can be used as a model system, for example, to test antitumor efficacy.

Some terms used in the specification, the examples, and the appended claims have been described below for convenience.

The term "explant" refers to a collection of organ-derived cells isolated from the body and propagated in synthetic media. An explant from an organ having both an epileptic component and an epithelial component generally refers to an explant comprising both components in a single explant derived from the organ.

The term "tissue" refers to a similarly divided group or layer of cells that together perform a specific specific function.

The term “organ” refers to a tissue composed of two or more contiguous layers that maintain some form of cell-cell and / or cell-stromal interaction to form a microstructure. Thus, in the present invention, micro-organ cultures were prepared from organs such as mammalian skin, mammalian pancreas, liver, kidney, duodenum, esophagus, bladder, cornea, prostate, bone marrow, thymus and spleen.

The term "epilepsy" refers to the supportive tissue or substrate of an organ.

As used herein, the term "microorgan culture" refers to an isolated cell population, such as an explant, that retains the microstructure of the organ or tissue from which the cell is isolated. That is, isolated cells form a three-dimensional structure that mimics / maintains the orientation of the original organism and the actual tissue from which spatial interactions such as cell-cell, cell-substrate and cell-stromal interactions and explants originate. Thus, such interactions, such as between the interstitial layer and epithelial layer, are preserved in the explanted tissue and are provided with self-secreting factors, peripheral secretion factors, and other extracellular stimuli that maintain important biological interactions, for example, the explant's biological function. Long term viability can be achieved under conditions where proper nutrient and waste transport occurs through the sample.

The micro-organ culture of the subject retains the microstructure of the organ or tissue from which the cell or tissue explant is isolated. As used herein, the term “microstructure” refers to at least about 50%, preferably at least about 60%, more preferably at least about 70%, even more preferably at least about 80%, of the cells of a cell population, Most preferably, at least about 90% or more of the cells remain in physical and / or functional contact in vitro with at least one or more cellular or non-cellular materials that are in physical and / or functional contact with in vivo and at least By isolated cell population or tissue explant, forming a cell culture of at least about 1 layer, more preferably at least about 5 layers, most preferably at least about 10 layers. Preferably, it is desirable to maintain at least one biological activity of the organ or tissue from which the cells of the explant are isolated.

As used herein, the term "isolated" refers to explants isolated from their natural environment in an organism. The term includes the entire physical separation from the natural environment, such as removal from a donor animal, such as a mammal, such as a human or miniature pig. For example, the term “isolated” refers to a population of cells that have been explanted, cultured as part of an explant or implanted in the form of an explant. As used to mean a cell population, the term "isolated" includes a cell population resulting from the proliferation of cells in the micro-organ culture of the present invention.

The term "extracellular layer" refers to the outermost of the embryonic primitive layers of the embryo, from which the epidermis and epidermal tissue, such as nails, head and skin glands, nervous system, external sensory organs, mucous membranes of the oral cavity and anus, etc. do.

The term "epithelial" includes the cell envelope of internal and external body surfaces (skin, mucosa and serum) and glands and other structures derived therefrom such as corneal cells, esophageal cells, epidermal cells and hair follicle epithelial cells. Examples of other epithelial tissues include the olfactory epithelium, which is a pseudolayered epithelium present in the olfactory region of the nasal cavity and containing the olfactory receptors; Gland epithelium, meaning the epithelium consisting of secretory cells; Squamous epithelium, meaning an epithelium consisting of squamous platelets. The term epithelium may also refer to tissue indicating metastasis between metastatic epithelium, such as stratified squamous and columnar epithelium, which is characteristically found in hollow organs that undergo significant mechanical changes due to contraction and expansion. The term "epithelialization" refers to healing by growth of epithelial tissue on the epidermal surface.

The term "skin" refers to the outer protective covering of the body consisting of the dermis and epidermis, and is to be understood to include sweat glands and sebaceous glands as well as hair follicle structures. The term "skin" is also used throughout this specification, which is to be understood as representing the properties of the skin appropriately in the context in which the term is used.

The term "epidermal" refers to the outermost non-vascular layer of the skin, derived from the extracellular layer of the embryo, varying in thickness from 0.07 mm to 1.4 mm. On the palm and plantar surfaces, the epidermis is divided into five layers from the inside to the outer layer: the basal layer of vertically arranged columnar cells, the polar or spinous layer of flattened polyhedral cells with short processes or spines, flattened granular cells. And a granular layer consisting of a transparent layer consisting of several layers of transparent permeable cells in which the nucleus is not pronounced or absent, and a keratin layer consisting of flattened keratinocytes. In general, no clear layer is present on the epidermis of the body surface. "Epidermal" is a cell or tissue similar to the epidermis, but may also be used to mean any tumor that occurs in the non-skin area and is formed by the inclusion of epidermal factors.

"Dermis" means a layer of skin deeply beneath the epidermis, which is based on a dense basis of vascular connective tissue and includes nerves and sensory terminal organs. Hair roots, sebaceous glands and sweat glands are structures of the epidermis deeply embedded in the dermis.

The term "sam" refers to specialized cell aggregates that secrete substances regardless of their regular metabolic needs. For example, "sebaceous glands" are complete secretory glands in the dermis that secrete oily substances and sebum. The term "glands" refers to glands that secrete sweat and are located in the dermis or subcutaneous tissue, which are opened by tubes on the body surface. Ordinary glands or exocrine glands are distributed on the outermost layer of the body surface to facilitate cooling by evaporation of secretions; Apocrine glands are not emptied directly on the skin but are emptied into the top of the hair follicles and are found only in certain areas of the body, such as around the anus and in the armpit.

The term "parent" (or "hair") refers to a filamentous structure, specifically a granular epidermal structure that occurs in the papillae, consisting of keratin and embedded in the dermis, produced only by a mammal and characteristic of the group of animals. The term also means a collection of such simulations. "Follicles" are one of the coronary indentations of the epidermis that surround the hair, from which the hair grows. "Follicle epithelial cells" refer to epithelial cells surrounded by the dermis in the hair follicles, such as stem cells, external ectoderm cells, stromal cells and internal endodermal cells. Such cells may be nonmalignant normal cells or transformed / innocent cells.

"Alopecia" generally means no hair in bald areas, such as areas of skin that normally exist. Various forms of alopecia are known in the art. For example, alopecia areata usually means reversible hair loss in very obvious areas, including beards or scalp, drug alopecia refers to hair loss due to drug ingestion, and alopecia areata or masculine baldness Completely determined, androgen-dependent, means the loss of the scalp hair starting from behind the forehead and progressing symmetrically, leaving only the hair's rough perimeter.

The term "proliferative skin disease" as used throughout this specification means any skin disease / disease characterized by unnecessary or abnormal proliferation of skin tissue. These diseases are generally characterized by epidermal cell proliferation or incomplete cell proliferation, such as X-related scleroderma, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermal keratosis and seborrheic dermatitis. For example, epidermal dysplasia is an incomplete form of the epidermis, for example, a wart epidermal dysplasia, which is a disease caused by the same virus as the common wart virus or a virus related to this virus. Another example refers to the loose state of the epidermis, where blisters and blisters are formed at the trauma site or spontaneously.

As used herein, the term "psoriasis" refers to a hyperproliferative skin disease that alters skin control mechanisms. Specifically, lesions are formed that involve primary and secondary changes in epidermal proliferation, skin inflammatory response, expression of regulatory molecules such as lymphokines and inflammatory factors. Morphological features of psoriasis skin include increased turnover of epidermal cells, epidermal hypertrophy, abnormal keratinization, invasion of inflammatory cells into the dermis, polymorphonuclear leukocyte infiltration into the epidermis, and thus increased basal cell circulation. In addition, hyperkeratinocytes and aberrant keratinocytes may be present.

As used herein, the terms "proliferative" and "proliferation" refer to cells that cause mitosis.

The term "progenitor cell" refers to an undifferentiated cell capable of proliferating and generating more progenitor cells that possess the ability to produce multiple parental cells, whereas the parental cell is capable of producing differentiated or differentiated daughter cells. have. The term "progenitor cells" as used herein also includes cells that are sometimes referred to in the art as "stem cells". In a preferred embodiment, the term "progenitor cells" refers to generalized blast cells that fully acquire and differentiate individual characteristics, as their offspring (progeny) often appear in different ways, such as by gradual differentiation of embryonic cells and tissues. Means. For example, "hematopoietic progenitor cells" (or stem cells) refer to progenitor cells that occur in the bone marrow and other blood-related organs, such as erythrocytes, lymphocytes, and other blood cells that produce such differentiated progeny.

As used herein, “transformed cell” means a cell that has spontaneously converted to an unlimited proliferation state, ie, a cell that has acquired the ability to proliferate through an infinite number of divisions in culture. Transformed cells may be characterized by terms such as angiogenesis, anaplasia and / or hyperproliferation in terms of loss of proliferation control.

As used herein, the term "infinite proliferative cell" means a cell that can be modified by chemical and / or recombinant means capable of retaining the ability of the cell to multiply through an infinite number of divisions in culture. do.

The term "carcinoma" refers to a malignant new proliferation consisting of epithelial cells that tend to infiltrate surrounding tissues and develop metastases. Examples of carcinomas include "basal cell carcinoma", an epithelial tumor of the skin that rarely metastasizes and retains the potential for local invasion and destruction; "Squamous cell carcinoma", meaning a carcinoma that develops in squamous epithelium and retains cubic cells; "Carcinosarcomas" including malignant tumors consisting of cancerous and sarcomaous tissues; Carcinomas represented by columnar or banded vitreous or mucous matrix isolated or surrounded by a network or tendon of small epithelial cells, and "cyst carcinomas" occurring in the mucous glands of the mammary gland, salivary glands and respiratory tracts; "Epidermal carcinoma", meaning cancer cells that tend to differentiate in the same way as epidermal cells, that is, to form polar cells and cause keratinization; "Nasal pharyngeal carcinoma", meaning a malignant tumor that develops in the epithelial lining of the space behind the nose; "Renal cell carcinoma" belonging to carcinoma of the renal parenchyma consisting of various arrays of coronary cells. Another cancerous epithelial proliferation, meaning a papilloma-derived benign tumor and having a papilloma virus as a causative factor; And "epidermoidomas," meaning brain or meningoid tumors formed by the incorporation of ectoderm factor upon closure of the nerve sulcus.

As used herein, "mutant animal" refers to any animal containing heterologous nucleic acid, for example, one or more cells introduced through human intervention, such as mutation techniques known in the art, preferably mammals other than humans. , Birds or amphibians. Nucleic acids are introduced into cells by direct or indirect introduction into cell precursors through deliberate genetic manipulation, such as microinjection or recombinant viral infection. The term genetic engineering relates to the introduction of recombinant DNA molecules without involving classical crossbreeding or in vitro fertilization. This molecule may be DNA that is integrated into the chromosome or replicates extrachromosomally. The term also encompasses mutant animals in which the recombinant gene is silent, eg, as a FLP or CRE recombinase dependent construct as described in the art. Mutant animals also include constitutive and conditional "knockout" animals. The term "animal except humans" of the present invention includes vertebrates such as rodents, primates except humans, pigs, sheep, dogs, cattle, chicks, amphibians, reptiles and the like. Animals other than humans are preferably selected from rodents including reduced pigs or rats and mice, most preferably mice. As used herein, the term "chimeric animal" refers to an animal in which a recombinant gene is found or expressed in some cells although the recombinant is not whole cells of the animal.

I. Establishment of micro-organ cultures

The main feature of the microorgan cultures of the present invention according to the present invention is the ability to preserve the cellular microenvironment found in vivo of certain tissues. The present invention is based, in part, on the discovery that in certain circumstances the growth of cells in different tissue layers of organ explants, such as mesenchymal and epithelial layers, can be activated to proliferate and mature in culture. In addition, the cell-cell and cell-epileptic interactions provided to the explant itself are sufficient to support cell homeostasis, such as maturation, differentiation and separation of cells in explant cultures, thereby prolonging the microstructure and function of the tissue for an extended period of time. Keep it.

One example of a physical contact between a cell and a noncellular matrix (epilepsy) is a physical contact between an epithelial cell and its base layer. One example of physical contact between cells and other cells includes actual physical contact maintained by intercellular cell joints such as, for example, crevices and dense joints. Examples of functional contact between one cell and another cell include electrical or chemical communication between cells. For example, cardiomyocytes communicate with other cardiomyocytes via electric shock. In addition, many cells communicate with other cells through chemical communication, such as local proliferation hormones (such as periphery and autosecretory signal hormones) or hormones (endocrine signaling hormones) that are delivered to more distant locations by the vascular system. Examples of peripheral secretory signals between cells include signals produced by various cells in the digestive tract (also known as enteroendocrine cells), such as pyloric D cells, which secrete somatostatin that inhibits gastrin release by nearby pyloric gastric (G) cells. There is.

The microstructures of the present invention can be very important for the maintenance of explants in minimal media, such as in minimal media without exogenous serum or growth factors, as a result of periphery secretion factors and specific cell interactions in explants. This is because autosecretory factors allow tissue to be maintained in minimal medium and are not intended to be limited to a particular theory.

In addition, the term "maintenance, in vitro, their physical and / or functional contact" refers to physical and / or non-cellular material with at least one cell or non-cellular material in which at least one cell is not in physical and / or functional contact in vivo. Or separate cell populations that form a functional contact state. One example of such an occurrence is the proliferation of at least one cell in an isolated cell population.

In a preferred embodiment, the cell populations that make up the explant are isolated from the organs in a manner that preserves the natural affinity of one cell for the other cells, such as in an explant but preserves another cell layer. For example, in skin micro-organ cultures, keratinocytes in the epidermis remain bound to the epilepsy and preserve normal tissue structures, including hair follicles and glands. This basic structure is common to all organs, including all organs including epithelial components. Moreover, this binding facilitates intercellular communication. Among the animal cells, various types of traffic occur. This is particularly important in differentiated cells where the induction occurs through interactions between one tissue or cell (induction) and another tissue or cell (response), resulting in the cells undergoing a change in the direction of differentiation. Induction interactions can also occur in embryonic and mature cells to induce differentiation as well as to establish and maintain morphogenesis patterns (Gurdon (1992) Cell 68: 185-199).

In addition, the micro-organ cultures prepared according to the present invention preserve the normal tissue structure even when cultured for an extended period of time. This includes maintenance of hair follicles, sweat glands and sebaceous glands (Examples VIII and FIGS. 10A-10C) in the skin micro-organs in vitro according to normal development in vivo, or maintenance of islet pancreatic islets in the pancreas normally occurring in vivo ( See Examples IV, V and VI). Because these cultures can be kept in a controlled, uniform state and are very similar to tissues in vivo, they provide a unique opportunity to observe, measure, and control natural phenomena and the disruption of natural phenomena resulting from disease, aging, or trauma. to provide. In addition, the rapid availability of the technology to study each cell at the identified site on the culture makes it possible to observe the function of each component of the organ and the interaction with each other as well as with the entire organ.

