CN1582330A

CN1582330A - In-vitro micro-organs and uses realated thereto

Info

Publication number: CN1582330A
Application number: CNA018239064A
Authority: CN
Inventors: E·N·米特拉尼
Original assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 2001-10-23
Filing date: 2001-10-23
Publication date: 2005-02-16
Also published as: KR20040070343A; KR100970195B1; JP2005506084A; KR20090060381A; CA2464460A1; IL161472A0

Abstract

Micro-organ cultures which include isolated populations of cells having specific characteristics are described. Salient features of the subject micro-organ cultures include the ability to be maintained in culture for relatively long periods of time, as well as the preservation of an organ microarchitecture which facilitates, for example, cell-cell and cell-matrix interactions analogous to those occurring in the source organ. The micro-organ cultures of the invention can be used in methods for delivering gene products to recipient subjects, for identifying cell proliferative and cell differentiating agents, and 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 of cell proliferation, cell differentiation and viral infectivity. In other embodiments, the micro-organ cultures can be used for transplantation.

Description

External micro-organs and associated uses thereof

Background of invention

Finished the eukaryotic cell cultivation in early days in nineteen fifties.From then on, use various substratum widely and clear and definite fill-in, as somatomedin and hormone, and indefinite fill-in, cultivated conversion and primary cell widely as serum and other body extract.For example, can be by a lot of passages such as caryogram double somatocyte or clone unlimited as that set up, the inoblast that conventional cultivation is obtained by animal skin.Yet epithelial cell has morphology different with inoblast and hyperplasia performance, more is difficult to cultivate.And, in identical culture, growing with inoblast when epithelial cell, inoblast makes epithelial cell growth excessive usually.

Though the cell two-dimensional growth is preparation, observe and the research cells in culture, allow the high-speed outgrowth method that makes things convenient for of cell, its lacks interactional characteristic between the cell-cell of whole tissue in body and the cell-matrix.

Interact in order to study this function and morphology, a few studies person has probed into use three-dimensional substrates such as collagen gel (Douglas et al., (1980) In Vitro 16:306-312; Yang et al., (1979) Proc.Natl.Acad.Sci.; 76:3401; Yang etal. (1980) Proc.Natl.Acad.Sci.77:2088-2092; Yang et al., (1981) Cancer Res.41:1021-1027); Cellulose sponge, (Leighton et al., (1968) Cancer Res.28:286-296) of (Leightonet al., (1951) J.Natl Cancer Inst.12:545-561) or collagen parcel separately; Gelfoam, Gelfoam (Sorour et al., (1975) J.Neurosurg.43:742-749).

In order to make epithelial cell, various culture condition have been adopted with the growth of clone's competitive mode.For example, (United States Patent (USP) 5,282,859 on (Rheinhardt et al. (1975) Cell 6:331-343) and semi-synthetic collagen stroma on the fibroblastic feeder layer of lethal exposure; European patent application 0361957) cultivated epithelial cell, especially skin epithelial cell (keratinocyte).In some cases, the substratum that is used for this cell growth can add bio-extract, comprise hypophysis extract and serum and growth fill-in, as Urogastron and Regular Insulin (Bosseau et al. (1992) J.Dermatol.Sci 3 (2): 111-120; United States Patent (USP) 5,292,655).

The a lot of trials of skin have been carried out in growth in vitro.These attempt typically comprising the step that the keratinocyte in the epidermis and the inoblast in the corium and adipocyte are separated.After separating, keratinocyte is grown in the mode that allows the layering epidermis to form usually.Yet the epidermis that this mode prepares lacks hair follicle and sweat gland.And in this cultivation, the natural relation between epidermis and the corium no longer keeps.The cultural method of having taked comprises that keratinocyte is positioned over (Sugihara et al. (1991) Cell.Dev.Biol.27:142-146 on the collagen and fibroblastic corium substrate that synthesizes or obtained by epidermis collagen and fibroblastic replaceable source in growth (Rheinwald et al. (1975) Cell 6:331-343) on the unvital inoblast or with keratinocyte; Parenteau et al. (1991) J.CellBiochem.45 (3): 245-251).Yet, in some cases, do not implement the separation of keratinocyte, whole organ is positioned in the cultivation.The trial of vitro culture organ is limited to cultivates organ (Li et al. (1991) Proc.Natl.Acad.Sci.88 (5): 108-112) in containing the substratum of serum.

Most existing external epidermis models lack hair follicle, sweat gland and sebiferous gland and (for the summary that epidermic cell is cultivated, see Coulomb et al. (1992) Pathol.Biol.Paris 40 (2): 139-146).The skin model of the gel support that comprises Li et al. ((1992) Proc.Natl.Acad.Sci89:8764-8768) of exception, wherein diameter 2 * 5mm ²With the thick skin explant of 2.0mm, containing in the presence of the substratum of serum, vigor kept several days.

Except the defective of primary cellular defect, other method of bio-reactor and cultivation mammalian cell provides and allows cell to be assembled into tissue, and the ability of the condition of true tissue is also very limited in the complete organism of simulated three-dimensional space form.Owing to similar reason, traditional tissue culture method has limited cultured tissue and has shown and think to the very crucial function hyperspecialization of the special bioactive molecules secretion of mammalian cell differentiation and research and pharmacy interest or the ability of differentiation state.Different with microorganism, higher organisms such as mammiferous cell self forms high-grade many cells tissue.Although do not know this cutter system really of self assembling, but under research situation so far, have been found that cell development is that tissue depends on the cell location of (identical or different cell type) or the existence or the shortage of other anchoring base and/or some substrate (factor) each other, as hormone, autocrine or paracrine.In a word, conventional cultural method can not reach enough low shear pressure simultaneously, and enough three-dimensional space degree of freedom and critical cell interaction (each other or substrate) sufficiently long period is to allow the splendid modeling of in-vivo tissue structure.

Therefore, need produce and keep the in vitro method of organ each several part in cultivation, wherein the natural iuntercellular relation of cultured cells keeps prolonging for some time.Simulate the whole tissue of organizing in vivo cytodifferentiation, hyperplasia and the cell function found and organ model will to understand organ keep state of health and thus anomalous event how can practicality be arranged by converse mechanism.

Summary of the invention

The invention provides the external micro-organs culture that satisfies above-mentioned needs.The prominent feature of micro-organs culture theme is included in the ability of keeping long relatively for some time in the cultivation, as, at least about twenty four hours, preferably at least seven days or longer, and the ability that keeps organ microarchitecture (microarchitecure), its promotes for example to be similar to the cell-cell that takes place among the source organ and the interaction of cell-matrix.

Typically, at least one cell has the hyperplasia ability in the cell mass of micro-organs culture.Micro-organs cells in culture group can be in equilibrium state generally, be that the ratio of hyperplasia and loss cell approximately is 1 in the cell mass, or micro-organs cells in culture hyperplasia speed can be lost greater than them, the ratio that causes hyperplasia and loss cell in the cell mass is greater than 1, the cell mass that obtains as tumor tissues, or as, in explant, induced outgrowth progenitor cell.

The preferred organ of separable micro-organs culture cell comprises lymphoid organ, as thymus gland and spleen; The digestive organ is as intestines, liver, pancreas, gall-bladder and bile duct; Lung; Reproductive organ is as prostate gland and uterus; Breast is as mammary gland; Skin; The urinary tract organ is as bladder and kidney; Cornea; Relevant organ with blood such as marrow.In certain embodiments, isolating micro-organs culture cell mass can be wrapped in the poly-unit, passes to the experimenter as cell or cellular products such as gene product.The present invention also relates to isolating conditioned medium in the micro-organs culture of the present invention.

In one embodiment of the invention, the micro-organs culture comprises cell mass, and it is the part of organ.Preferably, the micro-organs explant comprises epithelium and phoirocyte.In one embodiment of the invention, the organ explant derives from pancreas, and the microarchitecture of the initial pancreas of originating with this explant basically as the microarchitecture of cell mass is identical, and comprises the pancreas epithelial cell, as islet cells and pancreas phoirocyte.

In another embodiment of the invention, the micro-organs explant derives from skin, and is identical with the microarchitecture of skin in the body basically as the microarchitecture of cell mass, and comprises the skin epithelium, as epidermic cell and skin connective tissue cell, as dermal cell.The micro-organs culture that the skin explant obtains with can comprise the stratum basale that supports epidermic cell, comprise that the extracellular matrix of dermal cell and at least one cave in, as at least one hair follicle and body of gland.

In another embodiment of the invention, the micro-organs culture comprises the isolated cells group of virus infection, as hepatitis virus, and as hepatitis B or hepatitis C, or human papillomavirus (HPV), as HPV-6, HPV-8 or HPV-33.When virus infection, the micro-organs culture can be used for the method for the communicable inhibitor of identifying virus.This method comprises that the method according to this invention separates the micro-organs explant, and explant derives from the organ of virus infection, or virus infection in vivo and produce the cell mass of virus infection in explant afterwards.This explant then can contact with candidate agent, as the reagent of test antiviral activity with measure infectivity level (as viral load, newly infectivity etc.) and compare with there not being under the candidate agent viral infectivity level in the presence of candidate agent.Candidate agent exists down, and the reduction of viral infectivity level is the sign of viral infectivity inhibitor.

The present invention also relates to prepare the method for micro-organs culture.This method comprises from Mammals donor experimenter separates the micro-organs explant, and this explant has to be provided to the isolated cells group can keep at least approximately size of twenty four hours in the minimal medium.The micro-organs explant is positioned in the cultivation.Typically, explant comprises the isolated cell group of the microarchitecture with the organ that separates this explant.In one embodiment of the invention, at least one cell of explant has the hyperplasia ability.The cell of theme micro-organs culture can be in equilibrium state, be that the ratio of hyperplasia and loss cell is 1 in the cell mass, or the micro-organs cells in culture can be greater than the speed hyperplasia of their losses, the ratio that causes hyperplasia and loss cell in the cell mass comprises the cell mass that tumor tissues obtains greater than 1 as explant.

The preferred organ of separable micro-organs culture cell comprises lymphoid organ, as thymus gland and spleen; The digestive organ is as intestines, liver, pancreas, gall-bladder and bile duct; Lung; Reproductive organ is as prostate gland and uterus; Breast; Skin; The urinary tract organ is as bladder; Kidney; Cornea; Relevant organ with blood such as marrow.In each embodiment, the explant of cultivation is kept the microarchitecture of organ.The micro-organs culture can be the part of tissue, as the part of pancreatic tissue, comprises the β islet cells, as the part of skin, comprises epidermis and dermal cell and other skin specific structural features, as hair follicle.

Cell in the micro-organs explant can be modified with express recombinant protein the organ normal expression that this albumen can or can not originated by this explant.For example, normally produce by pancreas and can be increased by the theme transgenic method and as correct the gene product that lacks, comprise Regular Insulin, amylase, proteolytic enzyme, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, rnase, deoxyribonuclease, triglyceride level enzyme, phospholipase A ₂, elastoser and amylase; Equally, the gene product that is normally produced by liver and can replaced gene therapy replenish comprises thrombin, as blood coagulation factor VIII and factors IX, and UDP glucuronyl transferase, ornithine transcarbamylase and cytopigment p450 enzyme; Normally the gene product that is produced by thymus gland comprises serum thymic factor, Thymus humoral factor, thymopoietin and extrasin alpha ₁

Micro-organs culture of the present invention can be used in gene product is passed in the method for recipient subjects.The isolated cell group who provides from the donor experimenter is provided this method, and this cell mass has the microarchitecture of the organ or tissue that separates this cell and is provided at the isolated cells group's that can keep about at least twenty four hours in the minimal medium the surface-area and the ratio (asurface area to volume) of volume.But coding is followed the transfered cell group with the recombinant nucleic acid of directly expressing the expectation gene product, produces the transgenic cell group in the micro-organs explant, as the transgenosis explant.The transgenosis explant can give recipient subjects.Donor experimenter and recipient subjects can be of the same race or not of the same race.

Micro-organs culture of the present invention also can be used in identifies the cell of inducing given organ, comprises in the outgrowth compositions and methods of progenitor cell.This method comprises that generation cultivates according to micro-organs explant of the present invention, and wherein explant comprises a cell with hyperplasia ability at least.After in being positioned over cultivation, explant contact candidate compound as the compound of test cell hyperplasia ability, and is determined at the candidate compound existence level of hyperplasia down.Then the candidate compound mensuration level that has hyperplasia is down compared with there not being under the candidate compound hyperplasia level.Candidate compound exists down, and the increase of hyperplasia level is the sign of hyperplasia reagent.The use similar approach can the outgrowth inhibitor of identification of cell.Especially, when using aforesaid method to determine that there is the mensuration level of hyperplasia down in candidate compound, it can be compared with there not being under the candidate compound hyperplasia level.There is the sign of agents for inhibition of cell proliferation when the hyperplasia level reduces down in candidate compound.

Another method that micro-organs culture of the present invention can be used for is to identify the reagent of inducing or suppressing one or more cell type differentiation of given organ, or keeps the compositions and methods of specific differentiation state (prevention is dedifferented).This method comprises the micro-organs explant that produces interested organ, and cell mass is formed the explant with this organ microarchitecture, and as herein and the following stated, aleph is 1.5mm at least approximately ^-1, comprise that at least one has differentiation capability or differentiation and cell that have the ability of dedifferenting.In case after cultivating, this cell mass contact candidate compound is also measured this compound existence cytodifferentiation level down.The mensuration level that there is cytodifferentiation down in candidate compound is compared with there not being under the candidate compound cytodifferentiation level.The level increase of candidate compound selecting cell differentiation is the sign of cytodifferentiation reagent.Use the inhibitor that similar approach can the identification of cell differentiation.Especially, when using aforesaid method to determine that there is the mensuration level of cytodifferentiation down in candidate compound, it can be compared with there not being under the candidate compound cytodifferentiation level.There is the sign of cytodifferentiation inhibitor when the cytodifferentiation level reduces down in candidate compound.

Another aspect of the present invention provides to be identified and from the method for organ separate stem cells or progenitor cell.This method provides isolated cell group explant from organ usually in cultivation.As described here, the feature of explant be (i) in cultivation, keep the microarchitecture of the organ that this explant originates, (ii) at least about 1.55mm ^-1Surface-area and bulk index (aleph) and (iii) at least one has the progenitor cell or the stem cell of hyperplasia ability.In order to expand dispersive cell mass in the explant, the reagent of progenitor cell or stem cell proliferation is induced in this explant contact, as somatomedin or other mitogen.Subsequently, can separate the progenitor cell that expands from explant.Can rely on their proliferation response to identify this explant subgroup.In other embodiments, this ancestral/stem cell can be in cultivation spontaneous hyperplasia, even when not adding exogenous agent.In other embodiments, separable this reagent is reacted and outgrowth progenitor cell or stem cell from explant, as separating from the direct mechanical of newly sprouting of residue explant or dissolving all or part of explant and separate the cell mass that expands subsequently.