Examples of micro-organ cultures made in accordance with the present invention are described in the Examples below, and group the cell populations grouped in such a way that they can comprise a plurality of layers to preserve the natural affinity of one cell for another. It may include. The proliferation of each cell or group of cells can be observed and then subjected to autoradiography or immunofluorescence.

The accompanying examples are merely to illustrate in more detail, to demonstrate that the culture system of the subject provides replication of in vitro epithelial and epilepsy factors in a system corresponding to a physiological state. Importantly, the cells replicated in this system are properly spaced to form morphologically and histologically normal epidermal and dermal components.

In addition to isolating explants retaining the cell-cell, cell-substrate and cell-stromal structures of the original tissue, the size of the explants may, for example, extend the micro-organ culture for an extended period of time, such as 7 to 21 days or longer. If desired, it is important for the viability of the cells contained. Thus, the size of the tissue explants allows the appropriate nutrients and gases, such as O ₂ , to diffuse into all cells of the three-dimensional microorganisms and the cell waste to diffuse out of the explants, resulting in cytotoxicity associated with localization of waste in the microorganisms and concomitant Choose to minimize death. Thus, the size of explants is determined in accordance with the required minimum level of accessibility for each cell without special delivery structures or synthetic substrates. As described herein, it has been found that such accessibility can be maintained if Alep, an index calculated from the thickness and width of the explant, is at least greater than about 1.5 mm ⁻¹ .

As used herein, "alep" is a surface area to volume ratio represented by the formula 1 / x + 1 / a ≧ 1.5 mm ⁻¹ , where x is the tissue thickness (mm) and a is the tissue width (mm). In a preferred embodiment, the allef of the explant is preferably in the range 1.5 to 25 mm ⁻¹ , more preferably in the range 1.5 to 15 mm ⁻¹ , even more preferably in the range 1.5 to 10 mm ⁻¹ , in particular 1.5 to 6.67 mm ^−1. Range, 1.5 to 3.33mm ^-1 is preferred.

Thus, the present invention is such that the surface area to volume index of the tissue explant is maintained within a predetermined range. This predetermined range of surface area-to-volume index allows cell access to nutrients by diffusion and cell access to waste disposal means in a manner similar to monolayer cells. This level of accessibility can be achieved and maintained when the surface area to volume index (indicated herein as "alep or alleop index") is at least about 1.5 mm ^-1 or greater. Since the three-dimensional change causes radiometric changes in volume and surface area, it is ignored for measuring surface area versus volume index. However, when measuring aleph, a and x should be presented in the smallest two dimensions of the tissue section.

Examples of alephs are given in Table I, where, for example, tissue having a thickness of 0.1 mm and a width of 1 mm has an Aleph index of 11. In Example I, x was 0.3 mm and a was 4 mm so that the aleph was 3.48. In Example III x was changed and a was fixed at 4 mm. As illustrated in FIG. 6, proliferative activity decreases significantly as the thickness of the explant increases. Thus, the number of proliferative cells in micro-organ cultures was about 10 times less than in tissues of similar origin with a thickness of 300 μm at a thickness of 900 μm. The Allef Index of a tissue having a thickness of 900 μm is 1.36 mm ⁻¹ , which is less than the minimum value suggested by the present invention, while the Allef Index of a tissue having a thickness of 300 μm is 3.58 mm ⁻¹ , which is within the range of the present invention. Was.

Table 1

Several "alep" values that are surface area-to-volume ratio indices as a function of a (width) and x (thickness) (mm ^-1 )

Also, without wishing to be bound by a particular theory, many of the factors provided by a three-dimensional culture system can contribute to the success of this system:

(a) A three-dimensional substrate of explant size selected using, for example, the Allef calculation can provide adequate surface area-to-volume ratios for adequate diffusion of nutrients to all cells of the explant and adequate diffusion of cell waste from all cells of the explant. To provide.

(b) Due to the three-dimensionality of the substrate, various cells continue active proliferation, unlike cells in monolayer cultures, which proliferate until confluent and exhibit contact inhibition and stop growth and division. The contribution of growth factors and regulatory factors to cell replication of explants may be partially responsible for regulating cell proliferation stimulation and differentiation in culture, such as for micro-organ cultures that are stationary in total volume.

(c) The three-dimensional substrate has a spatial distribution of cellular components that is nearly similar to that found in corresponding tissues in vivo.

(d) Cell-cell and cell-substrate interactions can establish a local microenvironment that aids cell maturation. It has been found that maintenance of differentiated cell phenotype requires appropriate cell interactions as well as growth / differentiation factors. The present invention effectively mimics the tissue microenvironment.

As described in the following exemplary embodiments, micro-organ cultures derived from animals (including humans), such as skin, pancreas, liver, kidney, duodenum, bladder, bone marrow, thymus or spleen-derived micro-organ cultures are isolated. Were grown in culture for up to 21 days. However, it is also within the scope of the present invention to maintain the culture for an extended period of 21 days or more.

II. Source of explants used in micro-organ cultures

Micro-organ cultures of the subject include, for example, skin and mucous membranes (eg, oral mucosa, gastrointestinal mucosa, nasal tubes, respiratory tracts, cervix and corneas); Pancreas; liver; Gallbladder; Bile ducts; lungs; prostate; Womb; cable; Bladder tissue; And explants isolated from blood-related organs such as thymus, spleen and bone marrow. Thus, in vitro culture equivalents of such organs can be prepared. The tissue forming the explant may be diseased or normal (eg, healthy tissue). For example, the organ from which the micro-organ explant of the present invention is isolated may be a hyperproliferative disease such as psoriasis or keratosis; Proliferation of virus infected cells, such as hepatitis virus infected cells, or papilloma virus infected cells; Neoproliferative diseases such as basal cell carcinoma, squamous cell carcinoma, sarcoma or Wilms' tumor; Or fibrotic tissue, such as sclerosis liver or pancreatic tissue from pancreas with pancreatitis.

Examples of animals capable of isolating cells of the invention include humans and other primates, swine, such as fully homologous or partially homologous pigs (eg, miniature pigs and mutant pigs), rodents, and the like.

III. Growth badge

There are many tissue culture media that can be used to culture cells derived from animals. Some of these are complex and some are simple. Micro-organ cultures are expected to be able to grow in complex media, but we have confirmed that the culture can be maintained in simple media such as Dulbecco's minimum essential media. In addition, the culture may be grown in a medium containing serum or other biological extracts, such as the pituitary extract, but it has been confirmed herein that no serum or other biological extract is required. In addition, organ cultures can be maintained without serum for extended periods of time. In a preferred embodiment of the invention, no growth factor is added to the medium during maintenance of the in vitro culture.

Points related to growth in minimal media are important. Currently, most media or systems for extending and growing mammalian cells use feed cells that contain undetermined proteins or provide the proteins needed to maintain the growth.

As used herein, the term "minimum medium" refers to a medium in which the chemical component is evident, containing only the nutrients necessary for cells to survive and proliferate in culture. Generally, minimal media is free of biological extracts such as growth factors, serum, pituitary extracts, or any other material that is not necessary to support the survival and proliferation of cell populations in culture. For example, minimal media generally includes at least one amino acid, at least one vitamin, at least one salt, at least one antibiotic, at least one indicator used for determining hydrogen ion concentrations, such as phenol red, glucose, and cells. Other miscellaneous components necessary for survival and proliferation. Minimal medium is serum free. Various minimal media are available as the minimum essential media from Gibco BRL (Gettersburg, Maryland, USA).

However, growth factors and regulatory factors need not be added to this medium, but addition of these factors or inoculation of other special cells can be used to enhance, alter or modulate proliferation and cell maturation in culture. Proliferation and activity of cells in culture include various growth factors such as insulin, growth hormone, somatomedin, colony stimulating factor, erythropoietin, epidermal growth factor, liver erythropoietin (hepatopoietin), and hepatocyte growth. Factors can affect Other factors that control proliferation and / or differentiation include prostaglandins, interleukins and naturally occurring negative growth factors, fibroblast growth factors, and transforming growth factor-β based components.

Micro-organ cultures can be maintained in any suitable culture vessel, such as a 24-well or 96-well microplate, and maintained at 37 ° C., 5% CO ₂ . The culture can be shaken for aerobic improvement, with a shake rate of, for example, 12 rpm.

With regard to the culture vessel in which the micro-organ culture (if any) of the subject is provided on or in the stomach, it should be noted that in preferred embodiments such vessels may generally be of any material and / or form. Various materials that can be used to form the container include nylon (polyamide), dark (polyester), polystyrene, polypropylene, polyacrylate, polyvinyl compounds (e.g. polyvinylchloride), polycarbonate (PVC), polytetra Fluoroethylene (PTFE; Teflon), thermox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), intestinal thread suture, cellulose, gelatin, dextran and the like. All of these materials can be woven into a mesh. If micro-organ cultures are to be transplanted by themselves in vivo, it would be desirable to use biodegradable substrates such as polyglycolic acid, bowel suture or gelatin. If the culture should be maintained for a long time or cryopreserved, non-degradable materials, such as nylon, darkon, polystyrene, polyacrylate, polyvinyl, teflon, cotton, and the like may be desirable. A convenient nylon net that can be used according to the invention is Nitex, a nylon filtration net with an average pore size of 210 μm and an average nylon fiber diameter of 90 μm (# 3-210 / 36, Tetko, Inc. N.Y.). Still other embodiments are described below.

In an exemplary embodiment, pancreatic micro-organs containing the island of Langerhans are prepared as a culture of the present invention. This culture is then prepared in encapsulated form to avoid immunorejection. Three general (exemplary) methods of encapsulation may be used. First, the tubular membrane is wound around a frame containing micro-organ explants. The membrane is connected to the polymer graft and then the device to the blood vessel. Glucose and insulin can diffuse freely back and forth through the membrane and control membrane permeability to block the passage of antibodies and lymphocytes, thereby maintaining normal blood glucose in animals undergoing pancreatic resection treated with this device (Sullivan et al. (1991)). Science 252: 718).

As a second method, hollow fibers containing pancreatic explants are fixed (if any) to the polysaccharide alginate. When placed in the abdominal cavity of diabetic animals, the blood glucose level can be lowered and good tissue compatibility can be observed (Lacey et al. (1991) Science 254: 1782; see Example VI). Thus, fibers can be prespun and then loaded into micro-organ explants (Aebischer et al. US Pat. No. 4,892,538; Aebischer et al. US Pat. No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110: 39-44; Jaeger et al. (1990) Prog. Brain Res. 82: 41-46; and Aebischer et al. (1991) J. Biomech Eng. 113: 178-183).

Third, micro-organ islet explants can be injected into microcapsules composed of alginates or polyacrylates (eg, Lim et al. (1980) Science 210: 908; O'Shea et al. (1984) Biochim. Acta 840: 133; Sugamori et al (1989) Trans Am. Soc. Artif. Intern.Organs 35: 791; Levesque et al. (1992) Endocrinology 130: 644; and Lim et al (1992) Transplantation 53: 1180). .

Finally, it should be noted that the culture medium in which the micro-organ culture of the present invention is maintained can be collected and used as a synthetic medium source. The term "synthetic medium" refers to a supernatant free of cultured cells / tissue, and after a certain time the medium will be denatured, including certain perisecreting and / or autosecretory factors produced by the cells and secreted into the culture. Contact with the culture cells to the extent. Examples of such products are insulin, various growth factors and hormones. Such synthetic media can be used as culture media for culturing other types of cells and tissues. Alternatively, the synthetic medium may be used as a novel cell product source such as a growth factor. Such products can be purified from fractionated synthetic media and then purified or substantially purified.

IV. Determination of Biological Properties of Micro-Organ Cultures

The micro-organ cultures of the present invention obtained from normal tissues have been observed to maintain homeostasis with the proliferation of constitutive cells without the entire tissue being grown.

Methods of measuring cell proliferation are known in the art and the most common method is to measure the DNA synthesis characteristic of cell replication. Numerous methods are known in the art for measuring DNA synthesis, either of which may be used in accordance with the present invention. In one embodiment of the invention, DNA synthesis was measured by detecting immunofluorescence using radiolabels ( ³ H-thymidine) or labeled nucleotide analogues (BrdU).

Micro-organ cultures can be prepared and maintained through the proliferation of mature cells as well as the active participation of precursor cells, such as embryo cells in some cases. This micro-organ culture was found to provide a suitable environment for preserving, identifying, separating and facilitating natural evolution of the precursor cells. For example, immature cells in the basal layer were observed to be mature keratinocytes in skin micro-organ cultures, and likewise embryonic pancreatic cells could provide mature pancreatic epithelial cells in micro-organ cultures. The maturation of progenitor cells and their subsequent functionalization as mature cells may induce secretion of certain products, such as specific keratin in epidermal cells, insulin in pancreatic epithelium, Glut2 and glucagon, and albumin and VIII factors in liver micro-organ cultures. Can be measured and monitored.

Micro-organ cultures prepared according to the present invention preserve the normal tissue structure present in vivo. As mentioned above, examples include the maintenance of hair follicles, sweat glands and sebaceous glands in skin micro-organs in vitro, and maintenance of glucagon-secreting cells present in the pancreatic micro-organs following normal expression in vivo. Because these cultures can be maintained under uniform and controlled conditions and are very similar to the microstructure of organs in vivo, they can monitor and measure and control the confusion of natural phenomena and natural phenomena caused by disease, aging or trauma. Provide unique opportunities. In addition, the rapid availability of the technology to study each cell at the identified site on the culture makes it possible to observe the function of each component of the organ and the interaction with each other as well as with the entire organ.

In addition, the micro-organ culture of the subject can be maintained in culture for an extended time. Preferably, the culture can be maintained for at least about 24 hours or more, more preferably for at least about 2 days, even more preferably for at least about 5 days, even more preferably for at least about 7 days, even more preferably Can be maintained in culture for at least about 2 weeks or longer. The micro-organ culture of the present invention is generally maintained for at least 7 days. In one example, skin micro-organ cultures of human, mouse, guinea pig and rat skin have been incubated for at least about 21 days.

As used herein, the term "culture maintainable" is at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90% or more, most Preferably, a cell population of tissue explants in which 95% or more cells maintain viability in culture after a certain period of time.

In a preferred embodiment, the ratio of cell proliferation to cell loss of cells present in the micro-organ culture, such as cell loss by killing or stripping, is equal to 1, ie the number of cells lost and the number of proliferating cells are the same. In another embodiment of the invention, the ratio of cell proliferation to cell loss of cells present in the micro-organ culture is greater than 1, such that the cells proliferate at a faster rate than the cells lost. In the latter case the micro-organ culture is thought to contain a population of cells to be amplified.