Another method that micro-organs culture of the present invention can be used for is the method that promotes the recipient subjects wound healing.This method comprises that from donor experimenter isolated cell group this cell mass has at least approximately 1.5mm ^-1Aleph and this cell mass is applied to the wound of recipient subjects.Donor experimenter and recipient subjects can be of the same race or not of the same race.In one embodiment, the tissue of isolating cell is that the wound of skin and recipient subjects is a ulcer, the ulcer relevant as diabetes.

Remove non-return pointing out, enforcement of the present invention will be adopted cytobiology, cell cultures, molecular biology, genetically modified organism, microbiology, recombinant DNA and immunologic routine techniques, and they are in art technology.Explained this technology in the document fully.For example see MolecularCloning:A Laboratory Manbal, second edition, Sambrook, Fritsch and Maniatis chief editor (Cold Spring Harbor Laboratory Press:1989); DNACloning, Volumes I and II (D.N.Glover chief editor, 1985); Oligonucleotide Synthesis (M.J.Gait chief editor, 1984); Mullis etc.United States Patent (USP) 4,683,195; Nucleic Acid Hybridization (B.D.Hames﹠amp; S.J.Higgins edits .1984); Transcription And Translation (B.D.Hames ﹠amp; S.J.Higgins edits .1984); Culture of Animal Cells (R.I.Freshney, Alan R.Liss, Inc., N.Y.); Gene TransferVectors for Mammalian Cells (J.H.Miller and M.P.Calos chief editor, 1987, Cold Spring Harbor Laboratory); Methods inEnzymology, 154 and 155 volumes chief editors such as () Wu, Immunochemical Methods InCell And Molecular Biology (Mayer and Walker chief editor Academic Press, London, 1987); With Handbook of Experimental Immunology, VolumesI-IV (D.M.Weir and C.C.Blackwell, chief editor., 1986).

By following detailed description book and claims, other features and advantages of the present invention will be clearly.

The accompanying drawing summary

Fig. 1 describes decision Aleph, and wherein x=tissue thickness and a=organize the graphic representation of micro-organs of the size of width.

Fig. 2 is after showing the cultivation different time sections, the histogram of hyperplasia in the cavy micro-organs culture of BrdU marker determination.

Fig. 3 shows to cultivate after 1-8 days the histogram of hyperplasia in people's skin of back micro-organs culture of BrdU marker determination.

Fig. 4 A-4D is the Photomicrograph of demonstration corresponding to the immunofluorescence of the replicating cell of mouse skin (amplifying 50 times) (Fig. 4 A), guinea pig skin (amplifying 75 times) (Fig. 4 B), people's foreskin (amplifying 50 times) (Fig. 4 C) and people's foreskin (amplifying 75 times) (Fig. 4 D).

Fig. 5 A-5C is the transverse section (amplifying 75 times) of people's epidermis micro-organs explant, has shown cultivating zero (Fig. 5 A) system structure of tissue in the time of three (Fig. 5 B) and six (Fig. 5 C) days.

Fig. 6 is that proof uses BrdU to mix different thickness (x) and the histogram of the influence of the epidermal hyperplasia of the cavy micro-organs culture that wherein (a) maintenance 4mm is constant.

Fig. 7 A-7B is the Photomicrograph (amplifying 75 times) that shows corresponding to the immunofluorescence of the proliferative cell in the micro-organs culture that comes from pancreas.

Fig. 8 is the histogram that shows the amount of insulin of the pig pancreas micro-organs culture release of growing up.

Fig. 9 is presented at when cultivating three days, four days and six days, in the proliferative cell in the micro-organs culture of colon, liver, kidney, duodenum and oesophagus ³The histogram that the H-thymidine mixes.

Figure 10 A-10C shows that hair follicle enlivens outgrowth Photomicrograph in the micro-organs culture of immunofluorescence assay.40 times of ratio of enlargement (Figure 10 A), 40 times (Figure 10 B) and 75 times (Figure 10 C).

Figure 11 is the histogram of hair shaft size distribution when being presented at little cultivation and beginning and finish.

Figure 12 is presented at the histogram that the mitotic division in the micro-organs cultivation in the guinea pig skin culture suppresses under the 2.5ng/ml TGF-β existence.

Figure 13 is the graphic representation of micro-organs explant of the treatment of chronic skin ulcer, and the incomplete tangent plane that has shown tissue slice makes that keep can maneuverable in vivo structure.

Figure 14 is the photo with the mouse surface behind micro-organs culture replacement a slice normal skin; Can observe in healing, the implant new hair shaft and the implant of generation and incorporate normal mouse skin (amplifying 10 times) into.

After Figure 15 is (with the plasmid transfection culture of the plain enzyme reporter gene of coding fluorescence) transfection, the diagram that luciferase reporter gene is expressed in guinea pig skin micro-organs culture.

Figure 16 is with the plasmid that shows luciferase reporter gene, behind the cation lipid mediation transfection culture, and the diagram that luciferase gene is expressed in rat lungs and thymus gland micro-organs culture.

Figure 17 handles the final hair follicle activatory diagram in back with FGF in the micro-organs culture of the present invention.

Figure 18 is the diagram that the transgenosis luciferase gene is expressed in micro-organs explant of the present invention.

Detailed Description Of The Invention

The present invention relates to three-dimensional extraorgan's explant-culture system. This culture systems can be used for little device Official's explant external near body in long-term hyperplasia under the environment found of complete organ. Here retouch This culture systems of stating provides hyperplasia and suitable cell maturation to keep and the intracorporeal organ copy (counterpart) similar structure.

Micro-organs culture of the present invention provides vitro culture system, and it can keep tissue or device Official's a part and their function keep a period of time of prolongation. These culture systems provide External model, can make things convenient for and detect exactly Cell Differentiation, hyperplasia, cell function and Change the method for this cell characteristic and function. The gained culture has various application, scope from Transplant in the body or implant, to in-vitro screening cytotoxin compounds and pharmaceutical composition with " giving birth to The thing reactor " in preparation bioactive molecule and from organizing the isolate progenitor cell.

For example, in the mode that does not limit, specific embodiments of the present invention comprises (i) micro-organs Marrow is cultivated implant and is used for the marrow that the replacement chemotherapy process destroys; (ii) the micro-organs liver is planted Enter thing for increasing the liver function of liver cirrhosis patient; (iii) theme micro-organs culture (as What the science of heredity of growth changed the pancreas micro-organs of the insulin of expression recombinant gene expression) is thin Born of the same parents; The dental repair thing that (iv) is connected with the micro-organs culture of oral mucosa.

Still in the non-limiting embodiments of other example, theme micro-organs culture can be used for The various widely compounds of in-vitro screening, such as cytotoxin compounds, growth/regulatory factor, medicine Thing preparation etc. So far, compound to be detected is kept and contacted to the micro-organs culture external. Can measure the activity of cytotoxin compounds, for example, by its destruction or kill in the explant The ability of cell.

This can easily be estimated by important staining technique. Contain by the cell of analyzing explant Amount is as estimating growth/regulatory factor by total cell concentration and noble cells amount. This can make Finish with standard cell lines and/or histological techniques, comprise using and adopt the restriction specific type thin The immunocytochemical technique of the antibody of extracellular antigen. Can estimate that various medicines are in three dimension system Middle Normocellular effect of cultivating. For example, can detect and increase medicine that red blood cell forms pair The effect of marrow micro-organs culture. Can detect affects cholesterol metabolic as reducing the cholesterol product The medicine of giving birth to is to the effect of liver micro-organs. Also can adopt the micro-organs of abnormal structure to cultivate Thing is as promoting the research of hyperplasia or new hyperplasia disorder. For example, growth of tumour cell is invaded The micro-organs explant of organ can be used as the model system of the effect of test example such as anti-tumor agent comprising salmosin System.

For convenience, collected certain that adopts in specification, embodiment and the claims here A little terms.

Term " explant " refers to the cell aggregation from organ, takes from health and is grown in artificial In the culture medium. When mentioning the explant of the organ with substrate and epithelial components, this term is logical Often refer to that two kinds of compositions in the single explant of that organ all contain.

Term " tissue " refers to similar specialized cells group or layer, and they are carried out together, and some is special Function.

Term " organ " refers to two or more adjacent tissue layers, and wherein organized layer keeps some shapes Cell-the cell of formula and/or the interaction of cell-matrix are to produce microarchitecture. Among the present invention, the micro-organs culture is prepared by this organ, moves such as mammal skin, lactation Thing pancreas, liver, kidney, duodenum, oesophagus, bladder, cornea, prostate, marrow, Thymus and spleen.

Term " matrix " refers to supporting tissue or organ matrix.

Term used herein " micro-organs culture " refers to the cell mass that separates, as has branch Explant from the microarchitecture of the organ or tissue of this cell. That is to say separation thin Born of the same parents form the interactional three-dimensional structure of simulation/retaining space together, such as cell-cell, and cell The true tissue that-matrix and cell-matrix interact and this explant is originated and complete The location of organism. Therefore, kept in the tissue of outer planting such as between matrix and the epithelial layer this Plant interaction, so that critical cell interaction provides the biology of for example having kept explant The autocrine of function and paracrine factor and other extracellular stimulus thing, and provide in the sample and deposit Long-term surviving power under the condition of enough nutrition and refuse transportation.

Theme micro-organs culture has the little of organ or tissue that the cell or tissue explant separates Architecture. Term used herein " microbody system " refers to the cell or tissue explant that separates The group, wherein at least about 50%, preferably at least about 60%, more preferably at least about 70%, still More preferably at least about 80% and most preferably at least about 90% or more cell mass in external dimension Held they and their at least one cell of nature and/or functional cohesion or acellular in vivo The nature of substrate and/or functional cohesion, and form at least approximately one deck, more preferably at least about five Layer and choosing is arranged most about ten layers or more cell are cultivated at least. Preferably, the cell of explant Keep the biologically active of their organ or tissue of at least a separation.

Term used herein " separation " refers to separate from the natural environment of organism Explant. This term comprises the total natural separation from its natural environment, as from donor animal Take out, such as mammal such as people or piggy. For example, term " separation " refers to the thin of explant Born of the same parents group cultivates as the part of explant, or transplants with the explant form. When being used for mentioning During cell mass, term " separation " comprises that the proliferative cell by micro-organs culture of the present invention gets The cell mass that arrives.

Term " ectoderm " refers to the outermost layer of three primitive layers of embryo; By it derive corium and Epidermal tissue such as nail, skin and hair and body of gland, nervous system, external receptor and oral cavity and The mucous membrane of anus.

Term " epithelium " refer to inside and outside body surface Amphiesms (epidermis, mucus With serosity), comprise body of gland and other structure of deriving therefrom, as cornea, oesophagus, Epidermis and follicular epithelium cell. The epithelial tissue of other example comprises olfactory epithelium, and it is nasal cavity The vacation of regio olfactoria is layer covering epithelium again, and contain the acceptor of sense of smell; Galandular epithelium refers to that secretion is thin The epithelium that born of the same parents form; Scaly epithelium refers to the epithelium that flat like cell forms. The term epithelium also Can refer to the transition epithelium, it be special find owing to contraction and swelling are changed by huge machinery The indent organ that becomes, as represent the tissue of transition between layering squamous and the columnar epithelium. Term " on Skin forms " refer to that the epithelial tissue growth covers and strip off the surface and heal.

Term " skin " refers to health outer protection covering, is made up of corium and epidermis, should Be interpreted as to comprise sweat gland and sebaceous glands, and hair follicle structure. Run through the application, can use shape Hold word " epidermis ", and when being used for using their content, being construed as and being often referred to skin The attribute of skin.

Term " epidermis " is meant the outermost and the no vascular lamina of skin, is derived from embryonic ectoderm, and thickness is 0.07-1.4mm.In palm and plantar surface, from inside to outside it comprises five layers: the stratum basale of being made up of the mast cell of arranged vertical; The projection that the band of being made up of flat polyhedral cell is short or the spinose cell or the spinous process layer of spinous process; The GCL of forming by flat granulocyte; By the transparent layer that basic unit's hyaline cell is formed, the unclear or shortage of the nuclear in the hyaline cell; With the stratum corneum of forming by flat cutin cytode.In the epidermis of general body surface, lack transparent layer usually." epiderm-like " is and the similar cell or tissue of epidermis, but also can be used in reference to any tumour that forms with embedding epidermis composition that no epidermis position exists.

" corium " or " corium " is meant the skin layer that is deep to the epidermis below, is made up of the close layer of vascular reticular tissue, and contains the nerve and the end-organ of feeling.Root of hair and sebiferous gland and sweat gland are buried epidermal structures in corium.

Term " body of gland " is meant the cell aggregate of the material that special secretion or drainage metabolism usual with it need have nothing to do.For example, " sebiferous gland " is the holocrine secretion body of gland of secreting grease material and sebum in the corium.Term " sweat gland " is meant the body of gland of secretion sweat, is positioned at corium or subcutis, by the catheter opening on the body surface.Common and eccrine sweat gland distributes at most at body surface, and promotes cooling by the evaporation of secretory product; The apocrine secretion sweat gland is discharged to hair follicle top, rather than directly is discharged on the skin, only finds at some body regions, as around the anus and armpit.

Term " hair " (or " hair ") is meant filamentary texture, and particularly the special epidermal structure of being made up of Keratin sulfate and grown by the crater nipple in the corium by the Mammals generation, is the feature of that class animal only.This term also refers to the aggregate of this hair." hair follicle " is meant that the epidermis tubulose that holds hair caves in, and hair is longer from here; " follicular epithelium cell " is meant the epithelial cell that corium centers in the hair follicle, as stem cell, outer root sheath cell, stroma cell and internal root sheath cell.This cell can be normal non-malignant cell, or conversion/immortalized cell.

Term " alopecia " typically refers to baldness, does not have hair as the skin region of normal presence hair.Various forms of alopecia known in the art.For example, alopecia is meant the hair forfeiture, and is reversible usually, and the zone in that strictness limits generally includes beard or scalp; Medicine (mediacamentosa) alopecia is meant owing to take in drug induced hair forfeiture; Male pattern alopecia, or male pattern baldness are meant the hair forfeiture of unusual decision and androgen-dependent, move back decline beginning and symmetrical the progress to finally only staying the thin hair periphery from forehead usually.

Run through the application, term " disorder of hyperplasia skin " is meant that unnecessary or paraplasm is any disease/disorder of the skin of sign with skin histology.The typical feature of these situations is that epidermal cell proliferation or cytodifferentiation are incomplete, comprises for example chain scales of skin that peel off, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis (epidermolytichyperkeratosis) and the seborrheic dermatitis of X.For example, epidermodysplasia is a kind of epidermis developmental defect of form, and as " epidermodysplasia verruciformis (dermodysplasiaverruciformis) ", it is owing to the virus with common wart is equal to or the caused situation of closely-related virus.Another example is " epidermolysis ", and it is meant spontaneous or forms the epidermis relaxed state of blister and bleb in wound site.

Term used herein " psoriasis " is meant the hyperplasia skin disorder that changes the skin regulation mechanism.Especially, formed infringement, it comprises former and the change of secondary epidermal hyperplasia, the expression of skin inflammation reaction and adjusting molecule such as lymphokine and inflammatory factor.The morphological feature of psoriasis skin is that epidermic cell upgrades more, and epidermis thickens, and dyskeratosis, inflammatory cell infiltration skin corium and polymorphonuclear leukocyte infiltration epidermal area cause that the basal cell cycle increases.In addition, there are hyperkeratosis and parakeratosis.

" outgrowth " used herein and " hyperplasia " are the mitotic division of phalangeal cell experience.

Term " progenitor cell " is meant a kind of undifferentiated cell, and it can hyperplasia and produces the progenitor cells with the ability that produces a large amount of parent cells more, and wherein parent cell can produce the daughter cell that differentiation maybe can be broken up successively.Term used herein " progenitor cell " is also intended to comprise that this area is called the cell of " stem cell " sometimes.In preferred embodiments, term " progenitor cell " is meant the generalization parent cell, the special differentiation of its descendants (offspring), and common direction difference, as obtain complete individual character, as the carrying out property variation that occurs in embryonic cell and tissue.For example, " hemopoietic progenitor cell " (or stem cell) be meant that the relevant organ with other blood of marrow occurs with produce for example differentiation offspring's of red corpuscle, lymphocyte and other hemocyte progenitor cell.

" transformant " used herein is meant the spontaneous cell that changes the indeterminate growth state into, and promptly they have obtained in cultivation the ability by unlimited merisis.Growth control about them is impaired, can use as neoplastic, and the feature of transformant described in anaplastic and/or outgrowth term.

" immortalized cell " used herein is meant to change by chemistry and/or recombination method and makes cell have in cultivation the cell by the ability of unlimited merisis.

Term " cancer " is meant that pernicious new growth forms tends to the epithelial cell that soaks into surrounding tissue and tend to shift.The cancer of example comprises " rodent cancer ", and it is rare transfer, has the local skin epithelial tumor of invading and destroying potentiality; " squamous cell carcinoma ", it is meant that tesselated epithelium produces and has the cancer of cuboid cell; " sarcocarcinoma ", it comprises the malignant tumour of being made up of cancer and sarcoma tissue; " adenocele cancer ", with by little epithelial cell nest band is separated or around hyaloid or Saliva Orthana matrix post or band be the cancer of sign, occur in mammary gland and sialisterium and respiratory mucus gland; " epidermoid carcinoma ", it is meant tends to the cancer cells that breaks up in the identical mode of epidermal differentiation mode, and promptly they tend to form spinose cell and experience angling; " nasopharyngeal carcinoma ", it is meant the malignant tumour of the lining epithelium generation of nose rear space; " renal cell carcinoma ", it belongs to arranges the carcinoma of renal parenchyma that changeable renal tubular cell is formed.Another kind of cancer epithelial growth is " papilloma ", and it is meant that the innocent tumour that derives from epithelium and papilloma virus are as origin cause of formation thing; " epidermoid ", it be meant intermediate sulcus (neutral groove) when closing embedding ectoderm composition and the brain and the meninges tumour that form.

Any animal when " transgenic animal " used herein, preferred non-human mammal, bird or Amphibians, wherein one or more zooblasts contain reliable human intervention and the heterologous nucleic acid that imports, as known in the art transgenic technology.By intentional genetic manipulation, as by fiber injection (micro injection) or by the injection recombinant virus, transfered cell precursor and directly or indirectly with the nucleic acid transfered cell.The term genetic manipulation does not comprise classical cross-breeding, or in vitro fertilization, but would rather be directly to import recombinant DNA molecules.This molecule can be incorporated in the karyomit(e), or it can be the DNA that duplicates on the non-chromosome.This term also comprises transgenic animal, and wherein this recombination is a silencer, for example described FLP in this area or CRE recombinase dependency construct.Transgenic animal with comprise composing type and " rejecting " with good conditionsi animal." non-human animal " of the present invention comprises vertebrates such as rodent, inhuman Primates, pig, sheep, dog, cow, chicken, Amphibians, Reptilia etc.Preferred non-human animal is a piggy, or is selected from rodent family and comprises rat and mouse, most preferably mouse.Term used herein " heterozygosis animal " is meant wherein finds recombination, or wherein recombinant chou animal some but be not the animal of expressing in whole cells.

I. the foundation of micro-organs culture

According to the prominent feature of micro-organs culture of the present invention and method is the ability that keeps the cellular environment that particular organization finds in vivo.The present invention part is based on a kind of discovery, and this discovery is under the environment of definition, the different tissues layer of organ explant as between the growth of cell of matter and epithelial lining can in cultivation, be activated and hyperplasia and maturation.And, cell-cell that explant itself provides and cell-matrix enough sustenticular cell homeostasiss that interacts, the maturation of cell in cultivating as explant, differentiation and separating, so the microarchitecture of sustentacular tissue and function prolong for some time.

The example of the physics contact between cell and the acellular substrate (matrix) is the physics contact between epithelial cell and its stratum basale.The example of the physics contact between a cell and another cell comprises that for example iuntercellular connects as the slit connection and closely is connected the actual physical contact of being kept.The example that cell contacts with another cell function comprises iuntercellular electronics and chemical communication.For example, the myocardial cell is by electricimpulse and other myocardial cell's communication.In addition, many other cells are by chemical messenger, are transferred to the hormone of farther position (internal secretion signal) as local diffusion (paracrine signal and autocrine signal) or by vascular system, with other cell communication.The example of iuntercellular paracrine signal is the signal that the various cells of digestive tube (known is enteroendocrine cell) produce, and as the secretion Somatostatin, pylorus stomach (G) cell discharges the pylorus D cell of gastrin near suppressing successively.

Do not wish to be bound by any particular theory, this microarchitecture may be in the minimal medium, as not containing external source serum or somatomedin, explant keep extremely important, because by the paracrine and the autocrine factor that the interaction between special cells in the explant produces, this tissue can remain in this minimal medium.

In addition, term " keep, external, their physics and/or functional contact " does not comprise the isolated cells group, wherein at least one cell development is and at least one cell or acellular substrate generation physics and/or functional the contact, and this physics and/or functional contact do not have in vivo.The example of this growth is at least one cell or isolated cells group hyperplasia.

In preferred embodiments, the cell mass of forming explant separates from organ with the natural avidity mode between another to keep a cell, for example keeps the layer of different cells, if exist in the explant.For example, in skin micro-organs culture, the keratinocyte of epidermis keeps relevant with matrix and keeps the microarchitecture of healthy tissues to comprise hair follicle and body of gland.This foundation structure is that all organs are general, for example, contains epithelial components.In addition, this relation promotes intercellular communication.The communication of a lot of types takes place between the zooblast.This is very important in the cell that is just breaking up, and induces here to be defined as a kind of (inducing) and another kind of (replying) tissue or intercellular interaction, and responsive cell experience differentiation direction changes as a result.In addition, inductive interacts to occur in embryo and mature cell and can act on and sets up and keep form formation pattern and induce differentiation (Gurdon (1992) Cell 68:185-199).

In addition, micro-organs culture prepared in accordance with the present invention keeps the microarchitecture of healthy tissues, even when cultivating for some time that prolongs.This comprise according to they in vivo normally a situation arises, keep hair follicle, sweat gland and sebiferous gland (seeing example VII A I and Figure 10 A-10C) in the vitro skin micro-organs, or according in vivo normally a situation arises, keep youth's lattice Han Shi islet cells (seeing EXAMPLE IV, V and VI) in the pancreas.Because these cultivations can maintain under the controlled and unified condition and still near in-vivo tissue, they provide observation, mensuration and control spontaneous phenomenon and the unique opportunity of the spontaneous phenomenon disorder that produced by disease, aging or wound.And, determine during research is cultivated the position each cell technology standby property for each composition of tissue they each other and and whole tissue between function when interacting experience is provided.

Described the example of micro-organs culture prepared in accordance with the present invention among the appended embodiment, and can comprise can comprise that multilayer makes and kept a cell and the mode aggregating cells group of natural avidity between another.The hyperplasia of each cell or groups of cells can be observed and follow the trail of to radioautography or immunofluorescence technique.

As further example only, appended embodiment proved the theme culture systems provide epithelium and matrix components can with replication in vitro in the physiological condition system relatively.Importantly be that the cell that duplicates in this system suitably is separated to form normal epidermis of morphology and histology and corium composition.

Cell-the cell that has kept original structure except separation, the explant of cell-matrix and cell-matrix microarchitecture, the size of explant is very important for the vigor of wherein cell, tends to keep for some time of prolonging as the micro-organs culture, as 7-21 days or longer.Therefore, organize explant to have and select to provide enough nutrition and gas such as O ₂Diffuse to each cell in the three-dimensional micro-organs, and cellular waste diffuses out explant and makes cytotoxicity and because the dead minimized size that occurs together of local refuse in the micro-organs.Therefore, the size of explant can decide near the needs of the minimum level of each cell when lacking special transferring structure or synthetic substrate.As described here, have been found that if the index-Aleph that is calculated by explant thickness and width is at least greater than about 1.5mm ^-1, can keep this accessibility.

" Aleph " used herein is meant formula 1/x+1/a 〉=1.5mm ^-1The surface-area that obtains and the ratio of volume; Wherein x=tissue millimeter thickness and a=organize the millimeter width.In preferred embodiments, the aleph of explant 1.5 to 25mm ^-1Scope in, more preferably 1.5 to 15mm ^-1Scope in and even more preferably 1.5 to 10mm ^-1Scope in, though also comprise aleph 1.5 to 6.67mm ^-1, 1.5 to 3.33mm ^-1Scope in.

Therefore, the invention provides in the scope that the surface-area of organizing explant and bulk index maintain selection.The range of choice of this surface-area and bulk index provide cell by with individual layer in the diffusion of cell similar fashion near the approach of nutrition and waste treatment.If surface-area and bulk index, being defined as " Aleph or Aleph index " here is about at least 1.5mm ^-1, can reach this accessibility level.When definite surface-area and bulk index, ignored the third dimension, all made a variation because the variation of the third dimension causes the radiometric analysis of volume and surface-area.Yet when determining Aleph, a and x should be defined as two minimum dimensions of tissue.

Table I provides the example of Aleph, and the tissue that wherein for example has 0.1mm thickness (x) and 1mm width (a) will have 11 Aleph index.In example I, tissue has x=0.3mm and a=4mm makes Aleph=3.48.In EXAMPLE III, it is constant in 4mm that x changes a.As shown in Figure 6, with the increase of explant thickness, proliferative activity reduces in fact.Therefore, under the 900 μ m thickness, the quantity of proliferative cell is approximately hanged down 10 times than the tissue that the similar source with 300 μ m thickness obtains in the micro-organs culture.Aleph index with tissue of 900 μ m thickness is 1.36mm ^-1, be lower than minimum value described here, yet the Aleph index with tissue of 300 μ m thickness is 3.58mm ^-1, it is fine drops on here in the restricted portion.

Table 1: as millimeter ^-1A (width) and the different value of the ratio index " Aleph " of the function-surface-area of x (thickness) and volume.

Width

x(mm) a＝1mm a＝2mm a＝3mm a＝4mm a＝5mm

0.1 11 10.5 10.33 10.25 10.2

0.2 6 5.5 5.33 5.25 5.2

0.3 4.3 3.83 3.67 3.58 3.53

0.4 3.5 3 2.83 2.75 2.7

0.5 3 2.5 2.33 2.25 2.2

0.6 2.66 2.16 2 1.91 1.87

0.7 2.4 1.92 1.76 1.68 1.63

0.8 2.25 1.75 1.58 1.5 1.45

0.9 2.11 1.61 1.44 1.36 1.31

1 2 1.5 1.33 1.25 1.2

1.2 1.83 1.3 1.16 1.08 1.03

1.3 1.77 1.26 1.1 1.02 0.96

1.6 1.625 1.13 0.96 0.88 0.83

2 1.5 1 0.83 0.75 0.7

Once more, do not wish to be bound by any particular theory, a large amount of factors that the dimensional culture system provides have the success that helps it:

(a) suitable surface-area and volumetric ratio that the explant size of suitably selecting, as use top Aleph to calculate, three dimensional matrix provide all cells that nutrition enough diffuses to explant and cellular waste enough to diffuse out the explant all cells.

(b) since the three-dimensional of matrix compare with the cell of monolayer culture, the continuous active growth of various cells, growing to of the cell of monolayer culture converges, and shows contact inhibition, and stops growing and divide.Hyperplasia during growth and regulatory factor may partly be responsible for stimulate cultivating to taking great pains to build up of replicating cell in the explant and adjusting cytodifferentiation, as in addition responsible micro-organs culture, this cultivation is static constant aspect overall volume.

(c) three dimensional matrix has kept the spatial distribution of cellular constituent, and it is in close proximity to the spatial distribution of being found in the copy tissue in the body.

(d) cell-cell and cell-matrix interact and can allow the foundation of the localization microenvironment that helps cell maturation.Have realized that keeping of noble cells phenotype not only needs growth/differentiation factor, and need suitable cell interaction.The present invention has effectively simulated tissue microenvironment.

As described in following illustrative embodiment, separated micro-organs culture, as derived from skin, pancreas, liver, kidney, duodenum, oesophagus, bladder, marrow, thymus gland or spleen, and growth reaches 21 days in cultivation from animal (comprising the people).Yet, keep cultivating prolongation for some time also within the scope of the invention above 21 days.

II. the explant of micro-organs culture is originated

Use is from for example: skin and mucous membrane (comprising oral mucosa, gastrointestinal tract mucous, nasal cavity, respiratory tract, uterine cervix and cornea); Pancreas; Liver; Gall-bladder; Bile duct; Lungs; Prostate gland; The uterus; Mammary gland; Bladder; Relevant organ with blood such as the isolating explant of thymus gland, spleen and marrow can obtain theme micro-organs culture.Therefore can produce the vitro culture equivalent of this organ.The tissue that forms explant can be morbid state or normal (as health tissues).For example, the organ of separable micro-organs explant of the present invention can be subjected to following influence: the hyperplasia disorder, as psoriasis or keratosis; The hyperplasia of virus infection is as virus infection or parillomarvirus infections; New hyperplasia disorder is as rodent cancer, squamous cell carcinoma, sarcoma or Wei Ermusi tumour; Or the fibrosis tissue, as from cirrhotic liver or suffer pancreatitic pancreas.