V. Application of Micro-Organ Cultures

Application examples in which the micro-organ culture of the present invention can be used include the following:

(a) identification of factors involved in homeostasis of normal tissues and organs;

(b) studying the effects on homeostasis of normal organ tissues and cells on environmental changes, including nutrient changes, and the presence of potential toxicants;

(c) understanding the pathways of change in tissues and cells of organs that arise during and during the onset or trauma;

(d) identification of repair mechanisms that reverse adverse effects in the changing environment associated with the onset or trauma;

(e) modulating the development of cells that differentiate during normal tissue homeostasis;

(f) regulating the development of granular structures present in organs such as hair follicles;

(g) organ replacement / transplantation that retains some of the individual organs but is insufficient for replacement or regeneration of damaged tissue, such as occurs in patients with chronic skin ulcers, various forms of diabetes or chronic liver damage;

(h) use as a tissue / organ equivalent for drug selection and cytotoxicity studies;

(i) use as a diagnostic analysis of proliferative disease;

(j) use as a source of new growth factors;

(k) use as a source of stem / progenitor cells;

(l) use as a source of inducing molecules;

(m) use as selection for inducing molecules;

In more detail, the methods of the present invention can be used to produce skin equivalents in the form of micro-organ cultures. As background art, it will be appreciated that a number of attempts have been disclosed to proliferate epithelial cells in a manner that mimics human skin for wound healing purposes, in particular for burn healing purposes. The skin consists of two tissues. These include (1) epilepsy or epidermis, including fibroblasts sparsely dispersed within the high-density collagen matrix as well as nerve, vascular and adipocytes, and (2) the epidermal basal layer of densely packed active proliferative immature epithelial cells. It is the epidermis. When the basal layer cells replicate, some of the young cells remain in the basal layer while other cells move out of the layer to increase in size and form an envelope that is virtually resistant to surfactants and reducing agents. In humans, cells in the basal layer take about two weeks to reach the marginal or outer layer, after which the cells die and detach. The skin includes various structures such as hair follicles, sebaceous glands and sweat glands. Hair follicles result from the differentiation of keratinocytes, which are densely packed with epidermal infiltration. Open ended vesicles, formed from this inclusion, collect and concentrate the secreted keratin to produce hairy filaments. Alternatively, the epidermal cells filled in the inner surface of the infiltration may secrete fluid (glands) or sebum (sebum gland). Control of formation and proliferation of such structures is unknown. Constant regeneration of healthy skin is achieved by a balanced process where new cells are produced and senescent cells die. In order to cope with anomalies that occur during aging and appear through diseases and traumas that disrupt this balance, an understanding of how accurate control can be performed is required.

In one embodiment of the invention, the microstructure of the micro-organ culture is similar or nearly identical to the microstructure of the skin in vivo, such as retaining epithelial / connective tissue structure. For example, in skin micro-organ cultures, keratinocytes of the epidermis remain bound to connective tissue and the normal tissue structure, including hair follicles, is preserved. Micro-organ cultures obtained from cutaneous tissue sections may also comprise a bottom plate supporting epidermal cells, an extracellular matrix comprising dermal cells and at least one inclusion, such as at least one hair follicle. The bond between dermal epithelial tissue and skin connective tissue facilitates intercellular communication. Moreover, full thickness skin can be propagated in a variety of ways allowing an air interface. Exposure to air of keratinocytes of explants promotes faster differentiation of keratinocytes and more extensive secretion of the keratin layer, which can be critical for skin permeation studies.

Finally, recent studies suggest that skin is an important active component of the immune system (Cooper et al. (1987) The mechanobullous disease.In: Dermatology in General Medicine, 3d.Ed., McGraw Hill, NY (pp. 610-626)). One of the major cell types present in the skin important for various immune activities is Langerhans cells. These cells can be prepared from new skin samples and added to three-dimensional skin cultures to obtain an immunologically complete tissue system. Proliferation of these cells for a long time in a culture state is difficult with conventional tissue culture techniques. The ability to proliferate these cells in a three-dimensional system will be very important in all aspects of the study, including infusion, cytotoxicity and disease mechanisms. This type of skin culture system will have the greatest impact on studies involving autoimmune diseases (eg, systemic lupus erythematosus, bullous pseudocystic ulcer, etc.) that have direct or indirect skin involvement. Thus, the micro-organ cultures of the present invention can be used to study proliferative / differentiating diseases with minimal immunological aspects of the disease. In addition, as an example, psoriasis skin explants can be used to induce drug selection assays to identify agents that can inhibit proliferation of hyperplastic epithelial cells.

Skin is simply one example of a tissue that can proliferate as a micro-organ culture with epithelial tissue supported by interstitial tissue. Other tissues, including epithelial tissues, can also be propagated as micro-organ cultures of the present invention. Epithelial tissue is found in all parts of the body where there is an interface between the organs and the environment, which continually circulates in a wound-free body to form coated tissue for all glass surfaces in the body, including the skin. In some cases, for example, epithelial cells of the pancreas are filled with numerous infiltrations and secrete enzymes into open spaces where organs can function. Lungs are also highly invasive organs, and each indentation in the lungs is present in epithelial cells, spreading air from the environment into the body. These epithelial cells are characteristic properties. The inner lining of the stomach also consists of specialized epithelial cells that contain not only a barrier but also a granular structure that selectively absorbs food. All epithelial cells are supported by connective tissue. Another organ that contains important cell-substrate interactions is the bone marrow.

That is, in another embodiment of the present invention, the microstructure of the micro-organ pancreatic culture is similar or nearly identical to that of the source pancreas in vivo, such as having an epithelial / connective tissue structure. For example, pancreatic micro-organ cultures include pancreatic epithelial cells, such as islet cells, and remain associated with pancreatic connective tissue. Thus, in pancreatic micro-organ cultures normal tissue structure is preserved and normal pancreatic epithelial cell products such as insulin and glucagon are produced.

In another embodiment, the present invention provides a method of producing a bone marrow-derived micro-organ culture, wherein the culture preserves the microstructure of the organ in vivo. As described in Example XV, bone marrow microorganisms were isolated from culture to induce a system corresponding to physiological conditions.

Bone marrow cultures of the present invention can be used to treat diseases or conditions that destroy healthy bone marrow cells or inhibit their functional activity. Implantation of the microorganisms of the present invention may be effective in the treatment of hematologic malignancies and other tumors, including bone marrow. The present invention in this aspect is effective in treating patients whose bone marrow has been aggravated by environmental factors (eg, radiation, toxins, etc.). Replanting of explants derived from the patient's own bone marrow is generally preferred, but such explants may be homologous, such as from other members of the same species, or heterologous, such as from other organisms. One example of a heterologous graft may be a micro-organ culture from reduced pigs for transplantation into humans.

In addition, prolonged proliferation of human hematopoietic progenitor cells is possible provided the necessary epilepsy-derived growth / regulatory factors are provided. This interaction is provided by the microorganisms of the present invention, making these explants the source of stem cells and progenitor cells. In general, hematopoietic progenitor cells of the bone marrow form colonies (“seeds”) in natural packets formed in the interstitial matrix of the bone marrow microorganisms. The major rate limiting factor in the growth of myeloid stromal cells is the relatively low mitotic index of fibroblasts contained in the myeloid stromal cells. Thus, in order to improve the growth of these cells and the placement of extracellular matrix components in these cells, explants may be contacted with agents such as hydrocortisone or other fibroblast growth factors.

If the bone marrow must be cultured for the purpose of treating a particular patient suffering from metastatic disease or hematologic malignancy, the abnormally proliferative cells must first be "removed" from the bone marrow obtained from the patient by physical or chemotherapy means prior to culturing.

Synthetic medium derived from the bone marrow micro-organ culture of the present invention can be used as a source of new or known lymphokines, such as a source of interleukin.

In one aspect, the present invention provides the use of a micro-organ culture of a subject for implantation into an organism. As used herein, the terms “administering”, “introducing” and “transplanting” are used interchangeably to refer to a cell population of the invention via a method or route that localizes the cells to a desired site. By placement in a subject, eg, a heterologous or homologous subject. The cell population may be administered to the subject via any suitable route for delivering the cells to the desired location of the subject where at least some of the cells maintain viability. At least about 5%, preferably at least about 10%, more preferably at least about 20%, even more preferably at least about 30%, even more preferably at least about 40%, most of the cells after administration to the subject Preferably at least about 50% or more should maintain viability. The period of viability of the cells after administration to the subject may be as short as several hours, such as from 24 hours to several days, weeks to months. Methods of administering the cell population of the present invention include transplanting cells into the intestine or wall peritoneum, such as retinal pockets, transplanting cells into organs of the receiving subject, such as the pancreas, liver, spleen, skin, or the like. . The micro-organ of the present invention can also be administered to a subject by implantation under a kidney coat or the like.

The term "subject" as used herein refers to a mammal, such as a primate, such as a human. As used herein, a "heterologous subject" is a subject into which cells of another species are to be introduced or to be introduced. A "homologous subject" is a subject into which a homologous cell is to be introduced or to be introduced. A donor subject is a subject that is placed in culture and / or provides a cell, tissue or organ to be transplanted into a recipient subject. The recipient subject may be a heterologous or isotopic subject. The donor subject may also provide cells, tissues or organs for reintroduction to themselves, ie for autotransplantation.

Microorganisms can be inserted into a rechargeable or biodegradable device to facilitate transplantation of cell populations where heterologous transplantation is used, e.g. swine-human transplantation, which is susceptible to immunogenic attacks by the host, After encapsulation, the recipient can be implanted. Gene products produced by such cells can be delivered, for example, via polymeric devices designed for the controlled delivery of compounds, including proteinaceous biopharmaceuticals, such as drugs. Various biocompatible polymers (including hydrogels) can be used, including biodegradable and non-degradable polymers, to create grafts capable of sustained release of gene products by the population of cells of the invention at specific target sites. The manufacture of such grafts is generally known in the art. Examples include Concise Encyclopedia of Medical & Dental Materials, ed. By David Williams (MIT Press: Cambridge, MA, 1990); Sabel et al., US Pat. No. 4,883,666; Aebischer et al., US Pat. No. 4,892,538; Abishire et al., US Pat. No. 5,106,627; Lim, US Pat. No. 4,391,909; And Sefton, US Pat. No. 4,353,888. Cell populations of the invention can be administered in the form of pharmaceutically acceptable carriers or diluents, such as sterile saline and aqueous buffer solutions. The use of such carriers and diluents is known in the art.

In one embodiment, micro-organ cultures of the invention can be used for wound healing. Repair of skin lesions is known to be a very complex process involving replication of epithelial cells as well as primitive epithelial cell migration in response to molecular signals derived from connective tissue at the base. Skin micro-organ cultures are described herein as a model of wound healing. The factors controlling healing can be carefully monitored under controlled culture conditions. In addition, when micro-organ cultures are isolated from natural blood sources, the healing process can be analyzed without consideration of blood system factors or cells that add complexity. Normal epidermis has low mitotic activity as it circulates cells every 200 to 300 hours. However, cuts in the epidermis cause rapid mitotic activity, causing the cells to divide up to 10 times faster, depending on the extent and symptoms of the wound (Pinkus H. (1951) J. Invest. Dermatol. 16: 383-). 386).

As demonstrated in Example II, skin micro-organ cultures show up to a 10-fold increase in proliferation for several days. In this example, the edge of the wound is comparable to micro-organ culture. This increase in proliferation is similar to the events associated with wounds, providing a unique opportunity to study the wound healing process. Moreover, epidermal explants of the present invention can be applied to chronic wounds (Example IX) and form viable grafts capable of growing hair, as demonstrated by in vivo experiments through subsequent examples. Example XI).

In addition, the epidermal microorganisms of the present invention can be used for the treatment of burn patients. The need for skin replacement therapy is evident for burn patients, and several research centers in the United States and Europe have used cultures of human keratinocytes and autografts that can permanently cover burn wounds and chronic ulcers. (Eisinger et al., (1980) Surgery 88: 287-293; Green et al., (1979) Proc. Natl. Acad. Sci. USA 76: 5665-5668; Cuono et al., (1987) Plast. Reconstr Surg. 80: 626-635). These methods are often unsuccessful and recent studies have indicated that abnormalities in one or more connective tissue components formed under the implanted epidermal layer may result in bullous and / or skin erosion of the healing graft (Woodley et al., (1988) JAMA 6: 2566-2571. The skin culture system of the present invention provides a skin equivalent of both the epidermis and the dermis to overcome the problems characteristic of cultures of conventionally used keratinocyte grafts.

In another embodiment, the micro-organ culture system of the present invention can provide a medium for introducing genes and gene products in vivo for use in gene therapy. For example, recombinant DNA techniques can be used to place genes missing in a patient under the control of a virus or tissue specific promoter. Such recombinant DNA constructs can be used to transform or transfect all cells or only specific cells of the micro-organ culture system of the present invention. Micro-organ cultures expressing the active gene product can be transplanted into an individual whose product is defective.

As such, the method of using the micro-organ culture of the present invention for gene therapy has many advantages. First, since the culture contains eukaryotic cells, the gene product will be properly expressed and processed into the active product in the culture. Secondly, the techniques of gene therapy are only useful when the number of transfected cells can be substantially increased to a degree that is of clinical value, relevance and usefulness. The cultures of the present invention allow for expansion and amplification of transfected cell numbers. will be.

In another embodiment of the invention, mutant micro-organ cultures can be used to provide for ease of gene introduction. For example, micro-organ cultures containing recombinant viral expression vectors can be used to deliver recombinant virus to cells in contact with the culture, such as by implantation, to mimic viral transmission in vivo. Therefore, this system is a method to perform gene transduction more effectively than the techniques of current DNA transfection.

Thus, the cells of the micro-organ cultures of the invention can be modified to express gene products. As used herein, the term "gene product" refers to proteins, peptides and functional RNA molecules. Generally, the gene product encoded by the nucleic acid molecule is the desired gene product to supply to the subject. Examples of such gene products are proteins, peptides, glycoproteins, and lipoproteins normally produced in the organs of a recipient subject. For example, gene products that can be supplied as gene replacements for defective organs present in the pancreas include insulin, amylase, protease, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, ribonucleases, des Oxyribonuclease, triacylglycerol lipase, phospholipase A ₂ , elastase and amylase; Gene products normally produced in the liver include coagulation factors such as coagulation factors VIII and IX, UDP glucuronyl transferase, ornithine transcarbanoylase and cytochrome p450 enzymes, and serum adenosine processing or low density lipoproteins. Adenosine deaminase for cellular uptake of foreign bodies; Gene products produced in the thymus include serum thymus factor, thymic fluid factor, thymopoietin and thymosin a1; Gene products produced in gut cells include gastrin, secretin, cholecystokinin, somatostatin and substance P. Alternatively, the encoded gene product is one that induces expression of the desired gene product by the cell (eg, a genetic material encoding a transcription factor that induces transcription of the gene product to be supplied to the subject). In another embodiment, the recombinant gene may provide for a heterologous protein, such as not natural to the cell being expressed. For example, various human MHC components may be provided to non-human microorganisms to support injection in human receptors. Alternatively, the mutant gene may also inhibit the expression or action of a donor MHC gene product normally expressed in micro-organ explants.

The nucleic acid molecule introduced into the cell must be in a form suitable for the expression of the gene product encoded by the nucleic acid in the cell. Thus, the nucleic acid molecule must contain the coding and regulatory sequences necessary for gene (or part thereof) transcription, and if the gene product is a protein or peptide, the promoter, enhancer and polyadenylation signals, as well as the coding, are required for translation of the nucleic acid molecule. And a N-terminal signal sequence for transporting or secreting a protein or peptide, such as a protein or peptide, to the cell surface.

Nucleotide sequences (eg, promoter and enhancer sequences) that regulate the expression of a gene product are selected according to the cell type to which the gene product is to be expressed and the required expression rate of the gene product. For example, a promoter known to specifically express a gene bound to a promoter for each cell type can be used. For example, a promoter specific for myoblast gene expression may bind to the gene so that the gene product is specifically expressed only in muscle. Muscle specific regulatory factors known in the art include the upstream region from dystrophin gene (Klamut et al., (1989) Mol. Cell Biol. 9: 2396), creatine kinase gene (Buskin and Hauschka, (1989) Mol. Cell Biol. 9: 2627), and upstream regions from the troponin gene (Mar and Ordahl, (1988) Proc. Natl. Acad. Sci. USA. 85: 6404). Negative response factors present in keratin genes mediate transcriptional inhibition (Jho Sh et al., (2001). J. Biol. Chem). Regulators that are specific for other cell types are known in the art (e.g., albumin enhancers for liver specific expression; insulin regulators for pancreatic islet cell specific expression; various neuronal cell specific regulators such as neurons). Dystrophin, neuroenolase and A4 amyloid promoter). Alternatively, regulatory factors may be used that can induce constitutive gene expression in a variety of cell types, such as viral regulatory factors. Examples of viral promoters commonly used to induce gene expression include promoters from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40 and retrovirus LTR. Alternatively, regulatory factors may be used that allow for inducible expression of the bound gene. The use of inducible regulatory factors (such as inducible promoters) can regulate the production of gene products in cells. Examples of very useful inducible regulatory systems that can be used in eukaryotic cells include hormonal regulators (e.g. Mader, S. and White, JH (1993), Proc. Natl. Acad. Sci. USA 90: 5603-5607), Synthetic ligand modulators (eg Spencer, DM et al., 1993) Science 262: 1019-1024) and ionizing radiation modulators (eg Manome, Y. et al. (1993) Biochemistry 32: 10607-0613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1014-10153. Another developable tissue specificity or inducible regulatory system can also be used in accordance with the present invention.

There are a number of methods known in the art for introducing genetic material into cells that can be used for the cell modification of the present invention. In one embodiment, the nucleic acid may be in the form of a naked nucleic acid molecule. At this time, the nucleic acid molecule to be introduced into the cell to be modified consists only of the nucleic acid encoding the gene product and the essential regulator. In addition, nucleic acids (including essential regulatory elements) encoding gene products may be included in plasmid vectors. Examples of plasmid expression vectors are CDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). In another embodiment, the nucleic acid molecule to be introduced into the cell is also included in the viral vector. At this time, the nucleic acid encoding the gene product is inserted into the viral genome (or partial viral genome). Regulatory factors that induce the expression of a gene product may be embedded in the viral genome with the nucleic acid (ie, bound to a gene inserted into the viral genome) or supplied by the viral genome itself.

Naked nucleic acids can be introduced into cells using calcium phosphate mediated transfection, DEAE-dextran mediated transfection, electroporation, liposome mediated transfection, direct injection and receptor mediated uptake.

Naked nucleic acids, such as DNA, can be introduced into cells by forming precipitates containing nucleic acids and calcium phosphate. For example, HEPES buffered saline solution is mixed with a solution containing calcium chloride and nucleic acid to form a precipitate, which is then incubated with the cells. Glycerol or dimethyl sulfoxide shock steps may be added to increase the amount of nucleic acid taken up by a particular cell. CaPO ₄ -mediated transfection may be used to stably (or transiently) transfect cells, which method may only be applied to in vitro modification of cells. Protocols of CaPO _4- mediated transfection are described in Current Protocols in Molecular Biology, Ausubel, FM et al. (Eds.) Greene Publishing Associates, (1989), Section 9.1 and Molecular Cloning: A Laboratory Manual, 2nd Edition , Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.32-16.40 or other standard experiments.

Naked nucleic acids can be introduced into cells by forming a mixture of nucleic acids and DEAE-dextran and culturing the mixture with the cells. A dimethylsulfoxide or chloroquine shock step may be added to increase the amount of nucleic acid uptake. DEAE-dextran transfection is applicable only to in vitro cell modifications and can be used to temporarily introduce DNA into cells, which is undesirable for constructing stably transfected cells. Thus, this method can be used for short term production of gene products and is not the best method for long term production of gene products. Protocols of DEAE-dextran mediated transfection are described in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Section 9.2 and Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.41-16.46 or other standard experiments.

Naked nucleic acids can also be introduced into cells by incubating the cells and nucleic acids together in a suitable buffer and treating the cells with a high voltage electric pulse. The efficiency of introducing nucleic acids into cells by electropenetration is influenced by the applied electric field, the length of the electric pulse, the temperature, the form and concentration of the DNA and the ionic composition of the medium. Electropenetration can be used to stably (or temporarily) transfect various cell types. Useful protocols for electropenetrating cells are described in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Section 9.3 and Molecular Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.54-16.55 or other standard experiments.

Another method by which naked nucleic acids can be introduced into cells is liposome mediated transfection (lipofection). At this time, the nucleic acid is mixed with a liposome suspension containing cationic lipids. This DNA / liposomal mixture is then incubated with the cells. Liposomal mediated transfection can be used to stably (or transiently) transfect cells in in vitro culture. This protocol is described in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Section 9.4 and other standard experiments. In vivo gene delivery has also been performed using liposomes. See, eg, Nicolau et al. (1987) Meth. Enz. 149: 157-176; Wang and Huang (1987) Proc. Natl. Acad. Sci.USA. 84: 7851-7855; Brigham et al. (1989) Am. J. Med. Sci. 298: 278; And Gould-Fogerite et al. (1989) Gene 84: 429-438.

Naked nucleic acids can also be introduced into a cell by injecting the nucleic acid directly into the cell. In the case of in vitro culture of cells, DNA can be introduced by microinjection. Since each cell is microinjected, this approach is very labor intensive when transforming multiple cells. However, microinjection is the best method for the production of mutant animals (described in detail below). In such cases, DNA is stably introduced into fertilized oocytes that can develop into animals. The resulting animal will contain cells carrying the DNA introduced into the oocytes. Direct injection has also been used to introduce naked DNA directly into cells in vivo (eg, Acsadi et al. (1991) Nature 332: 815-818; Wolff et al. (1990) Science 247: 1465-1468). ). Delivery devices (eg, “gene guns”) can be used to inject DNA into cells in vivo. Such devices are commercially available (eg Biorad products).

Naked nucleic acids can form complexes with cations, such as polylysine, that bind to ligands of cell-surface receptors that are taken up by receptor mediated cellular uptake (eg, Wu, G. and Wu, CH (1988), J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267: 963-967 and US Pat. No. 5,166,320). The binding of nucleic acid-ligand complexes to receptors facilitates the uptake of DNA by receptor mediated cellular uptake. To avoid degradation of the complex by intracellular liposomes, DNA-ligand complexes bound to adenovirus capsids that naturally degrade endosomes and release material into the cytoplasm can be used (eg, Curiel et al. (1991). Proc. Natl. Acad. Sci. USA. 88: 8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2122-2126). Receptor-mediated DNA uptake can be used to introduce DNA into cells in vitro or in vivo, and additionally by using a ligand that binds to a receptor that is selectively expressed only in the target cell of interest, the DNA targets only a particular cell type. There is also a feature to choose from.

In general, when introducing naked DNA into cells in culture (eg, using one of the transfection techniques described above), only a small amount of cells (about 1 out of 10 ⁵ ) will generally produce the transfected DNA itself. Into the genome of the DNA (ie, the DNA remains episomal in the cell). Therefore, in order to identify cells which have absorbed heterologous DNA, it is advantageous to transfect the cells with a nucleic acid of interest and a nucleic acid encoding a selectable marker. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. Selectable markers can be introduced through a plasmid such as the gene of interest or through a separate plasmid.

A preferred method of introducing a nucleic acid encoding a gene product into a cell is to use a viral vector containing a nucleic acid encoding a gene product, such as cDNA. Infection of cells with viral vectors has the advantage that a large amount of cells can accommodate the nucleic acid, so there is no need to select a cell containing the nucleic acid. In addition, a molecule encoded in a viral vector, such as a cDNA contained in a viral vector, is effectively expressed in cells that have absorbed the viral vector nucleic acid, so that the viral vector system can be used in vitro or in vivo.

It is well established that defective retroviruses are useful for gene delivery for gene therapy purposes (Miller, A.D. (1990) Blood 76: 271). Recombinant retroviruses can be produced by inserting a nucleic acid encoding the gene product into the retroviral genome. In addition, a portion of the retroviral genome may be removed to confer replication deficiency on the retrovirus. Such replication defective retroviruses are packaged into virions that can be used to infect target cells with the use of helper viruses according to standard techniques. Recombinant virus construction and methods for cell infection in vivo or in vitro using the virus are described in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Section 9.10-9.14 and other standard experiments. Examples of suitable retroviruses are pLJ, pZIP, pWE and pEM known in the art. Examples of suitable packaging virus cell lines are ψCrip, ψ2 and ψAm. Retroviruses have been used to introduce various genes into a variety of different types of cells, such as epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and / or in vivo (e.g., Eglitis et al. 1985) Science 230: 1395-1398; Danosand Mulligan (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 3014-3018 Armentano et al., (1990) Proc. Natl. Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8039-8043; Feri et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89 Kay et al. (1992) Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150: 4104-4115; US Patent No. 4,868,116; US Patent 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Publication WO 92/07573). Retroviral vectors require the division of target cells so that the retroviral genome (and heterologous nucleic acid inserted therein) is integrated into the host genome in order to stably introduce the nucleic acid into the cell. Thus, it may be necessary to stimulate replication of target cells.

Although the genome of adenoviruses encodes and expresses the gene product, it can be manipulated to be inactivated in terms of its ability to replicate in normal lytic virus life cycle. See, eg, Berkner et al. (1988) BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 431-434; And Rosenfeld et al. (1992) Cell 68: 143-155. Suitable adenovirus vectors from adenovirus strain Ad type 5 dl324 or other adenovirus strains (eg, Ad2, Ad3, Ad7, etc.) are known in the art. Recombinant adenoviruses do not require dividing cells to enhance their effectiveness as gene transfer mediators, so that various cell types such as airway epithelium (Rosenfeld et al. (1992) cited above), endothelial cells (Lemarchand et al. (1992) Proc Natl. Acad. Sci. USA 89: 6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90: 2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2581-2584. In addition, introduced adenovirus DNA (and heterologous DNA inclusions) remain episomal rather than integrated into the genome of the host cell, resulting in insertional mutagenesis in situations where the introduced DNA begins to integrate into the host genome. Potential problems (eg, retroviral DNA) can be avoided.

In addition, the carrying capacity of the adenovirus genome for heterologous DNA is significantly greater (up to 8 kilobases) compared to other gene transfer vectors (Berkner et al., Cited above; Haj-Ahmand and Graham (1986) J. Virol. 57: 267). Most of the replication defective adenovirus vectors currently in use are those in which all or part of the virus E1 and E3 genes are deleted and retain about 80% of the adenovirus genetic material.

Adeno-associated virus (AAV) is a naturally occurring defective virus that requires other viruses such as adenovirus or herpes virus as a helper virus for efficient replication and a productive life cycle (Muzyczka et al. Curr. Topics In Micro.And Immunol. (1992) 158: 97-129). In addition, it is one of the few viruses that can integrate their DNA into non-dividing cells, which shows a high stable integration rate (eg Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7 Samulski et al. (1989) J. Virol. 63: 3822-3828; and McLaughlin et al. (1989) J. Virol. 63: 1963-1973. Vectors containing only about AAV 300 base pairs can be packaged and integrated. The space for heterologous DNA is limited to about 4.5 kb. Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260 can be used to introduce DNA into cells. Various nucleic acids have been introduced into various cell types using AAV vectors (eg, Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470; Tratschin et al. (1985) Mol. Cell Biol. 4: 2072-2081; Wondisford et al. (1988) Mol.Endocrinol. 2: 32-39; Tratscin et al. (1984) J. Virol. 51: 611-619; and Flotte et al. (1993) J. Biol. Chem. 268: 3781-3790).

The efficiency of certain expression vector systems and methods of introducing nucleic acids into cells can be assessed by standard techniques commonly used in the art. For example, cell-introduced DNA can be detected by filter hybridization techniques (eg, Southern blotting), and RNA produced by transcription of introduced DNA can be, for example, Northern blotting, RNase blocking or reverse transcriptase-polymerization. It can be detected by enzyme chain reaction (RT-PCR). Gene products may be determined by appropriate assays, such as immunological detection of produced proteins using specific antibodies, or by functional assays such as enzymatic assays, which measure the functional activity of gene products. If the gene product to be expressed by the cell cannot be easily analyzed, the expression system can be optimized by binding the reporter gene to the regulatory factor and vector used first. Reporter genes encode gene products that are easily detectable and can be used to assess the efficacy of this system. Standard reporter genes used in the art include genes encoding β-galactosidase, chloramphenicol acetyl transferase, luciferase, GFP / EGFP and human growth hormone.

If the method used to introduce nucleic acids into a cell population modifies most cells so that the gene product is efficiently expressed by the cells (eg, as is often the case with viral expression vectors), then the population of modified cells will be modified. It can be used as is without further separation or secondary cloning of each cell present in. That is, gene products can be produced sufficiently by the cell populations so that no further separation of cells is necessary. Alternatively, in order to isolate cells expressing gene products efficiently, it is also desirable to propagate a single modified cell into a homogeneous cell population with the same modification. Such homogeneous cell populations can be prepared by separating modified single cells by limiting dilution cloning and then expanding single cells in culture into clonal cell populations according to standard techniques.

As used herein, the term "mutant cell" refers to a cell into which a nucleic acid sequence is inserted or introduced that is partially or wholly heterologous, such as foreign, to the cell to be inserted or introduced. The mutant cell may also be a cell into which a nucleic acid homologous to the endogenous gene of the cell is inserted. In such cases, homologous nucleic acids are designed to be inserted or inserted into the cell genome in such a way that it can modify the genome of the cell into which it is inserted. For example, a homologous nucleic acid inserts a homologous nucleic acid to knock out a particular phenotype or to insert that is different from the trend of the native gene. Nucleic acid inserted into a cell may include one or more transcriptional regulatory sequences and other nucleic acids such as introns that may be required for optimal expression of the selected nucleic acid.

As another aspect of the present invention, the micro-organ culture of the present invention can be used to help diagnose and treat malignant diseases and diseases. For example, biopsies of organs (eg, skin, kidneys, liver, etc.) can be taken from patients suspected of hyperproliferative or neoplastic disease. When biopsy explants are cultured according to the methods of the present invention, proliferative cells of the explants are expanded clonally during the culture. This increases the chance of investigating the disease, thereby increasing the accuracy of the diagnosis. In addition, micro-organ cultures of patients can be used to select cytotoxic compounds and / or pharmaceutical compounds in vitro, thus killing the most potent compounds, ie malignant or diseased cells but not affecting normal cells. Compounds that do not fall can be identified.