The animal example of separable cell of the present invention comprises people and other Primates, pig, as inbreeding pig (as piggy and transgenic pig), rodent etc. wholly or in part.

III. growth medium

The tissue culture medium (TCM) that exists for the cell of cultivating from animal is arranged in a large number.Some complexity in these, some are simple.Although expectation micro-organs culture can be grown, shown that here culture can remain in the simple culture media, as Dulbecco ' s minimum essential medium in complicated substratum.In addition, although culture can be grown, shown that here serum or other any bio-extract do not need in the substratum that contains serum or other bio-extract such as hypophysis extract.In addition, organ cultures can lack for some time of keeping prolongation under the serum.In the preferred embodiment of the invention, in the maintenance process of vitro culture, do not comprise somatomedin in the substratum.

Very important about the main points of growing in the minimal medium.At present, indefinite albumen that prolonged most substratum of mammalian cell growth and system's fusion or use the feeder cell required albumen of this growth that provides support.Because this indeterminate proteic existence can disturb the termination that is intended to of theme micro-organs culture to use, exist under the situation that reduces to minimum and cultivate explant so be desirably in indeterminate albumen usually.

Wording used herein " minimal medium " is meant the chemically clear and definite substratum of the nutrition that the cell survival that only comprises in the cultivation and hyperplasia are required.Typically, minimal medium does not contain bio-extract, as somatomedin, serum, hypophysis extract or be not cell mass survival and other required material of hyperplasia in support cultivating.For example, minimal medium generally includes at least a amino acid, at least a VITAMIN, at least a salt, at least a microbiotic, at least a indicator, as phenol red, be used for determining hydrogen ion concentration, other various compositions that glucose and cell survival and hyperplasia are required.Minimal medium does not contain serum.Various minimal mediums can be from GibcoBRL, Gathersburg, and MD buys from commercial with minimum essential medium.

Yet, although somatomedin and regulatory factor do not need to join in the substratum, add this factor, or inoculate hyperplasia and cell maturation that other special cell can be used for strengthening, changing or regulate cultivation.Cell growth in the cultivation and the active influence that can be subjected to various somatomedins, as Regular Insulin, tethelin, somatomedins, G CFS, erythropoietin, Urogastron, liver erythrogenin (liver generates plain (hepatopoietin)), and pHGF.Other factor of regulating hyperplasia and/or differentiation comprises prostaglandin(PG), interleukin and naturally occurring anti-somatomedin, the member of fibroblast growth factor and transforming growth factor-beta family.

The micro-organs culture can maintain in any suitable containers as 24 or 96 hole microplates, and can maintain 37 ℃, at 5%CO ₂In.Can shake culture improve ventilation, hunting speed is 12rpm for example.

About the culture vessel of theme micro-organs culture (choosing wantonly) is provided, should notice that in preferred embodiments this container can be any material and/or proterties usually.Can use a large amount of differing materials to form container, include but not limited to: nylon (polymeric amide), terylene (polyester), polystyrene, polypropylene, polyacrylic ester, polyvinyl compound (as polyvinyl chloride), polycarbonate (PVC), tetrafluoroethylene (PTFE; Teflon), thermanox (TPX), nitrocellulose, cotton, polyoxyethylene glycol acid (PGA), cat catgut suture (cat gut sutures), Mierocrystalline cellulose, gelatin, dextran etc.Any in these materials can be woven net.Itself wait to implant when the micro-organs culture, preferably use for example polyoxyethylene glycol acid of biodegradable matrix, cat catgut suture material or gelatin.When culture is treated prolonged preservation or freezing preservation, can preferably non-biodegradable material such as nylon, terylene, polystyrene, polyacrylic ester, polyethylene, tetrafluoroethylene, cotton etc.The nylon wire that makes things convenient for that can use according to the present invention is the nylon filtering net that Nitex-has mean pore size 210 μ m and average nylon fiber diameter 90 μ m (#3-210/36, Tetko, Inc., New York).Other embodiment also has been discussed below.

In the embodiment of example, the pancreas micro-organs preparation that contains youth's lattice Han Shi (Langerhans) islet cells is as culture of the present invention.The feasible immunological rejection of avoiding of culture of capsule form then is provided.Can use three encapsulated general (example) methods.First, the tubular film coiling contains the shell of micro-organs explant.This film is connected with the transplanting polymkeric substance, and this device is connected with blood vessel successively.By operation to membrane permeability, make that allowing glucose freely to spread with the Regular Insulin front and back passes through film, yet blocking antibody and lymphocyte pass through, and the pancreatectomy animal blood glucose amount for the treatment of with this device can keep normally (Sullivan etc. (1991) Science 252:718).

In second method, the hollow fiber (choosing wantonly) that contains the pancreas explant is fixed in the polysaccharide alginate.When this device is positioned over diabetic animal by intraperitoneal, histocompatibility (Lacey et al. (1991) the Science 254:1782 that micromicro reduces and can be observed on the blood-glucose; Also see example VI).Therefore, textile fibres and with the back loading micro-organs explant (United States Patent (USP) 4,892,538 of Aebischer etc. in advance; The United States Patent (USP) 5,106,627 of Aebischer etc.; Hoffman etc. (1990) Expt.Neurobiol.110:39-44; Jaeger etc. (1990) Prog.Brain Res.82:41-46; With (1991) J.Biomech Eng.113:178-183 such as Aebischer).

The 3rd, micro-organs pancreas islet explant can be positioned in the microcapsule of being made up of alginate or polyacrylic ester (sees for example (1980) Science 210:908 such as Lim; O ' Shea etc. (1984) Biochim.Biochys.Acta 840:133; Sugamori etc. (1989) Trans Am.Soc.Artif.Intern.Organs 35:791; Levesque etc. (1992) Endocrinology 130:644; With (1992) Transplantation 53:1180 such as Lim).

At last, the substratum that should note keeping micro-organs culture of the present invention can be collected the source as conditioned medium.Term " conditioned medium " is meant supernatant, and as not containing cultured cells/tissue, it is that the cell that makes substratum be changed to comprise that the contact cultured cells produced after for some time produces some paracrine and/or the autocrine factor of justacrine in cultivate.The example of this product is Regular Insulin, various somatomedin and hormone.This conditioned medium can be as the cell of other type and the substratum of tissue culture.Alternatively, can adopt source such as the somatomedin of conditioned medium as new cellular products.This product can be from conditioned medium fractionation and purifying or basic purifying.

IV. measure the biological property of micro-organs culture

Shown from the micro-organs culture of the present invention of healthy tissues and kept homeostasis, the hyperplasia that the companion constitutes, organized whole does not grow.

The method of measuring hyperplasia is that well known in the art and most generally including determines with the cellular replication to be that the DNA of feature is synthetic.There are a lot of methods of the DNA synthetic of mensuration this area, can use any according to the present invention.In embodiments of the invention, with the nucleotide analog (BrdU) of radio-labeling (3H-thymidine) or mark, determined that with the immunofluorescence detection DNA is synthetic.

The formation of micro-organs culture and keep not only and can pass through the mature cell hyperplasia can also be by the active participation of precursor cell, and being included in is embryonic cell in some cases.Shown that the micro-organs culture is present in the proper environment that the preservation of these precursor cells, evaluation, separation and promotion evolve naturally.For example, the immature cell of observing stratum basale becomes ripe keratinocyte in skin micro-organs culture.Similarly, the embryonic pancreas cell can provide the ripe pancreas epithelial cell in the micro-organs culture.The secretion of measuring special product can be monitored the maturation of precursor cell and they subsequently as the function of mature cell, as the Keratin sulfate in the epidermic cell, and Glut 2 and glucagon in the pancreas epithelium, the Factor IX in the liver micro-organs culture.

Micro-organs culture prepared in accordance with the present invention kept presenting in the body in the healthy tissues microarchitecture.As above statement, this comprises keeping of hair follicle, sweat gland and sebiferous gland in the vitro skin micro-organs, according to keeping of Regular Insulin and glucagon secretory cell in normal circumstances in the body and the pancreas micro-organs.Because these cultures can maintain under the controlled and unified condition and still be very similar to the microarchitecture of intracorporeal organ with them, so they provide the unique opportunity of the spontaneous phenomenon disorder of observation, mensuration and control spontaneous phenomenon and disease, aging or wound generation.And, determine during research is cultivated the position each cell technology standby property for each composition of tissue they each other and and whole tissue between function when interacting experience is provided.

In addition, theme micro-organs culture can be kept for some time of prolongation in cultivation.Preferably, the micro-organs culture can be kept at least approximately twenty four hours in cultivation, and more preferably about at least two days, still more preferably about at least five days, still more preferably about at least seven days, still more preferably about at least two weeks or longer.Micro-organs culture of the present invention typically can be kept in cultivation seven days at least.In order to illustrate, in cultivation, kept approximately at least two ten one days from the skin micro-organs culture of people, mouse, cavy and rat skin.

Wording used herein " can be kept in cultivation " and be meant in the cell mass of organizing explant about at least 60%, preferably about at least 70%, more preferably about at least 80%, still more preferably about at least 90%, most preferably 95% or manyly after cultivating the certain period, maintain vigor.

In preferred embodiments, the hyperplasia of cell and loss cell equal one as ratio dead or the formation slough in the micro-organs culture, i.e. the quantity of hyperplasia equals the quantity of loss cell.In another embodiment, the ratio of the hyperplasia of cell and loss cell is greater than one in the micro-organs culture, i.e. hyperplasia speed is greater than loss cell.Under one situation of back, be interpreted as the micro-organs culture and comprise just the expanded cells group.

V. the application of micro-organs culture

The example application of micro-organs culture of the present invention comprises following:

(a) factor that comprises in the normal homeostasis of appraisement organization and organ;

(b) research comprises and changes nutrition and have the effect of potential toxic reagent to the normal homeostasis of the tissue of organ and cell about changing environment;

(c) understand morbidity or wound begin with process in the tissue of the organ that triggers and the approach of cell change;

(d) identify that reverse is fallen ill or the repair mechanism of the detrimental action of the environment change that wound is relevant;

(e) the cells whose development rule of breaking up in the normal homeostasis process of tissue;

(f) intraorganic specialized structure is as the growth rule of hair follicle;

(g) the replenishing/transplant of organ wherein kept the each several part of individual organ, but be not enough to replace or the destructive tissue of regenerating as taking place among chronic skin ulcer, various forms of diabetes or the chronic liver function depletion patient;

(h) as the tissue/organ equivalent of drug screening and Study of cytotoxicity;

(i) as the diagnostic method of hyperplasia disorder;

(j) source of the new somatomedin of conduct;

(k) as the source of ancestral cells;

(l) as the source of inducing molecule;

(m) as the screening thing of inducing molecule;

In order to further specify, the present invention can be used for producing the skin equivalent of micro-organs culture form.By background technology, should note having described for wound treatment the purpose of burn treatment particularly, a lot of trials that epithelial cell is grown in the mode of anthropomorphic dummy's skin.Skin is formed by two types.These are: (1) matrix or corium comprise loose inoblast and nerve, blood vessel and the adipocyte that is dispersed in the high-density collagen stroma; (2) epidermis comprises and the basal layer of epidermis of tight parcel enlivens outgrowth immature epithelial cell.When basal layer cell duplicated, some young cells were retained in stratum basale, and other are to external migration, and size increases and final coating of growing for anti-washing composition and reductive agent.In the mankind, the cell that stratum basale grows spends and about 2 weeks arrives edge or skins, necrocytosis and coming off after this time.Skin contains various structures and comprises hair follicle, sebiferous gland and sweat gland.Hair follicle is formed by the differentiation keratinocyte that dense lining epidermis caves in.This last capsule of opening that forms of caving in is collected and concentrating secreted Keratin sulfate and produce hair.Replacedly, the epidermic cell that caves in of lining can be secreted fluid (sweat gland) or sebum (sebiferous gland).The formation of these structures and hyperplasia rule are unknown.The equilibrium process of new cell generation and senile cell death is finished the constant renewal of healthy skin.In order to hinder aging, and the anomalous event that takes place of the disease of destruction of balance and wound, need understand this accurate rule and how to take place.

In one embodiment of the invention, the microarchitecture of skin and basic identical with it has epithelium/reticular tissue structure as it in the microarchitecture analogue body of micro-organs culture.For example, in the skin micro-organs culture, the keratinocyte of epidermis keeps relevant with reticular tissue and has kept this microarchitecture of organizing comprising hair follicle.The micro-organs culture that derives from the skin histology section also can comprise the stratum basale that supports epidermic cell, comprises that the extracellular matrix of dermal cell caves in at least one, as at least one hair follicle.Relation between skin epithelium and the skin connective tissue promotes intercellular communication.And full thickness skin can be grown under the variety of way that allows the air contact.The keratinocyte of explant is exposed to air and promotes faster differentiation of keratinocyte and the more extensive secretion of stratum corneum, and it is very important for skin permeation study.

At last, should notice studies show that recently that skin is the intrinsic and active element of immunity system (Cooper etc. (1987) The mechanobullous disease.In:Dermatologyin General Medicine, 3d.Ed., McGraw Hill, NY (pp.610-626).Being responsible for one of main cell type of various immune active in the skin is Langerhans' cells.These cells can and add three-dimensional skin culture by the fresh skin specimen preparation and produce the complete tissue system of immunology.The tradition tissue culture technique makes the long-time growth in cultivation of these cells very difficult.The ability that these cells are grown in three dimension system will have very big importance in research comprises aspect all of immigration, cytotoxicity and disease mechanisms.Such skin culture systems will comprise that the autoimmune disorder that directly or indirectly comprises skin (systemic lupus erythematous, large blister quasi-Pemphigus etc.) has maximum effect to research.Therefore, micro-organs culture of the present invention can be used to study the hyperplasia/differentiation disorder under minimized condition aspect the disease immunology.In order to identify the reagent that can suppress hyperplastic epithelium hyperplasia, the drug screening test of example can come from uses psoriasis skin explant.

This skin only makes can be as the example pattern of micro-organs culture growth with epithelium that matrix organization supports.Other tissue comprises that epithelium can be as micro-organs culture growth of the present invention.All find epithelium at each position of health, occur the interface of organ and environment there.In not damaging body, comprise the covering tissue of all free surface of skin in continuous circulation of epithelial cell and the organizer.In some cases, as in pancreas, epithelial cell lining is various cave in and with enzyme secretion to open space, make organ can bring into play function.Lung is another example of organ of highly caving in, and each caves in the epithelial cell lining lung, is diffused into health by its air from environment.Again, these epithelial cells have the characteristic performance.The lining of intestines also is made up of special epithelial cell, and these epithelial cells not only form barrier but also contain the selective specialized structure that assimilates food.All epitheliums are supported by reticular tissue.Comprise that still interactional another organ of important cells-matrix is a marrow.

Therefore, in another embodiment of the invention, the microarchitecture in pancreas source or basic identical with it has epithelium/reticular tissue structure as it in the microarchitecture analogue body of micro-organs pancreas culture.For example, pancreas micro-organs culture comprises the pancreas epithelial cell, as islet cells, keeps relevant with the pancreas reticular tissue.Therefore in pancreas micro-organs culture, kept the microarchitecture of healthy tissues and produced Normal Pancreas epithelial cell product such as Regular Insulin and glucagon.

In another embodiment, the invention provides by marrow and produce the micro-organs culture, wherein culture has kept the microarchitecture of intracorporeal organ.As described in embodiment XV, in cultivation, separated the marrow micro-organs, obtain the system that can compare with physiological condition.

Marrow culture of the present invention can be used for the treatment of disease or the situation of destroying the healthy bone myelocyte or reducing their Functional Capability.May be effectively in the haematological malignancies that being implanted in of theme micro-organs comprises marrow and other neoplastic treatment.This aspect of the present invention has been subjected among the patient of environmental factors (as ray, toxin etc.) disadvantageous effect also effective at treatment marrow.Although the marrow of usually preferably replanting into patient oneself obtains explant, should notice that this explant can be allochthonous, as from congener another member, or xenogeneic, as from another organism.The xenogenesis implant of example can be the micro-organs culture that derives from piggy of implanting the people.

In addition, the ancestors' of artificial blood long term growth is possible, if provide the growth/regulatory factor in essential matrix source to them.This interaction is provided by the theme micro-organs, provides the source of explant as dried and progenitor cell.Usually, the hemopoietic progenitor cell of marrow plantation (" sowing ") is in the substrate formed natural parcel of marrow micro-organs.The raw velocity restrictive factor of growth of stromal cells is that the inoblast mitotic index that comprises in the marrow stromal cell is low relatively.Therefore, in the growth of wanting to increase these cells and the extracellular matrix components place to their processing, explant can contact these reagent such as hydrocortisone or other fibroblast growth factor.

If shift disease or haematological malignancies patient and cultivate marrow in order to treat some, the marrow that obtains of this patient should be with physics or the paraplasm cell of chemotherapy " removing " before cultivation so.

Conditioned medium from marrow micro-organs culture of the present invention can be used as source new or known lymphokine, as the source as interleukin.

In one aspect, the present invention includes the purposes that theme micro-organs culture is transplanted in organism.Term used herein " gives ", " importing " and " transplanting " can exchange use, is meant with causing cellular localization in the method or the approach of desired site cell mass of the present invention to be positioned among the experimenter, as allosome of the same race or xenogenesis experimenter.Can give the experimenter with cell mass with any suitable approach, this approach causes cell to be delivered to desired site among the experimenter, and at least a portion cell maintains vigor there.Preferably about at least 5%, preferably about at least 10%, more preferably about at least 20%, still more preferably about at least 30%, still more preferably about at least 40% and most preferably about at least 50% or more many cells after giving the experimenter, maintain vigor.The cell viability phase can lack as several hrs after giving the experimenter, and is as twenty four hours, to several days, long as several thoughtful several months.The method that gives cell mass of the present invention comprises implants internal organ or body wall peritonaeum with cell, greater omentum bag for example, cell be implanted to the interior of recipient subjects organ or on, as pancreas, liver, spleen, skin.Micro-organs of the present invention also can be by being implanted to as giving the experimenter under the kidney tunicle.

Term used herein " experimenter " is meant Mammals, as Primates, as the people." xenogenesis experimenter " used herein is the experimenter who is imported into or waits to be imported into another species cell." allogeneic experimenter " is the experimenter who is imported into or waits to be imported into the same species cell.The donor experimenter provides the experimenter of cell, tissue or organ, and they are to be placed in cultivating and/or transplant and give recipient subjects.Recipient subjects can be xenogenesis or allogeneic experimenter.The donor experimenter also can provide cell, tissue or the organ that imports them again, i.e. autotransplantation.

In order to promote to suffer host immune to learn the transplanting of the cell mass of attacking, as using xenotransplantation, to transplant as pig and human, micro-organs can insert or be encased to chargeable or biodegradable device, then is transplanted to recipient subjects.The gene product that this then cell produces can be by for example comprising that for controlled delivery compound such as medicine the poly-unit that protein biology reagent designs transmits.Various biodegradable polymkeric substance (comprising hydrogel) comprise biodegradable or abiotic degradable polymer, can be used for being formed on the implant that the particular target position continues to discharge cell mass gene product of the present invention.This area is known to usually during the generation of this implant.See, for example, Concise Encyclopedia of Medical ﹠amp; Dental Materials, ed.By David Williams (MIT Press:Cambridge, MA, 1990); The United States Patent (USP) 4,883,666 of the Sabel etc.; The United States Patent (USP) 4,892,538 of Aebischer etc.; The United States Patent (USP) 5,106,627 of Aebischer etc.; The United States Patent (USP) 4,391,909 of Lim; United States Patent (USP) 4,353,888 with Sefton.Cell mass of the present invention can give in can accepting carrier or thinner, as Sterile Saline and water-containing buffering liquid.The purposes of this carrier is well known in the art.

In one embodiment, can adopt micro-organs culture treatment wound healing of the present invention.The reparation of known skin lesion is very complicated process, comprise that initial epithelial cell is divided a word with a hyphen at the end of a line and to from its down the molecular signal of the reticular tissue epithelial cell of replying generation duplicate.Skin micro-organs culture is described the model as wound healing here.Under controlled culture condition, can carefully monitor the healing of factor control.And because the micro-organs culture separates from natural blood confession, the agglutination analysis can not be subjected to blood to bear the other complicacy of the factor or cell and carry out.Normal epidermis mitotic division activity is low, and the cell cycle is 200-300 hour.After haveing a superficial wound, the mitotic division bursts of activities takes place make cell fission reach 10 times soon, depend on the situation and the seriousness (Pinkus H. (1951) J.Invest.Dermatol.16:383-386) of wound.

Prove that as example II skin micro-organs culture shows that hyperplasia increases several days and reaches 10 times.In this embodiment, edge of wound can liken the micro-organs culture to.This hyperplasia increases the simulation incident relevant with injury and the unique opportunity of research wound healing process is provided.And, appended embodiment proof external epidermis explant of the present invention can be applicable to chronic wounds (example I X) and can form can make natural on-off cycles of hair growth vigor implant (embodiment XI) arranged.

In addition, theme epidermis micro-organs can be used in fire victim's the treatment.It need be tangible that fire victim's skin is replaced.Several centers of US and European have utilized the human keratinized cell homotransplant of cultivation and autotransplant forever to cover burn wound and chronic ulcer (Eisinger et al., (1980) Surgery 88:287-293; Green etc. (1979) Proc.Natl.Acad.Sci.USA 76:5665-5668; Cuono etc. (1987) Plast.Reconstr.Surg.80:626-635).These methods are unsuccessful usually, studies show that recently in the graft of healing, may exist and send out blister and/or skin fragility, because under the epidermal area of transplanting, form one or more reticular tissue compositions unusual (Woodleyet al., (1988) JAMA 6:2566-2571).Skin culture systems of the present invention provides the skin equivalent of epidermis and corium, and should overcome the problem characteristic of keratinocyte graft of the cultivation of current use.

Still in another embodiment, micro-organs culture of the present invention system can provide the carrier that imports vivo gene and gene product to be used for gene therapy.For example, use recombinant DNA technology, the defective gene of patient can place under the control of virus or tissue-specific promoter.The recombinant DNA construction body can be used for transforming or all or some cell of transfection theme micro-organs culture systems.The micro-organs culture of expression activity gene product can be implanted the defective individuality of that product.

The purposes of theme micro-organs culture in gene therapy has dramatic benefit.At first, because culture comprises eukaryotic cell,, gene product forms active result so can correctly being expressed and process in cultivation.Secondly, as long as cells transfected quantity can increase to clinical value, dependency and practicality substantially, gene therapy technology just effectively; The theme culture allows cells transfected quantity to enlarge and amplification.

In the further embodiment of the present invention, transgenosis micro-organs culture can be used to promote gene transfer.For example, not with ways to restrain, the micro-organs culture that comprises recombinant virus expression vector can be used for recombinant virus is transferred to the cell of contact culture, implants as passing through, and the analogue body inner virus is relayed thus.Therefore, this system can be than the more effective method of finishing gene transfer of current dna rotaring dyeing technology.

Therefore, can modify micro-organs culture cell of the present invention with the expressing gene product.Phrase used herein " gene product " is finger protein, peptide and functional r NA molecule.Usually, the gene product of nucleic acid molecule encoding is to wait to supply with experimenter's expectation gene product.The example of this gene product comprises normal albumen, peptide, glycoprotein and the lipoprotein that produces of recipient subjects organ.For example, the gene product that relies on the defective organ of gene substitution pancreas to provide comprises Regular Insulin, amylase, proteolytic enzyme, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, rnase, deoxyribonuclease, triglyceride level enzyme, phospholipase A ₂, elastoser and amylase; Normally the gene product that is produced by liver comprises thrombin such as blood coagulation factor VIII and factors IX, UDP glucuronyl transferase, ornithine transcarbamylase and cytopigment p450 enzyme and carry out the adenosine deaminase of processing of serum adenosine or low-density lipoprotein endocytosis; The gene product that is produced by thymus gland comprises serum thymic factor, Thymus humoral factor, thymopoietin and extrasin alpha ₁The gene product that the digestive tube cell produces comprises gastrin, secretin, cholecystokinin, somatostatin and P material.Replacedly, coded gene product is to induce the gene product of expectation by the product of cell expressing (transcription factor of inducing the gene product of waiting to supply with the experimenter to transcribe as the genetic material coding that imports).Still in another embodiment, recombination can provide foreign protein, as non-natural concerning the cell of expressing it.For example, various people MHC compositions can offer non-human organs and come engraft in backer's acceptor.Replacedly, transgenosis is that the donor mhc gene product that suppresses normally to express in the micro-organs explant is expressed or the active gene.

The nucleic acid molecule of transfered cell is the form that is fit to expression in the cell of the gene product of this nucleic acid encoding.Therefore, this nucleic acid molecule comprise the coding and gene (or its part) transcribe required adjusting sequence, when being albumen or peptide when gene product, the translation of nucleic acid molecule comprises promotor, enhanser and polyadenylic acid signal, and transport code albumen or the required sequence of peptide, for example albumen or peptide are transported to cell surface or excretory N-terminal signal sequence.

The nucleotide sequence (as promotor and enhancer sequence) that the regulatory gene product is expressed is selected based on the expectation expression level of the cell type for the treatment of the expressing gene product and gene product.For example, can use the known gene cell type specific that links to each other with this promotor expression promoter that provides.Sarcoplast genetic expression specificity promoter can be connected with gene of interest and the muscle specific expression of that gene product is provided.Muscle specific regulatory element known in the art comprises dystrophin gene (Klamut etc. (1989) Mol.Cell Biol.9:2396), creatine kinase gene (Buskin and Hauschka, Mol.Cell Biol.9:2627) and troponin gene (Mar and Ordahl (1989), (1988) upstream region Proc.Natl.Acad.Sci.USA.85:6404), the negative reaction element (2001) .J Biol Chem such as () Jho Sh of mediation transcriptional expression in the keratin gene.Other cell type specificity regulatory element known in the art (as, the albumin enhanser of liver specifically expressing; The Regular Insulin regulatory element that islet cells is specific expressed; Various neurocyte specificity regulatory elements comprise neural dystrophin, neural Hydratase, phosphoenolpyruvate and A4 amyloid promotor).Alternatively, can use the regulatory element that can instruct gene constructive expression in various different cell types, as viral regulatory element.The example of viral promotors is generally used for driving genetic expression and comprises those that derive from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40 and retrovirus LTRs.Alternatively, can use the regulatory element that provides connected gene induced type to express.Use induction type regulatory element (as inducible promoters) to allow to regulate the generation of gene product in the cell.The example that is used for eukaryotic potential effective induction type regulation system comprises that hormone regulatory element is (as seeing Mader, S. and White, J.H. (1993) Proc.Natl.Acad.Sci.USA 90:5603-5607), the synthetic ligands regulatory element is (as seeing Spencer, D.M. wait 1993) Science 262:1019-1024) and the ion irradiation regulatory element (as seeing Manome, Y. etc. (1993) Biochemistry 32:10607-10613; Datta, R. etc. (1992) Proc.Natl.Acad.Sci.USA89:1014-10153).Also can use other tissue specificity or the induction type regulation system that to develop according to the present invention.

Capable territory known with can be used in the genetic material transfered cell modifying cell of the present invention a large amount of technology.In one embodiment, this nucleic acid is the naked nucleic acid molecular form.In this case, this nucleic acid molecule that imports cell to be finished only is made up of the nucleic acid of this gene product of coding and essential regulatory element.Alternatively, the nucleic acid of encoding gene product (comprising essential regulatory element) is included in the plasmid vector.The example of plasmid expression vector comprises CDM8 (Seed, B. (1987) Nature329:840) and pMT2Pc (Kaufman, et al. (1987) EMBO are J.6:187-195).In another embodiment, the nucleic acid molecule of cell to be imported is included in the virus vector.In this case, the encode nucleic acid of this gene product inserts viral genome (or part viral genome).Instruct the regulatory element of gene product expression to comprise to insert the nucleic acid in the viral genome (promptly be inserted into virus genomic gene be connected) or can provide by viral genome itself.

Can use the transfection, electroporation of transfection, the mediation of DEAE-dextran of calcium phosphate mediation, liposome-mediated transfection, direct injection and receptor-mediated absorption with the nucleic acid transfered cell.

The precipitation that contains nucleic acid and calcium phosphate by formation can be with naked nucleic acid such as DNA transfered cell.For example, the HEPES buffer salt solution can mix formation precipitation, precipitation and cell incubation then with the solution that contains calcium chloride and nucleic acid.Can add glycerine or dimethyl sulfoxide (DMSO) vibration step and increase the nucleic acid amount that some cell is admitted.CaPO ₄The transfection of mediation can be used to stablize (or instantaneous) transfectional cell and be only applicable to cell in vitro modifies.At Current Protocols in MolecularBiology, Ausubel, F.M etc. (chief editor) Greene Publishing Associates, (1989), 9.1 joint and Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press such as Sambrook, (1989), can find the transfection scheme of CaPO4 mediation in 16.32-16.40 joint or other standard laboratory handbook.