Another aspect of the present invention provides a method of using the micro-organ culture of the present invention for the purpose of selecting various compounds such as cytotoxic compound growth / regulatory factors, pharmaceutical agents and the like. For example, the need for thorough testing of potentially toxic compounds is universally recognized, and the need to develop sensitive and reproducible short-term in vitro assays to evaluate drugs, cosmetics, food additives and pesticides is self-evident. will be. The micro-organ cultures described herein make tissue equivalents available as analytical substrates and provide the advantages of normal cell interaction in a system very similar to in vivo conditions.

For this purpose, the culture is maintained in vitro and then exposed to the test compound. The activity of a cytotoxic compound can be measured through its ability to modulate the phenotype (eg killing) of cells present in the explant. Such measurements can be readily performed using vivid staining techniques, marker expression, etc. For example, the effects of growth / regulatory factors can be assessed by analyzing the cell contents of the culture, such as total cell numbers and differentiated cell numbers. have. This assay can be performed using standard cytological and / or histological techniques, including immunocytochemical techniques using antibodies that define type specific cell antigens. The effects of various drugs on normal cells cultured in the system of the present invention can also be evaluated. For example, drugs that reduce the proliferation of psoriasis tissues can be identified.

As an exemplary embodiment of the method induced for detection of an agent that stimulates proliferation of cells in an explant, the method comprises separating a tissue explant from the subject, wherein the cells of the explant The population includes the microstructure of the organ or tissue from which the explant is isolated, for example, the explant contains at least one cell characterized by at least about 1.5 mm ⁻¹ allele and having the ability to proliferate. This explant is incubated with the candidate compound. The level of cell proliferation in the presence of the candidate compound is measured and compared with the level of cell proliferation measured in the absence of the candidate compound. A statistically significant increase in cell proliferation level in the presence of candidate compounds suggests that it can be used as a cell proliferative agent.

As used herein, the term "candidate compound" or "candidate" means an agent to be tested or tested for proliferation, anti-proliferation, differentiation, anti-differentiation or antiviral activity. Such agents can be, for example, small organic molecules, biological extracts, and recombinant products or compositions.

Methods of measuring cell proliferation are known in the art and the most common methods include measuring DNA synthesis, which is characteristic of cell replication. There are a number of methods known in the art for measuring DNA synthesis, either of which may be used in accordance with the present invention. In one embodiment of the invention, immunofluorescence detection using radiolabeled ( ³ H-thymidine) or labeled nucleotide analogues (BrdU) was used for the measurement of DNA synthesis.

Another embodiment provides a method of identifying a cell proliferation inhibitor. The method includes providing a tissue explant as described above, contacting the explant with a candidate compound, and measuring the level of cell proliferation in the presence of the candidate compound. The statistically significant decrease in the level of cell proliferation in the presence of candidate compounds suggests that it is a cell proliferation inhibitor.

In exemplary embodiments, enhancers and inhibitors of cell proliferation (also referred to herein as antiproliferative agents) can be used to control hair growth, for example according to the desired effect.

The growth of hard keratin fibers such as wool and hair depends on the proliferation of epidermal cells. Hair follicle stem cells in the house are very active, producing hair fibers through rapid proliferation and complex differentiation. There are three distinct stages of this hair circulation: growth (proliferation), degeneration (degeneration) and rest (rest). Epidermal stem cells of hair follicles are activated by epidermal papilla during the late resting period. This is called "protrusion activation". In addition, such stem cells are considered to be pluripotent stem cells that generate hair and hair follicle structures as well as sebaceous glands and epidermis. Cell proliferation agents and cell proliferation inhibitors are a means of changing the dynamics of the hair growth cycle, such as stopping the proliferation of hair follicle cells, specifically stem cells of hair follicles.

Inhibitors of hair follicle cell proliferation can be used as a method that can reduce human hair growth unlike conventional removal methods such as cutting, shaving or hair removal. For example, hair follicle inhibitors identified in accordance with the methods of the present invention can be used for the treatment of alopecia, such as hairy hyperplasia, characterized by rapid or dense abnormal growth of the hair. In exemplary embodiments, such inhibitors can be used to treat hirsutism, a disease indicated by abnormal hair loss. The use of such inhibitors may provide a way to prolong the hair loss period.

Inhibitors of hair follicle cell proliferation can also be used to protect hair follicle cells from cytotoxic agents that must be advanced to the S phase of the cell cycle. Treatment with such inhibitors induces hair follicle cells into a stationary phase to provide protective action, for example, by inhibiting the cells from progressing to the S phase to prevent mitotic catastrophe or apoptosis of hair follicle cells. For example, inhibitors of hair follicle cell proliferation can be used in patients undergoing chemotherapy or radiotherapy, which typically causes hair loss. Inhibitor treatment can protect hair follicle cells from death, which may be manifested by activation of a cell death program by inhibiting the progression of cell circulation during this treatment. After treatment is terminated, there is no need to inhibit hair follicle cell proliferation, so the inhibitor treatment can be eliminated.

However, in vitro models of hair growth are needed to begin to identify the molecular mechanisms underlying hair growth control and to test for potential drugs that affect hair. In one aspect of the invention, the method comprises between the epidermal layer of the hair follicle and the matrix components of the hair follicle (epidermal papilla), such as stem cells, exogenous cells, stromal cells and internal myocytes, which retain the microstructure of the hair follicles. Used to prepare hair follicle micro-organ explants bearing the interaction of. As demonstrated in the examples that follow, hair growth is observed in the micro-organ cultures in the absence of serum, such as in minimal media. Importantly, the present invention provides a hair follicle culture that provides hair follicles that are substantially at rest, such as at rest. As demonstrated below, resting hair follicle explants may be activated into growth phase follicles that are proliferative in in vitro culture, and in certain embodiments may be activated in a synchronous manner. Epidermal stem cells of hair follicles at the beginning of transient proliferation offer a unique opportunity to understand the activation of the growth phase, for example mediated by peripheral secretory factors and / or autosecretory factors produced in various tissues of the hair follicle organs.

In addition, the micro-organ culture of the present invention provides a system that can identify agents that modulate the activation or inactivation of hair follicles, such as those that can promote or inhibit hair growth. In one embodiment, resting phase (rest state) hair follicle explants, as described in Example XVIII below, are contacted with various test agents, and then the level of stimulation of hair follicles is measured. For example, the transition from the resting phase of hair follicle stem cells to growth phase can be monitored by observing the mitotic index of the hair follicle cells or using other similar methods that can measure proliferation. As an example, FIG. 17 may use thymidine incorporation to determine the relative level of stem cell activation present in an explant in the presence or absence of a test compound (FGF in this figure) and increased proliferation may result in hair growth promoting activity. This suggests that this is a test formulation.

In reverse analysis, growth phase micro-organ explants, such as, for example, activated Sencar explants or growth factor stimulated explants (eg, FGF-stimulated) described in the appended examples, are provided in culture. do. For example, test agents that inhibit the growth of hair follicle stem cells relative to untreated hair growth explants may be considered for use as telogenic agents, which further inhibit hair growth.

In yet another embodiment, inhibitors of cell proliferation (eg, tumor formation and growth) identified by subject analysis can be used to inhibit the growth of neoplastic or hyperproliferative cells. Preferred embodiments of the present invention relate to inhibition of epithelial tumor formation and growth (US Patent Application No. 08 / 385,185, filed Feb. 7, 1995). The regulation of cell proliferation changes and the interaction between cells and their surrounding cells results in tumor formation as a result of disorders that induce infiltration and metastasis. The disruption of the interrelationship between increased cell numbers resulting from cell division and departure from the cell cycle due to differentiation or cell death impedes the regulation of cell proliferation. In normal tissues, homeostasis is maintained by the division of each stem cell, so that only one of the two daughter cells is maintained in the stem cell compartment and another daughter cell is guaranteed to go through a differentiation pathway. Cairns , J. (1975) Nature 255: 197-200. Thus, regulation of cell proliferation is due to signals affecting these processes. These signals can be positive or negative and tumorigenicity is obtained by genetic changes affecting these regulatory points.

As described in Example IX and shown in FIG. 12, skin micro-organ cultures of the present invention were used to identify cell proliferation agents and inhibitors of cell proliferation. As described in Example IX, TGF-β was tested and found to act as an inhibitor of cell proliferation. Activin proteins that are members of the TGF-β superfamily have also been shown to inhibit the proliferation of epithelial cells. These results indicate that there may be another constituent of the TGF-β family that is involved in inhibiting proliferation of epithelial cells. The data suggest a role for proteins of the TGF-β family as important regulators of epithelial homeostasis and in inhibiting epithelial tumor formation and growth in vivo.

Another aspect of the invention relates to a method for identifying cell differentiation agents, ie compounds that cause cell differentiation. The method comprises at least one cell having a differentiation ability, including separating the cell population from the subject with a microstructure of the organ or tissue from which the cell is separated and having a surface area to volume index of at least about 1.5 mm ⁻¹ . . The cells are then added to the culture for at least about 24 hours and contacted with the candidate compound. The level of cell differentiation in the presence of the candidate compound is then measured and compared with the level of cell differentiation in the absence of the candidate compound. Statistically significant increases in cell differentiation levels in the presence of candidate compounds suggest that the candidate compounds are cell differentiation agents. Differentiation as described herein refers to a cell having a form and / or function inherent in the cell as well as obtained forms and / or functions that are different from or different from these. Typically these forms and functions are characteristic of mature cells. Differentiation of the cell populations of the invention can be monitored by measuring production and / or secretion of specific cell products.

In a similar manner, the present invention also relates to a method for identifying inhibitors of cell differentiation. According to the same protocol as described above, the level of cell differentiation in the presence of the candidate compound is measured and compared with the level of cell differentiation in the absence of the candidate compound. Statistically significant reductions in cell differentiation levels in the presence of candidate compounds suggest that they are inhibitors of cell differentiation.

In another embodiment, the subject culture allows for establishing an in vitro model for viral infection. For example, epithelial or squamous tissue can be isolated and include, for example, herpes viruses such as herpes simplex virus 1, herpes simplex virus 2; It can be infected with varicella-zoster virus or human papilloma virus, eg, human papilloma virus 1-58, eg, human papilloma virus, such as HPV-6 or HPV-8. Similarly, the liver model can be provided for hepatitis, for example as an explant infected with hepatitis virus, eg, hepatitis A virus, hepatitis B virus or hepatitis C virus. Virus infected tissue explants may be used to identify inhibitors of viral infectivity by the methods of the present invention. As noted above, certain micro-organ cultures are provided and optionally contacted with a virus that infects the cells to produce a virus infected cell population. Virus infected cells are then contacted with the candidate compound and the level of viral infectivity is measured in the presence of the candidate compound. The measured level of viral infectivity in the presence of the candidate compound is compared to the level of viral infectivity in the absence of the candidate compound. A statistically significant reduction in the infectivity level of the virus in the presence of the candidate compound suggests that it is an inhibitor of viral infectivity.

Methods of measuring viral infectivity are known in the art and vary depending on the type of virus used. For example, one method that can be used to measure viral infectivity levels is a method of measuring production levels in microorgan culture media with infected cells of microorgan cultures or gene products specific for the particular virus to be tested. . For example, to determine the level of infectivity of a hepatitis virus, such as hepatitis B virus, to cells in micro-organ cultures, production of hepatitis protein and hepatitis DNA can be quantified. In general, micro-organ culture media can be incubated with antibodies against selected viral proteins and SDS-polyacrylamide gels, immunoreactive proteins analyzed by various methods known in the art on ELISA. For example, in order to measure the production of hepatitis B virus surface antigen, the microorgan culture medium of hepatitis B virus and microorganisms originating from previously incubated microorganisms were sampled at regular intervals every day and the ELISA as described by the manufacturer. The surface antigen can be analyzed by the (Abbott) method. This method for quantification can be modified using serially diluted standard surface antigens (CalBiochem). Statistically significant reductions in the accumulation of hepatitis B virus surface antigens in the culture medium indicate that the candidate compounds tested are inhibitors of the hepatitis virus infection ability.

In addition to measuring the levels of HBsAg in micro-organ culture medium, the newly synthesized hepatitis B virus DNA from cell extracts from micro-organ culture origin was subjected to PCR amplification of DNA followed by HBV pre-S (HBsAg coding) region as a probe. It can be detected and quantified by Southern blot analysis using labeled primer pairs in Sambrook, J. et al. (1989) Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd ed., Vol. 2, pp. 10.14-10.15]. It can be relatively quantified by a viscometer and identified by the scintillation count of the corresponding band. Reduction of newly synthesized viral DNA indicates that the candidate compound tested is an inhibitor of hepatitis virus infectivity.

In another example, gag, pol and env protein products of retroviruses, such as human immunodeficiency virus (HIV), can also be measured by the techniques described above and other standard techniques known in the art. For example, intracellular pol protein expression in HIV-infected micro-organ cultures can be measured by incubating cell extracts with anti-pol antibodies or immunoreactive proteins analyzed on collected AIDS patient serum and SDS / polyacrylamide gels. have. To determine the infectivity of the herpes virus, eg, Epstein / Barr virus (EBV) in the micro-organ culture of the present invention, EBV DNA and EBV-induced nuclear antigen production can be analyzed using the methods described herein. Can be.

Micro-organ cultures of the invention can also be used to promote wound healing in a subject. Thus, the present invention further relates to a method of promoting wound healing in a recipient subject. The method includes separating from the donor subject a cell population having a surface area to volume index of at least about 1.5 mm ⁻¹ . Typically, the cell population is added in culture for at least about 24 hours. The cell population can then be applied to the wound of the recipient subject. In one aspect, the wound or lesion is a slow healing or chronic wound, eg, a wound associated with diabetes, eg, a burn, eg an ulcer. As demonstrated in Examples X and XI, the skin micro-organ cultures of the present invention can be used as micro explants to be applied to chronic wounds (Example X) and to survive hair transplants (Example XI). ) Can be formed.

Still, in another embodiment, the subject micro-organ explant is provided for test analysis for cytotoxicity or stimulation. In an exemplary embodiment, the subject method provides an in vitro test technique for visual and epidermal stimulants. Methods very similar to the above include the topical application of liquid, solid granules or gelled materials (eg, cosmetics) to the micro-organ cultures of the invention, followed by detecting the effect produced in the culture.

Currently, the potential ocular and skin irritation of many chemicals, household cleaning products, cosmetics, paints and other substances is evaluated by direct application to animal or human subjects. However, as most appreciated in the industry, the method is overwhelmingly unsuitable for public response. The method of the present invention provides another assay that does not need to cause animal sacrifice or permanent injury and also provides data in the desired format. In an exemplary embodiment, skin micro-organ cultures are induced in accordance with the present invention. Cultured explants are contacted with a test preparation, such as a cosmetic preparation, and cell viability is assessed at some time point after exposure. In a preferred embodiment, MTT assay (based on tetrazolium reduction by functional mitochondria) is used to score for viability.

The micro-organ cultures herein further identify the factors involved in normal homeostasis of tissues and cells and the normal homeostasis of tissues and cells against changes in the environment of the cells, including changes in nutrients and the presence of potentially toxic agents. To study the effects on and to study tissue and intracellular changes pathways induced during the early and progression of pathology or trauma, to identify repair mechanisms that reverse adverse effects in the changed environment associated with pathology or trauma, and to differentiate during normal homeostasis of tissues Control of the development and development of certain structures in the tissue (eg hair follicles), and parts of the individual organs are preserved but have a chronic skin ulcer or have a healing deficiency induced by an inadequate blood supply or local skin Feet in non-healing patients, such as in symptoms known as type or type II That may be used to perform the study of insufficient authority supplement sikineunde replace or restore damaged tissue as described.

Summary of the Invention

The present invention provides in-vitro micro-organ cultures that address the aforementioned needs. The main characteristics of the micro-organ culture of the subject include the ability to remain in the culture for a relatively long time, such as at least about 24 hours, preferably at least 7 days, as well as cell-cell interactions similar to those found in the source organ and Preservation of organ microstructures that facilitate cell-epileptic interactions and the like.

In general, at least one cell in the cell population of the micro-organ culture is proliferative. The cell populations present in the micro-organ cultures are generally at equilibrium, ie the ratio of cell proliferation to cell loss in the cell population is approximately 1, or proliferates faster than the cells in the micro-organ cultures are lost, ie For example, the ratio of cell proliferation to cell loss in a cell population may be greater than 1, such as in cell populations obtained from tumor tissue or in progenitor cell populations induced to proliferate in explants.

Preferred organs capable of isolating cells of micro-organ cultures include lymphoid organs such as the thymus and spleen; Gut organs such as the stomach, liver, pancreas, gallbladder and bile ducts; lungs; Reproductive organs such as the prostate and uterus; Breasts such as mammary glands; skin; Urinary tract organs such as bladder and kidney; cornea; And blood-related organs such as bone marrow. An isolated cell population of micro-organ cultures may in one embodiment be encapsulated in a polymer device, such as a device for delivering a cell or cell product, such as a gene product, to a subject. The present invention also relates to a synthetic medium derived from the micro-organ culture of the present invention.

In one embodiment of the invention, the micro-organ culture comprises a population of cells that are sections of organs. Preferably, the micro-organ explants comprise epithelial and connective tissue cells. In one embodiment of the invention, the organ explant is obtained from the pancreas, for example, the microstructure of the cell population is almost identical to the microstructure of the original pancreas from which the explant is derived, and the epithelial cells of the pancreas, such as islet cells and The connective tissue cells of the pancreas.

In another embodiment of the invention, the micro-organ explants are obtained from the skin, for example the microstructure of the cell population is almost identical to the microstructure of the skin in vivo, and skin epithelial cells such as epidermal cells and skin connective tissue. Cells, such as skin cells. Micro-organ cultures obtained from skin explants may also include a bottom plate supporting epidermal cells, extracellular matrix comprising skin cells and at least one inclusion, such as at least one hair follicle or gland.

In another embodiment of the invention, the micro-organ culture is a virus, such as hepatitis virus, in particular hepatitis B or hepatitis C virus or human papilloma virus (HPV), such as HPV-6, HPV-8 or HPV-33. It includes an isolated population of infected cells. Upon viral infection, micro-organ cultures can be used in methods for identifying inhibitors of viral infectivity. The method comprises the step of isolating micro-organ explants in accordance with the method of the present invention, wherein the explants are isolated from a viral infectious organ or subsequently infected with a virus in vitro and thus a population of virus-infected cells. To generate. This explant is then contacted with a candidate agent, such as an agent for testing antiviral activity, and the infectivity level (eg, viral load, new infectivity, etc.) present in the presence of the candidate agent is determined and then in the absence of the candidate agent. Compare with the infectious level exhibited by the virus. Decrease in viral infectivity levels in the presence of candidate agents suggests that they are inhibitors of viral infectivity.

The present invention also relates to a method for producing micro-organ cultures. The method includes separating from the mammalian donor subject a micro-organ explant having a size that provides an isolated population of cells that can be maintained in minimal media for at least about 24 hours. This micro-organ explant is then injected into the culture. In general, the explant includes an isolated cell population which retains the microstructure of the organ from which the explant is isolated. In one embodiment of the invention, at least one cell of the explant is proliferative. The cells of the micro-organ culture of the present invention are in equilibrium, ie the ratio of cell proliferation to cell loss in the cell population is 1, or proliferates faster than the cells in the micro-organ culture are lost, ie in tumor tissue. The ratio of cell proliferation to cell loss in the cell population, such as the obtained cell population, may be greater than one.

Preferred organs capable of isolating cells of micro-organ cultures include lymphoid organs such as the thymus and spleen; Gut organs such as the stomach, liver, pancreas, gallbladder and bile ducts; lungs; Reproductive organs such as the prostate and uterus; breast; skin; Urinary tract organs, such as the bladder; kidney; cornea; And blood-related organs such as bone marrow. In these examples, the microstructure of the organ should be maintained in all cultured explants. Such micro-organ cultures may be tissue sections, such as pancreatic tissue sections including β-islet cells, epidermal and dermal cells, and skin tissue sections including other skin specific structural features such as hair follicles.

Cells present in the micro-organ explants can also be modified to express recombinant proteins that may or may not be normally expressed in the organ from which the explants are derived. For example, gene products that are normally produced by the pancreas and that can be augmented (e.g. to compensate for deficiencies) by the mutagenesis methods of the present invention include insulin, amylase, protease, lipase, trypsinogen, chymotrypsinogen, carboxy Peptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospholipase A ₂ , elastase, amylase and the like, which are commonly produced in the liver and can be supplemented by alternative gene therapy Gene products of interest include blood coagulation factors such as blood coagulation factors VIII and IX, UDP glucuronyl transferase, ornithine transcarbamoylase, cytochrome p450 enzymes, and genes commonly produced by the thymus Products include serum thymic factor, thymic fluid factor, thymopoietin, thymosin a1.

The micro-organ culture of the present invention can be used in a method of delivering a gene product to a recipient subject. This method yields from the donor subject an isolated cell population having a surface area to volume that retains the microstructure of the organ or tissue from which the cell is separated and provides an isolated cell population that is maintainable in minimal media for at least about 24 hours or more. It includes a step. This cell population is then introduced with a recombinant nucleic acid that encodes and induces the expression of the desired gene product to produce a mutant cell population, such as a mutant explant, in the micro-organ explant. This mutant explant may then be administered to the recipient subject. The donor subject and the recipient subject may be allogeneic or heterologous.

Micro-organ cultures of the invention can also be used in methods for identifying agents that induce proliferation of cells of a given organ, such as progenitor cells. The method comprises producing a micro-organ explant culture comprising at least one cell that is proliferative in accordance with the present invention. This explant is contacted with a candidate compound, such as a compound to be tested for cell proliferative capacity, after culture, and then the cell proliferation level is measured in the presence of the candidate compound. The level of cell proliferation measured in the absence of the candidate compound is then compared with the level of cell proliferation measured in the presence of the candidate compound. Increasing cell proliferation levels in the presence of candidate compounds suggests that they are cytostatic agents. Cell proliferation inhibitors can also be identified in this manner. Specifically, when the measured level of cell proliferation in the presence of the candidate compound is measured according to the method described above, it can be compared with the level of cell proliferation in the absence of the candidate compound. Reduction of cell proliferation levels in the presence of candidate compounds suggests that they are cytostatic inhibitors.

Other methods that can use the micro-organ culture of the present invention include a method for identifying an agent that induces or inhibits one or more cell differentiation present in a given organ, or an agent that maintains a specific state of differentiation (prevents dedifferentiation). There is this. The method includes preparing a micro-organ explant from said organ, wherein the cell populations that make up the explant retain the microstructure of the organ, at least about 1.5 mm ⁻¹ allele, as described below, At least one cell that retains the ability to differentiate or has the ability to dedifferentiate as a differentiated state. Upon incubation the cell population is contacted with the candidate compound and the cell differentiation level is measured in the presence of the compound. The level of cell differentiation measured in the presence of the candidate compound is compared with the level of cell differentiation measured in the absence of the candidate compound. Increasing the level of cell differentiation in the presence of candidate compounds suggests that it is a cell differentiation agent. Inhibitors of cell differentiation can also be identified in this manner. Specifically, when the cell differentiation level measured in the presence of the candidate compound is measured according to the method described above, it can be compared with the cell differentiation level measured in the absence of the candidate compound. Reduction of cell differentiation levels in the presence of candidate compounds suggests that the candidate compounds are inhibitors of cell differentiation.

In another aspect of the invention, the invention provides a method for identifying and isolating stem cell or progenitor cell populations from organs. The method generally comprises the step of separating the explants of the cell population from the organ in culture. As described herein, explants may comprise (i) the ability to maintain the microstructure of the organ from which the explants originate, (ii) a surface area to volume index (alep) of at least about 1.55 mm ⁻¹ , And (iii) at least one progenitor or stem cell with proliferative capacity. The explants are contacted with agents that induce proliferation of progenitor or stem cells, such as growth factors or other mitotic factors, to amplify the isolated cell population present in the explant. The amplified progenitor cells can then be isolated from the explant. Such secondary populations of explants can be identified through their proliferative response. In another embodiment, progenitor / stem cells will spontaneously proliferate in culture without the addition of exogenous agents. In another embodiment, isolation of progenitor or stem cells derived from explants that proliferate in response to the exogenous agent may be achieved by direct mechanical separation of newly occurring buds from the explant, or by dissolution of all or part of the explant. The amplified cell population can then be isolated, for example.

Another method by which the micro-organ culture of the present invention can be used is a method for promoting wound healing of a recipient subject. The method includes separating a population of cells bearing at least about 1.5 mm ⁻¹ or more allele from the donor subject and then applying the population of cells to a wound in the recipient subject. The donor subject and the recipient subject may be allogeneic or heterologous. In one embodiment, the tissue from which the cells are isolated is the skin and the wound of the recipient subject is an ulcer, such as a diabetes related ulcer.

The practice of the present invention will employ methods of conventional cell biology, cell culture, molecular biology, mutation biology, microbiology, recombinant DNA and immunology, unless otherwise noted, which may be practiced by those skilled in the art. Such methods are described in detail in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. US Patent No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S. J. Higgins eds. 1984); Culture of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., N. Y.); Gene Trasfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods in Enzymology, Vols. 154 and 155 (Wu et al. Eds.), Immunochemical Methods in Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); and Handbook of Experimental Imunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986).

Other features and advantages of the invention will be apparent from the following detailed description and claims.

The invention, which has been outlined, will now be readily understood by reference to the following examples, which are included for the purpose of merely describing certain aspects and aspects of the invention and are not intended to limit the invention.

Micro-organ cultures of animal origin, including adult human skin, mouse, guinea pig and rat skin, are isolated and grown by incubating for up to 21 days. However, it is within the scope of the present invention to maintain culture periods in excess of 21 days.

In addition, it is within the scope of the present invention to form micro-organ cultures from a wide range of animals. Various animals are merely exemplary and are not limited to the samples provided below.

As described in the accompanying examples, micro-organ cultures are prepared from the skin and from organs including the mammalian pancreas, liver, kidney, duodenum, esophagus and bladder. Similarly, micro-organ cultures of epithelial origin of esophageal origin such as mammalian cornea, kidney, breast tissue and tissues of various intestines, as well as intestines and colon can also be prepared using the methods of the invention. Indeed, it is within the scope of the present invention to separate and maintain micro-organ cultures from any site that includes the esophagus / epileptic structures in the body.

Notwithstanding the above, subject micro-organ culture techniques have been used to preserve tissue explants in organ cultures of tissue origin that do not have specific lymphoid tissues, such as thymus and splenic explants, which do not have an esophageal / epileptic structure.

Example I

Preparation of Epidermal Micro-Organ Cultures

Fresh skin is obtained postoperatively and washed from the lower adipose tissue and cut into 0.4 x 5 cm flaps using a tissue cutter or other suitable cutting means and then under sterile conditions so that the dimensions of the final tissue sections are 4 mm wide and 0.3 mm thick. Cut horizontally into 300 μm fractions (see FIG. 1). These micrograndes are placed in 24-well microplates in 400 μl of DMEM under 5% CO ₂ at 37 ° C. with constant shaking at 12 rpm for a 1-8 day period. 20 micro explants are grown per well.

Example II

Proliferation Measurement of Mouse, Guinea Pig, and Human Epidermal Micro-Organ Cultures

Micro-organ cultures are prepared according to Example I and cell proliferation is determined by analyzing the amount of DNA synthesis as follows. After growing mouse skin and guinea pig skin for 2 days and human skin for 4 days, BrdU is added to the medium so that the final concentration is 100 μM for 3 hours followed by immobilization of cells in 4% formaldehyde. After immobilization, the cultures are stained with goat anti-BrdU antibody followed by anti goat FICT labeled IgG. Histological preparations are embedded in 4% formaldehyde in the following immobilization, cut into 3 μm pieces and stained with methylene blue.

Two to four days after incubation, the cell fraction capable of synthesizing DNA in vitro increased up to 10-fold compared to the value observed in vivo, after which the rate of DNA synthesis was gradually reduced but 10 days in culture. High during the following (see FIGS. 2, 3 and 4A-4D). Even on day 6 of cultivation, cells maintained a steady state of proliferation and differentiation to preserve tissue structure (see FIGS. 5A-5C).

Example III

Proliferation of Cells of Various Sizes in Micro-Organ Cultures

Guinea pig micro-organs are prepared according to Example I. The full thickness skin strips, 4 mm wide, are cut into explants of varying thickness, including pieces 300, 450, 600, 700, 900, 1200 and 3000 μm thick. These pieces are each added to wells containing serum-free medium for 2 days each. BrdU is added for 4 hours before terminating at a final concentration of 100 μM. The explants are then immobilized in 4% formaldehyde and stained with goat antibodies against BrdU followed by anti goat IgG FITC labeled secondary antibody preparations. The result of this experiment is demonstrated in FIG. BrdU infusion as a function of the number of cells / unit tissues is significantly reduced by increasing the thickness of the explant.

Example IV

Preparation of Pancreatic Micro-Organ Cultures and Measurement of Cell Proliferation

Following removal of the guinea pig pancreas, sections of 300 μm thick, 4 mm wide and 2 mm deep are cut in such a way that the pancreatic microstructure is maintained using a suitable tissue cutter. Micro explants are grown in culture for several periods of 2 to 18 days. Seven micro-organs are added to each of the 96 wells of the plate in 150 μl serum-free DMEM under 5% CO ₂ at 37 ° C. under constant shaking at 12 rpm. BrdU is added 3 hours before termination at a final concentration of 100 μM and the explants are then immobilized in 4% formaldehyde and stained with goat antibodies against BrdU followed by staining with anti goat FITC labeled IgG. 7A-7B show that cells in the pancreas-derived microorganisms are actively proliferating.