Compound by forming nucleic acid and DEAE-dextran and compound and the common incubation of cell can be with in the naked nucleic acid transfered cells.Can add dimethyl sulfoxide (DMSO) or chloroquine vibration step and increase the nucleic acid intake.The transfection of DEAE-dextran is only applicable to external modification cell and can be used for the instantaneous transfered cell with DNA, is not preferred for producing stable transfectional cell still.Therefore, this method can be used for short-term and produce gene product, but is not the system of selection that produces gene product for a long time.At Current Protocols in Molecular Biology, Ausubel, (chief editor) Greene Publishing Associates such as F.M., (1989), 9.2 joint and Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press such as Sambrook, (1989), can find the transfection scheme of DEAE-dextran mediation in 16.41-16.46 joint or other standard laboratory handbook.

Cell and nucleic acid common incubation and cell in suitable damping fluid are accepted high electric field pulse also can be with the naked nucleic acid transfered cell.The efficient of nucleic acid transfered cell is applied the influence of ion component of intensity, electricimpulse length, temperature, DNA conformation and the concentration and the substratum of electric field.Electroporation can be used to stablize the various cell types widely of (or instantaneous) transfection.At CurrentProtocols in Molecular Biology, Ausubel, (chief editor) Greene Publishing Associates such as F.M., (1989), 9.3 joint and MolecularCloning:A Laboratory Manual, second edition, Cold SpringHarbor Laboratory Press such as Sambrook, (1989), can find the scheme of electroporation of cells in 16.54-16.55 joint or other standard laboratory handbook.

Another method of naked nucleic acid transfered cell comprises liposome-mediated transfection (lipofection).Nucleic acid mixes with the liposome suspension of cation lipid.The DNA/ liposome compound is followed and the cell incubation.Liposome-mediated transfection can be used for stablizing the cell of (or instantaneous) transfection vitro culture.At Current Protocols in Molecular Biology, Ausubel, F.M. etc. (chief editor) Greene Publishing Associates, (1989) can find scheme in 9.4 joints and other standard laboratory handbook.In addition, use liposome to finish the vivo gene transmission.See for example (1987) Meth.Enz.149:157-176 such as Nicolau; Wang and Huang (1987) Proc.Natl.Acad.Sci.USA 84:7851-7855; Brigham etc. (1989) Am.J Med.Sci.298:278; With (1989) Gene 84:429-438 such as Gould-Fogerite.

Naked nucleic acid also can be by being injected directly into cell and transfered cell with nucleic acid.For the cell of vitro culture, can import DNA by microinjection.Because each cell carries out microinjection one by one, therefore when modifying a large amount of cell, this method is a work highly dense type.Yet microinjection is that the situation of a system of selection is preparation during transgenic animal (below more go through).In this case, DNA is stabilized the importing fertilized oocyte, and it then allows to grow is animal.The gained animal contains carries the DNA that imports this ovocyte.Direct injection also has been used for naked NDA is imported cells in vivo (as seeing (1991) Nature 332:815-818 such as Acsadi; Wolf etc. (1990) Science 247:1465-1468).Also can use the transfer device (as " particle gun ") that DNA is expelled to cells in vivo.This device is commercial can to obtain (as from BioRad).

Naked nucleic acid also can be compound with positively charged ion, and as polylysine, the ligand coupling of it and cell-surface receptor is taken in and (seen for example Wu, G. and Wu, C.H. (1988) J.Biol.Chem.263:14621 by receptor-mediated endocytosis; Wilson etc. (1992) J.Biol.Chem.267:963-967; With United States Patent (USP) 5,166,320).Nucleic acid-ligand complex promotes that with combining of acceptor DNA is taken in by receptor-mediated endocytosis.Aim at DNA-part compound acceptor and to comprise TfR and asialoglycoprotein acceptor.The DNA-ligand complex connects the adenovirus housing of corollary failure endosome, therefore material is discharged into tenuigenin and can be used for avoiding mixture (for example to be seen (1991) Proc.Natl.Acad.Sci.USA 88:8850 such as Curiel by lysosome degraded in the cell; Cristiano etc. (1993) Proc.Natl.Acad Sci.USA90:2122-2126).Receptor-mediated DNA takes in and can be used for DNA is imported external or intravital cell, and in addition, has the feature of increase, promptly uses the part of the acceptor of expressing on target cell interested in conjunction with selectivity, and DNA can selectivity aim at particular cell types.

Usually, during cell in naked DNA import to be cultivated (as with one of above-mentioned rotaring dyeing technology), only the small portion cell (about 10 ⁵/ one) typically in their genome, integrated the DNA (be DNA in cell, keep free type) of transfection.Therefore, in order to identify the cell of admitting foreign DNA, that the nucleic acid of coding selected marker is beneficial with nucleic acid transfectional cell interested.Preferred selected marker comprises those that medicine such as G418, homomycin and methotrexate resistance are provided.Selected marker can import on the plasmid identical with gene of interest and maybe can import on the plasmid that separates.

The preferred method of the nucleic acid transfered cell of encoding gene product is to use the nucleic acid that contains the encoding gene product such as the virus vector of cDNA.The cell of viral vector infection has most of cell and accepts nucleic acid, can remove the advantage of the needs of the cell that selection accepted nucleic acid.In addition, the molecule of coding in the virus vector, as cDNA contained in the virus vector, effective expression and virus carrier system can use in external or body in the cell of admitting virus vector nucleic acid.

Defective virus has the fine characteristic (summary is seen Miller, A.D. (1990) Blood 76:271) that is used for transgenosis for the gene therapy purpose.Can make up recombinant retrovirus inserts in this reverse transcription virus gene group its nucleic acid with coding gene of interest product.In addition, the part of reverse transcription virus gene group can be removed and obtain the replication defect type retrovirus.Then the replication defect type retrovirus is wrapped into virion, and it can be used for target cell infection with standard technique by using helper virus.At Molecular Biology, Ausubel, (chief editor) Greene Publishing Associates such as F.M., (1989) can find the scheme of preparation recombinant retrovirus and or cells in vivo external with this virus infection in 9.10-9.14 joint and other standard laboratory handbook.Suitable retroviral example comprises pLJ well known to those skilled in the art, pZIP, pWE and pEM.The example of suitable packaging virus system comprises Ψ Crip, Ψ 2 and Ψ Am.Retrovirus can be used for range gene is imported a lot of different cell types, comprise external and/or body in epithelial cell, endotheliocyte, lymphocyte, sarcoplast, liver cell, medullary cell (see for example Eglitis, wait (1985) Science 230:1395-1398; Danosand Mulligan (1988) Proc.Natl.Acad.Sci.USA 85:6460-6464; Wilson etc. (1988) Proc.Natl.Acad.Sci USA 85:3014-3018; Armentano etc., (1990) Proc.Natl.Acad.Sci.USA 87:6141-6145; Huber etc. (1991) Proc.Natl.Acad.Sci.USA 88:8039-8043; Feri etc. (1991) Proc.Natl.Acad.Sci.USA88:8377-8381; Chowdhury etc. (1991) Science 254:1802-1805; (1992) Proc.Natl.Acad.Sci USA 89:7640-7644 such as van Beusechem; Kay etc. (1992) Human Gene Therapy 3:641-647; Dai etc. (1992) Proc.Natl.Acad.Sci.USA 89:10892-10895; Hwu etc. (1993) J.Immunol.150:4104-4115; United States Patent (USP) 4,868,116; United States Patent (USP) 4,980,286; PCT applies for WO 89/07136; PCT applies for WO 89/02468; PCT applies for WO 89/05345; With PCT application WO 92/07573).With the nucleic acid stability transfered cell, retroviral vector need divide target cell for the reverse transcription virus gene group that is incorporated into host genome (with the exogenous nucleic acid that inserts wherein).Therefore, may must stimulate target cell to duplicate.

Can operate making its coding and expressing the gene of interest product the adenoviral gene group, but inactivation aspect its ability of in normal bacteriolyze viral life cycle, duplicating.See for example (1988) BioTechniques 6:616 such as Berkner; Rosenfeld etc. (1991) Science 252:431-434; With (1992) Cell 68:143-155 such as Rosenfeld.Those skilled in the art know the suitable adenovirus carrier from the 5d1324 type of adenopathy strain Ad or other adenopathy strain (as Ad2, Ad3, Ad7 etc.).The advantage of recombinant adenovirus is that they do not need to divide efficient gene and transmit the cell of carrier and can be used to infect various cell types widely, comprise airway epithelial (Rosenfeld etc. (1992) quote the front), endotheliocyte (Lemarchand etc. (1992) Proc.Natl.Acad.Sci.USA 89:6482-6486), liver cell (Herz and Gerard (1993) Proc.Natl.Acad Sci.USA 90:2812-2816) and myocyte (Quantin etc. (1992) Proc.Natl.Acad.Sci.USA 89:2581-2584).In addition, adenovirus DNA (with the foreign DNA that the wherein contains) unconformability that imports is in host genome, but keep free type, therefore having avoided becoming at the DNA that imports is incorporated under the situation of host genome (as retrovirus DNA), and the result's of insertional mutagenesis potential problems can take place.And, the adenoviral gene group to the carrying capacity of foreign DNA with respect to other gene delivery vector bigger (reaching 8 kilobase) (Berkner etc., quote the front; Haj-Ahmand and Graham (1986) J.Virol 57:267).Most replication-defective adenoviral vectors of current use have been deleted all or part of viral E1 and E3 gene, but keep nearly 80% adenovirus genetic material.

Adeno-associated virus (AAV) (AAV) is naturally occurring defective virus, needs another kind of virus, effectively duplicates and the productive life history as helper virus as adenovirus or simplexvirus.(summary is seen Muzyczka et al.Curr.Topics In Micro.And Immunol. (1992) 158:97-129).It also is that its DNA can be incorporated into Unseparated Cell and show that one of minority virus of high frequency stable integration (sees for example (1992) Am.J.Respir.Cell.Mol.Biol.7:349-356 such as Flotte; Samulski etc. (1989) J.Virol.63:3822-3828; With (1989) J.Virol.62:1963-1973 such as McLaughlin).Containing the AAV carrier of few as 300 base pairs can be packaged and can integrate.The space of foreign DNA is limited to about 4.5kb.Can be used for transfered cell as the AAV carrier of describing among (1985) Mol.Cell.Biol.5:3251-3260 such as Tratschin with DNA.Use the AAV carrier various nucleic acid to be imported different cell types and (for example see (1984) Proc.Natl.Acad.Sci.USA 81:6466-6470 such as Hermonat; Tratschin etc. (1985) Mol.Cell Biol.4:2072-2081; Wondisford etc. (1988) Mol.Endocrinol.2:32-39; Tratschin etc. (1984) J.Virol.51:611-619; With (1993) J.Biol.Chem.268:3781-3790 such as Flotte)).

Can use the conventional standard method of using in this area to estimate the effect of particular expression carrier system and with the method for nucleic acid transfered cell.For example, can detect the DNA of transfered cell with screening hybridization technique (as the Southern trace) and for example protect or reversed transcriptive enzyme-polymerase chain reaction (RT-PCR) can detect the RNA that importing DNA transcribes generation with Northern trace, Rnase.Can detect gene product with suitable test,, as use specific antibody, or, test as zymetology with the functional activation of functional trial detection gene product for example with the proteic immunology detection that produces.If interested treating is not easy to detect by the interested gene product of cell expressing, can use at first optimization expression system of the reporter gene that is connected with regulatory element and carrier to be used.The gene product that reporter gene coding can detect easily, and therefore can be used for estimating the effect of this system.The reporter gene of the standard that use this area comprises coding beta-galactosidase, E.C. 2.3.1.28, luciferase, GFP/EGFP and human growth hormone.

When being used for method with nucleic acid transfered cell group and causing most of cell modification and this gene product of cell effective expression (as usually as use the situation of virus expression carrier), can use the cell mass of modification, further do not separate or the group in the subclone of each cell.That is, enough gene products that cell mass produced are arranged, making does not need further cellular segregation.Replacedly, may preferably make and modify isogenous group from being equal to of single modification cell and grow and separate the cell of effective expression gene product.Restricted dilution separates single modification cell, with standard technique single cell is extended for the cell clone group subsequently in cultivation and can prepares this unified cell mass.

Phrase used herein " transgenic cell " is meant and has wherein inserted partially or completely xenogenesis that promptly a kind of cell of the nucleotide sequence of external source refers to the cell that is inserted into or imports.Transgenic cell also can be the cell that has inserted with cell native gene homologous nucleic acid.Yet in this case, the design homologous nucleic acid is so that the mode that the cellular genome that it inserts changes is inserted into or is inserted into cellular genome.For example, the homologous nucleic acid insertion that is inserted into position different with nature gene or homologous nucleic acid causes particular phenotype to be rejected.The nucleic acid that inserts this cell can comprise one or more transcriptional regulatory sequences and any other nucleic acid, and as intron, they can be that the optimum expression of selected nucleic acid is required.

Still in the present invention aspect another, theme micro-organs culture can be used for the diagnosis and the treatment of auxiliary malignant tumour and disease.For example, organ (as skin, kidney, liver etc.) biopsy can be taken from and be suspected the patient with hyperplasia or new hyperplasia disorder.If cultivate the explant of biopsy according to present method, the proliferative cell of explant is with asexual expansion in the culturing process.This will increase the chance that detects this disorder, and therefore increase the accuracy of diagnosis.And, in order to identify compounds effective, promptly kill malignant tumour or sick cell, and absolve Normocellular those, patient's micro-organs culture can be used for in-vitro screening cytotoxicity and/or medical compounds.Then, these reagent are used to patient's therapeutic treatment.

The present invention further aspect relates to the method for using theme micro-organs culture to screen various extensive compounds such as cytotoxic compound growth/regulatory factor, pharmaceutical agent etc.For example, recognize the needs of the chemicals of the potential poisonous character of thorough detection usually, and the needs that exploitation is estimated the responsive of medicine, makeup, foodstuff additive and sterilant and can be repeated the short-term in vitro tests clearly.Micro-organs culture described herein allows the using-system equivalent as testing substrate and the interactional advantage of normal cell in being very similar to the system of interior state being provided.

In this, keep culture and contact compound to be detected external.Can measure (comprise and killing) activity of cytotoxic compound by its phenotype of regulating cell in the explant.This can be by the estimations at an easy rate such as expression of important staining technique, mark.For example, the cell content by analyzing culture is as estimating with total cell count and differential cell counts to be meant/effect of regulatory factor.This immunocytochemistry that can use standard cell lines and/or histological techniques to comprise and use employing to limit the antibody of specific type cell antigen can be finished.Can estimate of the Normocellular effect of various medicines to cultivating in the cell of the present invention.For example, can identify the medicine that reduces psoriasis hamartoplasia.

In the exemplary of this method, be obtaining of the reagent that detect to stimulate hyperplasia in the explant, this method comprises from experimenter's chorista explant, wherein the cell mass of explant keep to separate the microarchitecture of the organ or tissue of this explant, is at least approximately 1.5mm of Aleph as the feature of explant ^-1And comprise that at least one has the cell of hyperplasia ability.Cultivate explant and contact candidate compound.Then measuring candidate compound exists the level of hyperplasia down and compares with there not being under the candidate compound level of hyperplasia.Significantly increasing on the horizontal statistics of hyperplasia under candidate compound exists is the sign of hyperplasia reagent.

Phrase used herein " candidate compound " or " candidate agent " are meant the reagent of detected or to be detected hyperplasia, anti-hyperplasia, differentiation, anti-differentiation or antiviral activity.This reagent can be for example ['s molecule, biology extract and recombinant products or composition.

The method of measuring hyperplasia is that well known in the art and most DNA that determine the cellular replication characteristic that generally include synthesize.There are a lot of methods of the DNA synthetic of mensuration this area, can use any according to the present invention.In one embodiment of the invention, with radio-labeling ( ³The H-thymidine) or the nucleotide analog of mark (BrdU), detect with immunofluorescence and determined that DNA is synthetic.

Another embodiment still provides the method for identification of cell hyperplastic inhibitory agent.The explant of organizing that provides as above is provided this method, and there are the level of hyperplasia down in this explant contact candidate compound and mensuration candidate compound.Significantly reducing on the horizontal statistics of hyperplasia under candidate compound exists is the sign of agents for inhibition of cell proliferation.

In illustrative embodiment, can use the toughener and the inhibitor (being also referred to as anti-hyperplasia reagent here) of hyperplasia, for example according to desired effects control natural on-off cycles of hair growth.

The growth of hard keratin fiber such as fine hair and hair depends on the dermal sheath cell hyperplasia.The hair follicle stem cells of sheath is very active, and produces hair fiber by quick hyperplasia and complex differentiation.Comprise three different stages hair cycle: regeneration (growth), degeneration (degeneration) and final period (static).The epidermal stem cells of hair follicle was activated by the corium projection in final late period.This term is called " activation of expanding ".And, think that this stem cell is a multipotential stem cell, not only produce hair and hair follicle structure, and produce sebiferous gland and epidermis.Hyperplasia nerve and agents for inhibition of cell proliferation provide change growth cycle of hair dynamics methods, for example induce the hair follicle cell hyperplasia static, particularly hair follicle stem cells.

The outgrowth inhibitor of hair follicle cell can use as the method that reduces people's hair growth, with cut off, scraping or alopecia convenient remove relative.For example, the hair follicle cell inhibitor that uses the inventive method to identify can be used for the treatment of with the quick or dense growth of paratrichosis, is the trichopathy of feature as hirsutism.In illustrative embodiment, it is the disorder of sign that this inhibitor can be used to handle hirsutism-with unusual crinosity.The purposes of this inhibitor can also provide the method that prolongs the depilation extended period.

The outgrowth inhibitor of hair follicle cell also can be used for the protective hair bladder cell and do not brought into play effect, as ray induction death, need enter the influence of the cytotoxic reagent of cell cycle S phase.Become static with this inhibitor for treating by causing hair follicle cell, as entering the S phase and therefore stop bladder cell experience mitotic division sudden change or programmed cell death that protection is provided by suppressing cell.For example, the hair follicle cell hyperplastic inhibitory agent can be used to suffer chemistry or radiotherapy to cause the patient of trichomadesis usually.During this treatment by suppressing the cell cycle progress, but inhibitor for treating protective hair bladder cell is not subjected to dead influence, otherwise the necrocytosis program animation cause can be dead.After treatment finished, the alleviation that the hair follicle cell hyperplasia suppresses to occur together also can be removed inhibitor for treating.

Yet, in order to begin to describe the characteristic of hair growth control molecular mechanism down, and the medicine that detects the potential impact hair, the external hair growth model that needs are suitable.In one aspect of the invention, subject methods is used to produce the hair follicle micro-organs explant that keeps the hair follicle microarchitecture, as interacting between the matrix components (corium projection) of follicular epithelium layer and hair follicle, as one or more stem cells, outer root sheath cell, stroma cell and internal root sheath cell.As proving among the appended embodiment, in these micro-organs cultures, even lack serum down as in minimal medium, observing hair growth.Importantly, the present invention also provides the hair follicle culture of the hair follicle that is in basic final period such as stationary phase.Following proof can activate final hair follicle explant and make regeneration hair follicle growth and in certain embodiments, with the method for synchronization in vitro culture.The early stage of short duration hyperplasia of hair follicle epidermal stem cells provides the paracrine of understanding for example various tissues generations of hair follicle organ and/or the regeneration period activatory unique opportunity that the autocrine factor mediates.

In addition, theme micro-organs culture is the system of reagent that identify to regulate hair follicle activation or inactivation, can promote or the reagent of hair growth inhibition as identifying.In one embodiment, following embodiment XVIII is described, and final (stationary phase) hair follicle explant contacts various detection reagent, and detects the irritation level of hair follicle.For example, by the mitotic index of observation hair follicle cell, or detect more outgrowth other similar approach and can monitor the transformation of hair follicle stem cells from final period to regeneration period.In order to illustrate, Figure 17 has shown that thymidine mixes to can be used for measuring and has lacked or have under the detection compound in (FGF among the figure) explant stem cell activatory level relatively that the hyperplasia increase is to have the sign that promotes the active detection reagent of hair growth.

In the phase negative test, provide the regeneration period micro-organs explant in cultivating, activatory Sencar explant described in appended embodiment, or factors stimulated growth explant (stimulating) as FGF.With respect to untreated regeneration explant, suppress the outgrowth detection reagent of hair follicle stem cells and can further consider the final reagent that is used as hair growth inhibition.

Still in other embodiments, the inhibitor of the hyperplasia that the theme test is identified can be used for suppressing the growth of knurl or proliferative cell, forms and growth as tumour.The preferred embodiments of the invention relate to the inhibition that epithelial tumor forms and grows.For the detailed description that the skin epithelial tumor forms, see the U.S. Patent Application Serial 08/385,185 of application on February 7 nineteen ninety-five.Cause that hyperplasia control break and the disorderly generation tumour as a result of the interaction between cell and its environment invading and shift form.Cell quantity that cell fission causes increases and causes hyperplasia control disorderly because the relation of the cell cycle that differentiation or necrocytosis cause between returning broken.In healthy tissues, by guaranteeing when each stem cell division, in two daughter cells only one be retained in the stem cell compartment, and another enters differentiation pathway and keep homeostasis (Cairns, J. (1975) Nature 255:197-200).Therefore the control of cell proliferation will be the result who influences the signal of these processes.These signals can be positive or negatives, and the acquisition that tumour forms is to be changed by the genetics that influences these reference mark to cause.

As described in the embodiment IX and shown in Figure 12, skin micro-organs culture of the present invention has been used for identification of cell hyperplasia reagent and agents for inhibition of cell proliferation.As described in embodiment IX, detect TGF-β and find to be used as agents for inhibition of cell proliferation.Albumen-the Nrolone Phenylpropionate that has also shown TGF-β subfamily member suppresses epithelial hyperplasia.These results show to have other member of TGF-'beta ' family of working in suppressing epithelial hyperplasia.In the data prompting TGF-'beta ' family as the albumen of the remarkable conditioning agent of epidermis homeostasis suppress that external epithelial tumor forms and growth in effect.

Another aspect of the present invention relates to the method that the identification of cell differentiation agents promptly causes the compound of cytodifferentiation.This method comprises that from experimenter's isolated cell group wherein cell mass has the microarchitecture of the organ or tissue that separates this cell, and surface-area and bulk index be 1.5mm at least approximately ^-1And comprise that at least one has the cell of differentiation capability.Then this cell is placed about at least twenty four hours and is contacted candidate compound in cultivation.Measuring candidate compound then exists down the cytodifferentiation level and compares with there not being under the candidate compound cytodifferentiation level.Significantly increasing on the horizontal statistics of cytodifferentiation under candidate compound exists is the sign of cytodifferentiation reagent.Differentiation used herein is meant the cell that obtains with the original morphology that has of this cell and/or function morphology different and/or except that it and/or function.Typically, these morphology and function are the features of mature cell.The differentiation that generation by measuring the specialized cells product and/or secretion can be monitored cell mass of the present invention.

In a similar manner, the present invention also relates to the method for identification of cell differentiation inhibitors.After last same approach, be determined at candidate compound and have the level of cytodifferentiation down and compare with there not being under the candidate compound cytodifferentiation level.Significantly reducing on the horizontal statistics of cytodifferentiation under candidate compound exists is the sign of cytodifferentiation inhibitor.

Still in another embodiment, the theme culture allows to produce the external model of virus infection.For example, can separate epidermis or flaser texture, and with the virus infection as simplexvirus, as herpes simplex virus 1, hsv 2, varicella zoster virus; Or human papillomavirus, as human papillomavirus 1-58 any, as HPV-6 or HPV-8.Similarly, can provide hepatic model for infecting, as the explant of hepatites virus infections, as hepatitis A virus, hepatitis B virus or hepatitis C virus.The explant of organizing of virus infection can be used for according to the communicable inhibitor of the inventive method identifying virus.As above, provide special micro-organs culture, and contact (choosing wantonly) cells infected produces the virus of the cell mass of virus infection.The cells contacting candidate compound of virus infection and in the presence of candidate compound, measure the level of viral infectivity then.Significantly reducing on the horizontal statistics of viral infectivity under candidate compound exists is the sign of viral infectivity inhibitor.

The method of measuring viral infectivity is known in the art and changes according to employed Virus Type.For example, a kind of method that can be used for measuring the viral infectivity level is by measuring the yield level in the cells infected of the specific gene product of detected specific virus in micro-organs culture or micro-organs culture base.For example, measure the hepatitis virus infectivity level of cell in the micro-organs culture, as hepatitis B virus, hepatitis protein yield and hepadna can be quantitative.Usually, micro-organs culture base can with anti-selected viral protein and the whole bag of tricks known in the art, as at the SDS-polyacrylamide gel, the common incubation of the antibody of the immune-reactive protein of elisa assay.For example, be to measure the output of hepatitis B surface antigen(HBsAg), can with before the interval sampling in several days with the micro-organs culture base of the micro-organs of the common incubation of hepatitis B virus and with the described ELISA of manufacturers (Abbott) method detection surface antigen.But use serial dilution standard surface antigen (CalBiochem) can improve quantitatively this method.Hepatitis B surface antigen(HBsAg) is accumulated significantly to reduce on the statistics and is shown that detected candidate compound is the communicable inhibitor of hepatitis virus in the substratum.

Except measuring the level of HbsAg in the micro-organs culture base, can use pcr amplified dna, the primer that uses mark among the HBV pre-S (coding HBsAg) subsequently is to carrying out the Southern engram analysis as probe, can the new synthetic hepatitis B virus DNA of micro-organs cells in culture extract be detected and quantitatively (see as Sambrook, (1989) Molecular Cloning-A Laboratory Manual such as J., Cold Spring HarborLaboratory, second edition, the 2nd volume, the 10.14-10.15 page or leaf).Relative quantification can be finished and confirms with the scintillation counting of respective strap with densitometry.The level reduction of new synthetic viral DNA shows that the candidate compound of test is the communicable inhibitor of hepatitis virus.

In another embodiment, use above-mentioned and known in the art other standard technique also can measure gag, pol and the env albumen of retrovirus such as human immunodeficiency virus (HIV).For example, can measure the pol albumen of expressing in the cell of the micro-organs culture that HIV infects by the common incubation of analyzing on cell extract and anti-pol antibody or blended AIDS patients serum and the SDS/ polyacrylamide gel of immune-reactive protein.For the simplexvirus in the mensuration micro-organs culture of the present invention such as the infectivity of Epstein-Barr virus (EBV), use method described here can analyze EBVDNA and EBV inductive nuclear antigen output.

Micro-organs culture of the present invention also can be used to promote experimenter's wound healing.Therefore, the invention further relates to the method that promotes the recipient subjects wound healing.This method comprises that from donor experimenter separating table area and bulk index be about at least 1.5mm ^-1Cell mass.Typically, cell mass is placed at least approximately twenty four hours in cultivation.Then cell mass can be applicable to the wound of recipient subjects.In one embodiment, wound or infringement are slowly healings or chronic, and the wound that occurs together as diabetes is as burn, as ulcer.Prove that as embodiment X and XI what little explant and can forming that skin micro-organs culture of the present invention can be used as chronic wounds to be applied to (embodiment X) can make hair growth has a vigor implant (embodiment XI).

Still in another embodiment, theme micro-organs explant provides detection cytotoxicity or pungency in a test.In the embodiment of example, subject methods provides vitro detection eyes and skin irritant technology.This method resembles toply very much, comprises to micro-organs culture topical application liquid of the present invention, solid particulate or gel-like material (as makeup), detects the effect that produces in cultivating subsequently.

At present, estimate the potential eyes and the skin irritation of a lot of chemicals, household cleaning product, makeup, coating and other material by directly applying to animal or human experimenter.Yet as industrial most understanding, this method does not satisfy the overpowering public and supports.Present method provides alternative test, and it does not need to sacrifice animal or animal is produced permanent injury, and the data of objective form also are provided.In illustrative embodiment, obtain skin micro-organs culture according to the present invention.The explant Contact test reagent of cultivating, as cosmetic formulations, for some time is estimated cell viability after contact.In preferred embodiments, MTT test (based on functional plastosome reduction tetrazolium dye) is used for the vigor scoring.

Micro-organs culture of the present invention can be used for the factor that appraisement organization and the normal homeostasis of cell comprise in addition, the research cellular environment changes and to comprise alteration in nutrition and to have the effect of potential toxic reagent to the normal homeostasis of cell of tissue, research morbidity or wound begin with process in the tissue that triggers and the approach of cell change; Identify the repair mechanism of the detrimental action of the environment change that reverse morbidity or wound are relevant; The cells whose development rule of breaking up in the normal homeostasis process of research organization and the growth rule of in-house specialized structure (as hair follicle); Replenish with organ, wherein kept the each several part of individual organ, but be not enough to replace or regeneration destructive tissue, as taking place among the chronic skin ulcer patient, wherein inappropriate blood supply causes the healing defective, or local skin can not heal under the situation as known I type or type ii diabetes.

Embodiment

To be more readily understood the invention of progressively describing now with reference to the following example, the embodiment that comprises is just to the purpose of setting forth some aspect of the present invention and embodiment, and is not intended to limit the present invention.