Example V

Preparation of Pancreatic Micro-Organ Cultures and Measurement of Insulin Secretion into Culture Media

Adult pig pancreatic micro-organ cultures are prepared as in the previous examples for skin. The pancreas is removed and cut with scissors to a suitable length of 2 mm and sliced into sections 300 mm thick and 4 mm wide. Micro-organ cultures are grown for 14 days in serum-free medium. Every two days, the medium is removed and fresh medium is added. The harvested media is analyzed for insulin content using standard radioimmunoassay methods.

Example VI

Transplantation of Pig Pancreatic Micro-organs into Heterologous Subjects

Adult pig pancreatic micro-organ cultures are prepared as in the previous examples for skin. The pancreas is removed and cut with scissors to a suitable length of 2 mm and sliced into sections 300 mm thick and 4 mm wide. The micro-organ cultures are then grown and incubated in serum-free medium for 0-5 days at different time points, then the pancreatic micro-organs are removed from the culture and transplanted into both the visceral and acid secreted mesoderm of the rat host. Micro-organs survive in vivo for more than 1 month and are widely vascularized. After 3 days, 5 days, 7 days and 14 days in vivo, extensive cell proliferation can be detected. Moreover, positive insulin staining is observed in vivo 4, 7 and 30 days after transplantation.

Example VII

Preparation of liver, kidney, duodenum, esophagus and bladder micro-organ cultures and measurement of cell proliferation in micro-organ cultures

Guinea pig micro-organ cultures from various epithelial tissue containing organs are prepared as in the previous examples for skin. The trachea is removed and cut with scissors to a suitable width of 2 mm, a length of 3 mm and cut into sections of 300 μm thickness. Microcultures are incubated for 3 days, 4 days and 6 days in serum free medium. 12 hours before the end of the experiment, ³ H-thymidine is added to the culture of the explant. At the end, the tissue is immobilized, washed several times and counted with a scintillation counter. The results of this experiment are shown in FIG. As shown in FIG. 9, all tissues show active proliferation and continue for 6 days as determined by uptake of ³ H-thymidine.

Example VIII

Proliferation of Hair Follicles in Micro-organ Cultures

Skin micro-organ cultures are prepared according to Example I and incubated for 2 days. BrdU is added 3 hours before the end of the culture. Cells are immobilized in 4% formaldehyde and stained with goat anti BrdU antibody followed by anti goat FITC labeled IgG. Intact hair follicles present in vivo under normal ambient conditions can be maintained under precisely controlled culture conditions and there is no need to add blood serum or any other exogenous factor. Hair follicle cells in these microorgans were found to proliferate vigorously for several days under the conditions of the method of the present invention as indicated by the majority of hair follicle cells incorporating BrdU (FIGS. 10A-10C). The size distribution of the hair shaft at time zero and after two weeks of the micro-organ guinea pig culture is shown in FIG. 11. Change the medium every two days. Hair shaft size is arbitrarily divided into small, medium and large. After 9 days of culture, the size distribution apparently shifted, reducing the percentage of small hair from 64% to 28%, while the large stems that were not present at the beginning of the culture accounted for 30% of the stem population.

Example IX

Assay Preparation to Measure the Effect of Candidate Compounds on Cell Proliferation

Cultures are prepared and maintained in synthetic medium under growth conditions similar to those described in Example I. Control samples are analyzed by immunohistochemistry to determine that micro-organ cultures are maintained in a manner similar to that occurring in vivo.

Two samples of skin microcultures are treated with 2.5 ng / ml TGF-β. Quantitative analysis of the number of BrdU labeled cells / explants is performed according to Example II. Inhibition of greater than 90% DNA synthesis is observed in the presence of TGF-β as compared to the control (FIG. 12).

Example X

How to promote the healing of chronic non-oil skin ulcers

According to the method, a small area of normal uninvolved skin grafts is removed from the patient and a microvitrograft of 4 mm thick and 0.3 mm thick is prepared as described in Example I. However, this formulation differs from Example I in that the cut is deliberately incomplete with a 0.3 mm piece so that one piece is held together as indicated in FIG. 13 and the upper epithelial layer comprises an interstitial layer. The design of the implant is oriented so that the nutrients can reach all the cells but the tissue fragments remain in an operable format. The patient's wound is cleaned and the surrounding skin edges are removed. The area without the skin is then covered with a micro explant, which is placed in the wound so that the non-segmented edge is up and the opposing piece is suspended in the intravenous fluid. Sufficient microexplants are prepared to substantially cover all wound areas. The treated area is then covered with a suitable dressing and left to heal.

Example XI

Proliferation of hair follicles in vivo

In vivo animal experiments are performed in which 1 cm ² area of skin is removed from the mouse and incompletely microsectioned leaving keratin of the entire skin area as described above. The micro-organs are replanted to their original position in the sutured mouse and left to heal. The grafts remain viable, feed into animal tissue and grow from the graft 1-2 weeks after the new hair shaft has been cultured (see FIG. 14).

Example XII

Human Pancreatic Skin Micro-Organ Cultures

Split-thickness psoriasis skin of 82-year-old elderly patients is obtained after dissection using a skin cutter. The skin is then cut into 0.5 × 5 cm flaps which are then laterally cut into sections of 300 μm using a tissue cutter or other suitable cutting device. These micro-organ sections are added to microplates in serum-free DMEM under 5% CO ₂ at 37 ° C. under constant shaking for 1-14 days. In some cases, growth factors are added to the culture medium. Change the badge every two days. Human psoriasis skin grows extensively as micro-organ cultures.

Example XIII

Liver micro-organ cultures infected with hepatitis virus

Human, rat, mouse and guinea pig livers are cut and cultured as described in Example VII as micro-organ cultures. Active proliferation in these micro-organ cultures is detected using BrdU incorporation as described herein. Hepatocytes in these micro-organ cultures were determined to be functional as measured by assays for urea (Sigma Chemical, Urea Detection Kit) and albumin production (ELISA) at least 14 days after culture.

Human liver micro-organ cultures prepared above are incubated with serum of patient origin positive for hepatitis B virus and hepatitis C virus. After 24 hours, the medium is removed and fresh DMEM is added in the presence and absence of 10% normal fetal bovine serum (FCS). Every two days, the culture medium is exchanged with fresh medium and the conditioned medium is tested for viral particles using antibodies to viral protein HB. A significant number of viral particles is detected after 4 days in this micro-organ culture in the presence of FCS.

Example XIV

Thymus and Spleen Micro-Organ Cultures

Mouse and rat micro-organ cultures of thymus and spleen origin are prepared essentially as for the skin as in the previous examples. Remove the trachea and cut with scissors to 2 mm wide and 3 mm long. These samples are then sliced into explants having a thickness of approximately 300 μm using a suitable tissue cutter in a manner that preserves the essential microstructure of the trachea. The microorgans are then incubated for 1, 3, 5 and 10 days in serum free medium. Active proliferation in these micro-organ cultures is detected using BrdU incorporation as described herein.

Example XV

Bone Marrow Organelle Culture

Micro-organ cultures of bone marrow origin are prepared by carefully removing the bone marrow intact from the thighs of rats and mice. Since the diameter of the bone marrow in the explant is only about 1 to 2 mm, the bone marrow is sliced into a micro-organ explant with a thickness of 300 μm using a direct tissue cutter. The method ensures that the microstructure of the bone marrow is preserved while maintaining the surface / volume index following long term culture. Active proliferation of bone marrow cells in these micro-organ cultures is detected using BrdU incorporation as described herein.

Example XVI

Delivery of Gene Products to Skin Micro-Organ Cultures

The high surface area-to-volume inherent in the micro-organ cultures of the invention allows for an easily accessible method for various gene delivery techniques. In this example, micro-organ cultures are transfected with foreign genes using electroporation and lipofection. Micro-organ cultures can be implanted into animals and survive and vascularize in vivo for at least about 30 days. This demonstrates the possibility of using tissue MC culture in an ex vivo gene therapy protocol. An additional benefit of MC culture can be implanted into defined locations in the body so that it can be easily removed later as needed. This is in contrast to the implantation of cell suspensions into the body where cells can migrate or are lost in normal tissue.

The guinea pig skin is incised and sliced into sections 2 mm wide and 300 μm thick. The skin is cultured as microorganisms in serum-free Dulbecco's minimal essential medium containing penicillin and streptomycin at the manufacturer's recommended concentrations. After 1 day incubation at 37 ° C. and 5% CO ₂ , the skin micro-organ cultures are washed with DMEM without antibiotics and 500 μl of medium on ice is added to a 0.4 cm gap disposable electroporation cuvette.

10 μg of plasmid DNA containing the indicated reporter gene is added as shown (each plasmid has a cytomegalovirus promoter that drives the expression of β-galactosidase (control) or luciferase reporter gene). The luciferase plasmid backbone is pRC-CMV (Invitrogen) in which the firefly is fused with the frame of the luciferase gene.

Samples are electroporated at 220 mV and various capacitances (high = 900 μF, medium = 500 μF, low = 250 μF) as shown in FIG. 15. NIH3T3 is treated at 250 μF with a bio-rad electroporation device. Samples are then incubated for two days in 24-well culture plates with DMEM containing additional 10% fetal bovine serum, penicillin, streptomycin and glutamic acid. The medium is removed and the sample is suspended in about 700 μl of cell culture lysis reagent (Promega). Tissue fragments are homogenized and then 20 [mu] l is added to 100 [mu] l of Luciferase Assay Reagent (Promega) and luminescence is detected three times with a Packard top count. As a positive control, NIH3T3 cells from 75 cm culture flasks were trypsinized and the microorgan cultures were also treated the same. As shown in FIG. 15, significant luciferase activity is detected at medium (500 μF) and low (250 μF) capacitance settings. For comparison, similar amounts of NIH3T3 immortal culture cells are electroporated with the same plasmid at 250 μF.

In another experiment, transfection of micro-organ explants is accomplished by lipofection, which is observed to be more efficient than electroporation. In particular, micro-organ cultures of guinea pig skin, newborn mouse skin, and rat lung origin are transfected with plasmids containing luciferase reporters. Briefly, micro-organ cultures are grown at 37 ° C. of 5.5% CO ₂ in DMEN containing 1% penicillin / streptomycin and 1% L-glutamine one day before transfection. The explants are aliquoted onto a 24-well plate containing 20 explants and 400 μl of medium per well. For transfection, micro-organ cultures are washed twice with Optitime and 10 μl of Lipofectin (Gibco BRL) + 2 μg DNA + Optitime are added to each well to a final volume of 500 μl. Optimem / Lipofectin / DNA solutions are prepared according to the instructions of the lipofectin manufacturer. The culture is then prepared for 5-6 hours at 37 ° C. in 5.5% CO ₂ . Optimem / Lipofectin / DNA medium is then replaced with 400 μl of DMEN containing 1% penicillin / streptomycin, 1% L-glutamine and 10% FCS and the culture is incubated overnight at 37 ° C. in 5.5% CO ₂ . The next morning, the micro-organ cultures are removed and washed twice with 1 × PBS and ground in 750 μL of 1 × cell culture lysis buffer (Promega) with a manual glass grinder. Luciferase activity of transgenic gene origin was detected using the Luciferase Assay System (Promega) and the results are shown in FIG. 18.

Example XVII

Delivery of Gene Products to Microcultures

Lungs and thymus of 8 week-old female Ruwis rat origin are dissected and processed into micro-organ cultures as described in Example XV. Micro-organ cultures are added to the culture wells and transfected with cationic lipid / luciferase encoding the plasmid DNA complex for 5-6 hours while incubating at 37 ° C. The cationic lipid / plasmid DNA solution is aspirated and the culture is then incubated in medium + 10% serum for 2 days, followed by luciferase reporter gene expression (indicated by any optical unit). The result of this experiment is shown in FIG. As demonstrated in FIG. 16, the lung expresses the transfected luciferase gene under this condition but not in the thymus. As expected, the negative control β-galactosidase transfected lung micro-organ culture (10 μl of cationic lipid concentrate) is near the machine background for light generation (23 light units).

Example VIII

Hair growth in vitro

New mouse skin is obtained postoperatively, washed from the lower adipose tissue and cut into 0.4 cm x 5 cm flaps and then laterally cut into 300 μm sections using a tissue cutter or other suitable cutting device. Micro-organs are added to microplates in DMEM under 5% CO ₂ at 37 ° C. under constant shaking for 1 to 14 days in the absence of serum. Certain micro-organ explants are contacted with growth factors such as FGF, which are added to the culture medium. The medium is changed every two days.

New “hair-free” skin can be induced to produce hair shafts when grown in MC culture. Micrographs of skin of 30-hour old mouse origin grown in micro-organ cultures for 3 days in the presence of 1 ng / ml EGF show hair shaft growth in ex vivo grafts that do not grow early in culture.

In another set of experiments, activation of resting follicles is observed. Senka mice provide a useful model for the study of hair follicle activation because the follicles are distributed in the same size and the cycle steps are widely characterized. Senka mice provide an in vivo model for antigen activation. Removal of clubs from resting follicles can lead to new hair formation, the first signs of which are well characterized. Skin of adult Senka mouse origin is obtained after surgery and removed from the lower adipose tissue and cut into 0.4 x 5 cm flaps and then laterally cut into 300 μl sections with a tissue cutter or other suitable cutting device. The microorgans are added to the microplates in DMEM in the absence of serum at 5% CO ₂ at 37 ° C. under constant shaking for 1-14 days. Activation of resting follicles, whether induced by club removal or growth factor treatment, is augmented by the proliferation of follicular stem cells. 17 depicts activation of resting ex vivo grafts as detected by thymidine incorporation.

Example IXX

Preparation of Pancreatic Islets for Transplantation

Various techniques have been developed for producing islet cells in large quantities from various mammalian sources because they occupy potentially implantable β cell masses to treat established diabetes type I. Two major drawbacks have so far emerged. It has proven difficult to obtain reproducible and reliable methods for preparing beta cells. In part due to the first reason and in part due to the fact that most β cells require support from the epilepsy underlying the islet cells in the normal pancreas. A series of attempts at maintaining pancreatic organs in vitro has not been successful until now. The techniques of the MC culture described herein have been successful in establishing micro-organ cultures of in vitro mice, rats, guinea pigs, and pig pancreas in synthetic culture media.

Pancreatic micro-organ cultures are currently grown in vitro for a period of up to one month. In culture, explants maintain their tissue microstructure and certain cell subpopulations proliferate actively as determined by BrdU incorporation and labeling. In addition, islet cells secrete insulin into the medium even after one month of in vitro culture.

Transplant experiments were performed in which the pig micro-organ pancreas cultures were transplanted into both visceral and acid-secreting mesoderm of the rat host. Ex vivo grafts are maintained for various periods of time in vivo up to several days. Explants are widely vascularized and bottled into tissue hosts.

Example XX

Preparation of Human Psoriasis Skin Micro-Organ Cultures

Psoriasis skin of divided thickness from the patient is obtained after dissection using an epidermal cutter. The skin is cut into 0.4 cm x 5 cm flaps and then laterally cut to a thickness of 300 μm with a tissue cutter. These micro-organ explants are incubated in DMEM (serum free) in microplates at 37 ° C. and 5% CO ₂ for 1-14 days. Examination of micro-organ explants at various time points indicates that the cells of the explants remain viable and proliferate.

All references and publications cited above are hereby incorporated by reference.

Equivalent

One skilled in the art can recognize or be confident that numerous equivalents can be used for routine experiments, certain assays and reagents described herein. Such equivalents are considered to be within the scope of this invention and are protected by the following claims.

Claims (67)

A genetically modified micro-organ explant comprising a cell population and expressing at least one or more recombinant gene products, wherein the appropriate nutrients and gases are maintained in vitro while maintaining the intrinsic microstructure of the organ from which the micro-organ explant is derived. The microorganism has a size selected to diffuse into the cells of the graft and the cell waste to diffuse out of the micro-organ explant, thereby minimizing the cytotoxicity and subsequent death of insufficient nutrients and waste in the micro-organ explant. A genetically modified micro-organ explant, characterized in that at least some cells of the cell population of the explant express at least one or more recombinant gene products. The genetically modified micro-organ explant of claim 1, wherein the at least one recombinant gene product is selected from the group consisting of recombinant protein and recombinant functional RNA molecules. The genetically modified micro-organ explant of claim 2, wherein the recombinant protein is normally produced in the organ from which the micro-organ explant is derived. The genetically modified micro-organ explant of claim 2, wherein the recombinant protein is not normally produced in the organ from which the micro-organ explant is derived. The genetically modified micro-organ explant of claim 2, wherein the recombinant protein is a marker protein. The method of claim 2 wherein the recombinant protein is insulin, amylase, protease, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospho Lipase A ₂ , elastase, amylase, blood coagulation factor, UDP glucuronyl transferase, ornithine transcarbamoylase, cytochrome p450 enzyme, adenosine deaminase, serum thymus factor, thymus factor, ty Morphoitine, thymosin a1, growth hormone, somatomedin, colony stimulating factor, erythropoietin, epidermal growth factor, hepatocytogenetic factor (hepatopoietin), hepatocyte growth factor, interleukin, negative growth factor, fibroblast Growth factor, transforming growth factor with β, gastrin, secretin, cholecystokinin, somatostatin, substance P and transcription factor , Genetically modified micro-organ explants. The genetically modified micro-organ explant of claim 1, which is maintainable in culture for at least about 24 hours or more. The method of claim 1, wherein the surface area to volume index is expressed by the formula 1 / x + 1 / a> 1.5 mm ⁻¹ , where 'x' is tissue thickness and 'a' is the tissue width in mm. Characterized by genetically modified micro-organ explants. The organ of claim 1, wherein the organ is selected from the group consisting of lymphatic organs, pancreas, liver, gallbladder, kidneys, digestive tract organs, respiratory tract organs, reproductive organs, skin, ureter organs, blood-related organs, thymus, spleen, Genetically modified micro-organ explants. The genetically modified micro-organ explant of claim 1, comprising epithelial tissue cells and connective tissue cells arranged in a microstructure similar to the microstructure of the organ from which the explant was obtained. The genetically modified micro-organ explant of claim 1, wherein the organ is a pancreas and the cell population comprises islets of Langerhans. 2. The genetically modified micro-organ explant of claim 1, wherein the organ is skin and the explant comprises at least one hair follicle and hair gland. The genetically modified micro-organ explant of claim 1, wherein the organ is diseased skin and the explant comprises a population of hyperproliferative or neoplastic cells from the diseased skin. The genetically modified micro-organ explant of claim 1, wherein the explant is maintainable in minimal medium. 2. The genetically modified microorganism of claim 1, wherein the microstructure retained by the explant comprises one or more cell-cell orientations and cell-epileptic orientations that appear between two or more tissues of the organ from which the explant is separated. Tracheal explants. The genetically modified micro-organ explant of claim 1, wherein at least a portion of the cell population is infected with a recombinant virus carrying a recombinant gene encoding a recombinant gene product. 18. The group of claim 16 wherein the recombinant virus is a group consisting of recombinant hepatitis virus, recombinant adenovirus, recombinant adeno-associated virus, recombinant papilloma virus, recombinant herpes virus, recombinant lentivirus, recombinant retrovirus, recombinant cytomegalovirus and recombinant simian virus. Will be selected from, genetically modified micro-organ explants. The trait of claim 1, wherein at least a portion of the cell population is selected from the group consisting of calcium phosphate mediated transfection, DEAE-dextran mediated transfection, electroporation, liposome mediated transfection, direct injection and receptor mediated uptake. Genetically modified micro-organ explant, which is transformed by heterologous nucleic acid through the conversion method. A synthetic medium formed by the genetically modified micro-organ explant of claim 1 and containing said recombinant gene product. A pharmaceutical preparation comprising the genetically modified micro-organ explant of claim 1. As a method of producing a micro-organ explant that expresses at least one recombinant gene product, (a) containing a population of cells, while maintaining the original microstructures from which the organs are derived, while at the same time adequate nutrients and gases diffuse into the cells of the micro-organ explants and cell waste diffuses out of the micro-organ explants, resulting in insufficient Isolating a portion of the organ with a selected size from the animal to minimize cytotoxicity and subsequent killing of one nutrient and waste accumulation; And (b) genetically modifying at least some cells of the cell population of some of the organs with a recombinant gene to express at least one or more recombinant gene products, the method of producing the genetically modified micro-organ explants . 22. The method of claim 21, wherein at least one recombinant gene product is selected from the group consisting of recombinant proteins and recombinant functional RNA molecules. 23. The method of claim 22, wherein the recombinant protein is normally produced in the organ from which the micro-organ explant is derived. 23. The method of claim 22, wherein the recombinant protein is not normally produced in the organ from which the micro-organ explant is derived. 23. The method of claim 22, wherein said recombinant protein is a marker protein. The method of claim 22, wherein the recombinant protein is insulin, amylase, protease, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospho Lipase A ₂ , elastase, amylase, blood coagulation factor, UDP glucuronyl transferase, ornithine transcarbamoylase, cytochrome p450 enzyme, adenosine deaminase, serum thymus factor, thymus factor, ty Morphoitine, thymosin a1, growth hormone, somatomedin, colony stimulating factor, erythropoietin, epidermal growth factor, hepatocytogenetic factor (hepatopoietin), hepatocyte growth factor, interleukin, negative growth factor, fibroblast Selected from the group consisting of growth factor, transforming growth factor with β, gastrin, secretin, cholecystokinin, somatostatin, substance P and transcription factor The method of producing a micro-organ explant of the genetic modification. The method of claim 21, wherein the genetically modified micro-organ explant is maintainable in culture for at least about 24 hours or more. 22. The method of claim 21, wherein the surface area to volume index is expressed by the formula 1 / x + 1 / a> 1.5 mm ⁻¹ , where 'x' is tissue thickness and 'a' is the tissue width in mm. Characterized by the method of producing a genetically modified micro-organ explant. The organ of claim 21, wherein the organ is selected from the group consisting of lymphatic organs, pancreas, liver, gallbladder, kidneys, digestive tract organs, respiratory tract organs, reproductive organs, skin, ureter organs, blood-related organs, thymus, spleen, Method for producing the genetically modified micro-organ explants. 22. The genetically modified micro-organ of claim 21, wherein the genetically modified micro-organ explant comprises epithelial tissue cells and connective tissue cells arranged in a microstructure similar to the microstructure of the organ from which the explant was obtained. Method for producing explants. 22. The method of claim 21, wherein the organ is pancreas and the cell population comprises islets of Langerhans. 22. The method of claim 21, wherein the organ is skin and the explant comprises at least one hair follicle and hair gland. 22. The method of claim 21, wherein the organ is diseased skin and the explant comprises a population of hyperproliferative or neoplastic cells derived from the diseased skin. The method of claim 21, wherein the genetically modified micro-organ explant is maintainable in minimal medium. The microstructure of claim 21, wherein the microstructure retained by the genetically modified micro-organ explant includes one or more cell-cell orientations and cell-epileptic orientations that appear between two or more tissues of the organ from which the explants are separated. , Method for producing the genetically modified micro-organ explants. 22. The method of claim 21, wherein at least a portion of the cell population is infected with a recombinant virus carrying a recombinant gene encoding a recombinant gene product. 37. The group of claim 36, wherein the recombinant virus is comprised of recombinant hepatitis virus, recombinant adenovirus, recombinant adeno-associated virus, recombinant papilloma virus, recombinant herpes virus, recombinant lentivirus, recombinant retrovirus, recombinant cytomegalovirus and recombinant simian virus. The method for producing a genetically modified micro-organ explant is selected from. 22. The trait of claim 21 wherein at least a portion of the cell population is selected from the group consisting of calcium phosphate mediated transfection, DEAE-dextran mediated transfection, electroporation, liposome mediated transfection, direct injection and receptor mediated uptake. Method for producing the genetically modified micro-organ explants that are transformed by heterologous nucleic acid through the conversion method. (a) a micro-organ explant comprising a cell population and expressing at least one or more recombinant gene products, wherein the appropriate nutrients and gases are retained while maintaining the intrinsic microstructure of the organ from which the micro-organ explant is derived. The microorganism has a size selected to diffuse into the cells of the graft and the cell waste to diffuse out of the micro-organ explant, thereby minimizing the cytotoxicity and subsequent death of insufficient nutrients and waste in the micro-organ explant. Providing a micro-organ explant in which at least some cells of the cell population of the explant express at least one or more recombinant gene products; And (b) transferring the gene product to the receptor, comprising transplanting the micro-organ explant into a receptor. The method of claim 39, wherein the micro-organ explant is from a receptor. The method of claim 39, wherein the micro-organ explant is from a donor subject. The method of claim 39, wherein the micro-organ explant is from a human. 40. The method of claim 39, wherein the micro-organ explant is from an animal other than a human. The method of claim 39, wherein the receptor is human. The method of claim 39, wherein the receptor is an animal other than a human. 40. The method of claim 39, wherein the at least one recombinant gene product is selected from the group consisting of recombinant protein and recombinant functional RNA molecule. 47. The method of claim 46, wherein the recombinant protein is normally produced in the organ from which the micro-organ explant is derived. 47. The method of claim 46, wherein the recombinant protein is not normally produced in the organ from which the micro-organ explant is derived. 47. The method of claim 46, wherein the recombinant protein is a marker protein. 50. The method of claim 49, wherein the recombinant protein is insulin, amylase, protease, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase, phospho. Lipase A ₂ , elastase, amylase, blood coagulation factor, UDP glucuronyl transferase, ornithine transcarbamoylase, cytochrome p450 enzyme, adenosine deaminase, serum thymus factor, thymus factor, ty Morphoitine, thymosin a1, growth hormone, somatomedin, colony stimulating factor, erythropoietin, epidermal growth factor, hepatocytogenetic factor (hepatopoietin), hepatocyte growth factor, interleukin, negative growth factor, fibroblast Selected from the group consisting of growth factor, transforming growth factor with β, gastrin, secretin, cholecystokinin, somatostatin, substance P and transcription factor Way. The method of claim 39, wherein the genetically modified micro-organ explant is maintainable in culture for at least about 24 hours. 40. The genetically modified micro-organ explant of claim 39, wherein the surface-to-volume index is 1 / x + 1 / a> 1.5 mm ^-1 where 'x' is tissue thickness and 'a' is tissue Width of mm). 40. The method of claim 39, wherein the organ is selected from the group consisting of lymphatic organs, pancreas, liver, gallbladder, kidneys, digestive tract organs, respiratory tract organs, reproductive organs, skin, ureter organs, blood related organs, thymus, spleen . 40. The method of claim 39, wherein the genetically modified micro-organ explants comprise epithelial tissue cells and connective tissue cells arranged in a microstructure similar to the microstructure of the organ from which the explant was obtained. The method of claim 39, wherein the organ is the pancreas and the cell population comprises islets of Langerhans. 40. The method of claim 39, wherein the organ is skin and the explant comprises at least one hair follicle and hair gland. 40. The method of claim 39, wherein the organ is diseased skin and the explant comprises a hyperproliferative cell or neoplastic cell population from the diseased skin. The method of claim 39, wherein the genetically modified micro-organ explant is maintainable in minimal medium. 40. The microorganism of claim 39, wherein the microstructure retained by the genetically modified micro-organ explant includes one or more cell-cell orientations and cell-epileptic orientations that appear between two or more tissues of the organ from which the explant is isolated. , Way. The method of claim 39, wherein at least a portion of the cell population is infected with a recombinant virus that carries a recombinant gene encoding the recombinant gene product. 61. The group of claim 60, wherein the recombinant virus is comprised of recombinant hepatitis virus, recombinant adenovirus, recombinant adeno-associated virus, recombinant papilloma virus, recombinant herpes virus, recombinant lentivirus, recombinant retrovirus, recombinant cytomegalovirus and recombinant simian virus. Which is selected from. 40. The trait of claim 39 wherein at least a portion of the cell population is selected from the group consisting of calcium phosphate mediated transfection, DEAE-dextran mediated transfection, electroporation, liposome mediated transfection, direct injection and receptor mediated uptake. And transformed with the heterologous nucleic acid via a conversion method. 40. The method of claim 39, further comprising encapsulating said genetically modified micro-organ culture before step (c). 40. The method of claim 39, wherein step (a) (i) contains a population of cells, while maintaining the original microstructure from which the organ is derived, while at the same time appropriate nutrients and gases diffuse into the cells of the micro-organ explant and cell waste diffuse out of the micro-organ explant, Separating a portion of the organ from the animal with a selected size to minimize cytotoxicity and subsequent death from insufficient nutrients and waste accumulation in the organ explant; And (ii) genetically modifying at least some cells of the cell population of some of said organs with recombinant genes such that at least one or more recombinant gene products are expressed. 40. The method of claim 39, wherein step (a) is performed by obtaining a micro-organ explant from the organ of a mutant animal expressing said recombinant gene product. Including the cell population, while maintaining the original microstructure from which the organs originate, the appropriate nutrients and gases diffuse into the cells of the micro-organ explants and the cell waste diffuses out of the micro-organ explants, resulting in insufficient nutrients in some of the organs. And separating a portion of the organ having a selected size from the mutant animal so as to minimize cytotoxicity resulting from waste accumulation and subsequent death, wherein at least some cells of the cell population of the organ part are at least one recombinant. A method for producing a micro-organ explant for expressing at least one recombinant gene product, which is to express a gene product. Including the cell population, while maintaining the original microstructure from which the organs originate, the appropriate nutrients and gases diffuse into the cells of the micro-organ explants and the cell waste diffuses out of the micro-organ explants, resulting in insufficient in the micro-organ explants. It has a size selected to minimize cytotoxicity and subsequent death of one nutrient and waste accumulation, wherein at least some cells in the cell population of the micro-organ explants express at least one recombinant gene product, A medical device comprising a polymer device encapsulating a genetically modified micro-organ explant expressing at least one recombinant gene product.