The micro-organs culture that has separated from animal comprises grownup's skin, mouse, cavy and rat skin and in cultivation the growth reach 21 days.Yet, culture keep surpass 21 days for some time also within the scope of the invention.

In addition, form the micro-organs culture also within the scope of the invention by various animals.The scope of animal only is an example, but the sample that is not limited to provide below.

As described in appended embodiment, the micro-organs culture comprises mammalian pancreas, liver, kidney, duodenum, oesophagus and bladder preparation by skin with by organ.Similarly, also can use method of the present invention preparation from Mammals cornea, kidney, breast tissue with except oesophagus the various tissue from intestines such as the micro-organs culture of the epithelium of small intestine and colon.Really, separate and keep all within the scope of the invention from the micro-organs culture that contains epithelium in the body/matrix microarchitecture at any position.

Theme micro-organs culture technique has been used for organizing explant in the long-term cultivation preservation of the tissue that does not have epithelium/matrix microarchitecture, as some Lymphoid tissue, as thymus gland and spleen explant.

Example I

The preparation of epidermis micro-organs culture

After fresh skin derives from surgical operation, remove the flap that it descends fatty tissue and is cut into 0.4 * 5cm, follow using-system knife mill or other suitable parting tool laterally are cut into 300 μ m under aseptic condition section, make the last segmentation of organizing have the big or small (see figure 1) of width 4mm and thickness 0.3mm.These micro-organs place 24 hole microplates, are not containing 400 μ lDMEM of serum, 5%CO ₂, in 37 ℃, with one to eight day time of 12rpm sustained oscillation.Every hole 20 little explants of growing.

Embodiment 2

Mouse, cavy and the outgrowth mensuration of people's epidermis micro-organs culture

Prepare the micro-organs culture according to example I, following analyzing DNA resultant quantity is measured hyperplasia.Mouse skin and guinea pig skin growth two days and human skin growth four days were added BrdU three hours with final concentration 100 μ M afterwards in substratum, subsequently with cell fixation in 4% formaldehyde.After fixing, with the anti-BrdU antibody of goat, the IgG with anti-goat FICT mark dyes to culture subsequently.Histological specimen is implanted in the following stationary liquid of 4% formaldehyde and is cut into 3 μ m thin slices and uses methylene blue staining.

Discovery is in vitro culture after two to four days, and the cell of synthetic DNA part is compared with observed value in the body to increase and reached 10 times, afterwards the DNA resultant velocity progressively increase but in cultivation, keep up to 10 days (see Fig. 2,3 and 4A-4D).Even cultivating the 6th day, cell is kept steady state hyperplasia and differentiation, makes to have kept to organize microarchitecture (Fig. 5 A-5C).

EXAMPLE III

The hyperplasia of the micro-organs cells in culture of all size

Prepare the cavy micro-organs as example I.The explant that the full thick skin bar of width 4mm is cut into variation in thickness comprises thickness 300,450,600,700,900,1200 and 3000 μ m.These thin slices were positioned in the hole that contains serum free medium two days one by one.Add BrdU after four hours, final concentration 100 μ M stop.Then explant is fixed on the goat antibody of also using BrdU in 4% formaldehyde, and two anti-preparations with anti-goat IgG FITC mark dye subsequently.Fig. 6 has shown this result of experiment.Increase with explant thickness, significantly reduce as the incorporation of the BrdU of the quantity effect of cell/unit organization.

EXAMPLE IV

The preparation of pancreas micro-organs culture and the mensuration of hyperplasia

Take out cavy pancreas, then use the suitable section of organizing knife mill and being cut into 300 μ m thickness, 4mm width and the 2mm degree of depth in the mode of keeping the pancreas microarchitecture.Little explant is grown in cultivation several times of from two to eight days.Seven micro-organs place each hole of 96 orifice plates, at 150 μ l serum-free DMEM, 5%CO ₂Down, 37 ℃, under the 12rpm sustained oscillation.BrdU added after three hours, and final concentration 100 μ M stop, and explant then is fixed in 4% formaldehyde and uses the goat antibody of BrdU, and the IgG with anti-goat FITC mark dyes subsequently.The cell that Fig. 7 A-7B shows in the micro-organs that derives from pancreas is just enlivening hyperplasia.

EXAMPLE V

Preparation pancreas micro-organs culture is also measured the Regular Insulin that is secreted in the substratum

Prepare the pig pancreas micro-organs culture of growing up as preceding embodiment to skin.Take out pancreas, it is dark and to be cut into 300 μ m thick, the section that 4mm is wide to be cut into about 2mm with scissors.The micro-organs culture was grown in serum free medium 14 days.Every three days, remove substratum and add fresh culture.Use standard radioimmunoassay method detects content of insulin in the substratum of collecting.

Example VI

Pig pancreas micro-organs is transplanted to the xenogenesis experimenter

Prepare the pig pancreas micro-organs culture of growing up as preceding embodiment to skin.Take out pancreas, it is dark and to be cut into 300 μ m thick, the section that 4mm is wide to be cut into about 2mm with scissors.Follow the different time sections that the micro-organs culture was grown 0 to 5 day in serum free medium, after the cultivation, from cultivate, take out pancreas micro-organs and transplanting internal organ and parietal mesoderm to the rat host.Micro-organs survive in vivo at least one month and the good vascularization that become.Behind body interior three, five, seven and the fortnight, can detect hyperplasia widely.In addition, transplant the back body and observed positive Regular Insulin dyeing in interior four, seven and 30 days.

Example VII A

The preparation of liver, kidney, duodenum, oesophagus and bladder micro-organs culture and

The mensuration of hyperplasia in the micro-organs culture

As the preceding cavy micro-organs culture that the embodiment preparation of skin is contained the organ of several epithelium.Take out this organ, be cut into about 2mm width, 3mm length and be cut into 300 μ m slabs with scissors.Little culture was cultivated in serum free medium three, four and six days.Preceding 12 hours of off-test is added in the explant culture ³The H-thymidine.After the end, fixing organization, flushing is counted several times and with scintillometer.This result of experiment has been shown among Fig. 9.As shown in Figure 9, institute shows active propagation in a organized way, as ³The absorption of H-thymidine is measured and is continued six days.

Example VII A I

Hair follicle hyperplasia in the micro-organs culture

Prepared skin micro-organs culture and incubation two days according to example I.Add BrdU and stop incubation after three hours.Cell fixation is in 4% formaldehyde and with the anti-BrdU antibody of goat, subsequently with the IgG dyeing of anti-goat FITC mark.The complete hair follicle that is present in the interior home of body can maintain under the culture condition of accurate control, does not need to add serum or any other exogenous factor.The hair follicle of discovery in these micro-organs healthy and strong hyperplasia several days under condition of the present invention, as the hair follicle cell quantity of mixing BrdU greatly as indicated in (Figure 10 A-10C).Figure 11 has shown the size distribution of cavy micro-organs culture zero-time and two all backs hair shafts.Change substratum every three days.That the hair shaft size is divided into arbitrarily is little, the neutralization big.After cultivating the Ninth Heaven, size distribution clearly shifts, and make the per-cent of little hair be reduced to 28% from 64%, and non-existent big hair shaft shows 30% hair shaft group when cultivating beginning.

Example I X

Measuring the test of the outgrowth influence of candidate compound pair cell prepares

The preparation culture also maintains as in the definition substratum under the described similar growth conditions of example I.Immunocytochemistry analysis of control sample is determined that the micro-organs culture is maintained at and is similar under the esoteric mode.

Handle the copy sample of skin micro-organs with 2.5ng/ml with TGF-β.Implement the quantitative analysis of the cell/explant quantity of BrdU mark according to example II.Compared with the control, in the presence of TGF-β, observe the synthetic inhibition of DNA and surpass 90% (Figure 12).

Embodiment X

Promote the method for chronic non-healing skin ulcer healing

According to this method, from the patient, take out small area normally, do not wrap up skin graft, as complete thick little explant of preparation width 4mm as described in the example I and thickness 0.3mm.Yet the different 0.3mm thin slices that are cut into that are with example I of this sample make not exclusively that deliberately a series of sections flock together as shown in figure 13, and top epidermal area comprises stratum corneum.The design of this explant is pointed to and is allowed nutrition to arrive all cells, but but tissue slice is kept operation format.Cleaning patient's wound is also removed the surrounding skin edge.Then cover the zone that lacks skin carefully with little explant, explant places and makes non-cutting edge be suspended in liquid in the wound towards outer and relative cutting blade on the wound.Prepare enough little explants and cover injured area substantially.Then cover the zone of treatment and allow healing with suitable dressing.

Embodiment XI

The hyperplasia of hair follicle in the body

Animal experiment in the injured body takes out 1cm from mouse ²The skin of area and incomplete microsection make the stratum corneum in intact skin zone be kept perfectly as mentioned above.Micro-organs is implanted its original position of sewing up mouse once more and is allowed healing.Implant maintains vigor, becomes to incorporate animal tissues into and cultivate the hair shaft (seeing Figure 14) that two all backs make new advances from the implant growth.

Embodiment XII

People's psoriasis skin micro-organs culture

Use dermatome after necrotomy, to obtain the psoriasis skin of 82 years old gerontal patient's incision thickness.Then skin is cut into 0.5 * 5cm flap, however the section that using-system knife mill or other suitable cutting appliance laterally are cut into 300 μ m.These micro-organs sections place microplate, at serum-free DMEM, 5%CO ₂, 37 ℃, sustained oscillation one is to fortnight.In some cases, in substratum, add somatomedin.Change substratum every three days.People's psoriasis skin such as the extensive hyperplasia of micro-organs culture.

Embodiment XIII

The liver micro-organs culture of hepatites virus infections

As described in example VII A, cut people, rat, mouse and cavy liver and cultivate the micro-organs culture.Using as described herein, BrdU mixes the hyperplasia of enlivening that detects in these micro-organs cultures.Cultivate after at least 14 days, determine that as urea (Sigma Chemical, urea detection kit) and albumin product (ELISA) mensuration the liver cell in these micro-organs cultures has function.

People's liver micro-organs culture for preparing above and hepatitis B and hepatitis c virus-positive patient's the common incubation of serum.After 24 hours, remove substratum and add the fresh DMEM that contains and do not contain 10% normal tire bovine serum (FCS).Every three days, change substratum with fresh culture and use virion in the antibody test conditioned medium of antiviral protein HBs.After 4 days, detecting virion quantity in these micro-organs cultures significantly increases and cultivation in the presence of FCS.

Embodiment XIV

Thymus gland and spleen micro-organs culture

Basically the mouse and the rat micro-organs culture that as the front embodiment of skin are prepared thymus gland and spleen.Remove organ and become the length of width and the 3mm of about 2mm with scissors cut.Then use and suitably organize knife mill these samples to be cut into the thick explant of about 300 μ m in the mode of the microarchitecture of reservation organ essence.Micro-organs was then cultivated in serum free medium 1,3,5 and 10 days.Mix the hyperplasia of enlivening that detects these micro-organs cultures as use BrdU described here.

Embodiment XV

Marrow micro-organs culture

Take out marrow carefully from the femur of rat and mouse and prepare myeloid micro-organs culture.Because the marrow diameter in this explant is about 1-2mm only, so the using-system knife mill directly is cut into the micro-organs explant with the thickness of 300 μ m with marrow.This method guarantees that marrow obeying long-term cultivation, keeps microarchitecture in the time of reservation table area/bulk index.Micro-organs was cultivated in serum free medium 3 days.Mix the hyperplasia of enlivening that detects medullary cell in these micro-organs cultures as use described here BrdU.

Embodiment XVI

Gene product passes to skin micro-organs culture

Micro-organs culture inherent high surface area specific volume of the present invention all allows to enter easily tissue concerning range gene transmits technology.In this embodiment, use electroporation and liposome transfection foreign gene transfection micro-organs culture.This micro-organs culture portable is given animal and was survived about at least 30 days in vivo and the formation blood vessel.This proof using-system MC cultivates the possibility at non-vivo gene treatment plan.MC cultivates more advantages position that to be its portables determine in the body makes that if desired can take out it future at an easy rate.This and cell can be divided a word with a hyphen at the end of a line in healthy tissues or the cell suspending liquid of " loss " is transplanted to health formation contrast.

The cutting guinea pig skin also is cut into width 2mm and the section of thickness 300 μ m.Cultivating skin in the serum-free minimum essential medium is micro-organs, the concentration that penicillin and Streptomycin sulphate are recommended with supplier.At 37 ℃ and 5%CO ₂Under cultivate one day after, with not containing antibiotic DMEM flushing skin micro-organs culture, and add the 0.4cm slit to and can handle in the electroporation cuvette, 500 μ l substratum are on ice.

Add as shown ten micrograms contain shown in the plasmid DNA (each plasmid has the cytomegalovirus promoter of driving beta-galactosidase enzymes (contrast) or luciferase reporter gene) of reporter gene.The luciferase plasmids main chain is the pRC-CMV (Invitrogen) that merges firefly luciferase gene in the framework.

Sample at 220mV with under the electric capacity that changes as shown in figure 15 electroporation (height=900 μ F, in=500 μ F, low=250 μ F).The NIH3T3 cell is handled with the Bio-Rad electroporation device under 250 μ F.Then this sample further contains in 24 hole culture dish among the DMEM of 10% bovine serum, penicillin, Streptomycin sulphate and L-glutamic acid and cultivated 2 days.Remove substratum, sample is suspended in about 700 μ l cell cultures cracking agents (Promega).The tissue homogenization then adds 20 μ l in 100 μ l luciferase detection reagent (Promega), detect luciferase in triplicate with Packard Top Count.As positive control, trypsin acting is in the NIH3T3 cell from the 75cm culturing bottle, with micro-organs culture equivalent processes.As shown in figure 15, under substratum (500 μ F) and low (250 μ F) capacitance settings, detect tangible uciferase activity.In order to contrast, with the unlimited culturing cell of NIH3T3 of identical plasmid electroporation similar quantity under 250 μ F.

In another experiment, with observing the transfection of finishing the micro-organs explant than the more effective liposome transfection of electroporation.Especially, with containing the micro-organs culture of the plasmid transfection of luciferase reporter molecule from guinea pig skin, newborn mice skin and rat lungs.In brief, before the transfection, the micro-organs culture is at 37 ℃, 5.5%CO ₂, contained among the DMEN of 1% penicillin/streptomycin and 1%L-glutaminate growth 1 day.Explant is positioned over 24 orifice plates, and every hole contains 20 explants and 400 μ l substratum.For transfection,, add 10 μ l Lipofectin (Gibco BRL)+2 μ g DNA+Optimem, cumulative volume 500 μ l to every hole with Optimem flushing micro-organs culture twice.Guidance according to lipofection manufacturers prepares Optimem/Lipofectin/DNA solution.Follow this culture at 37 ℃, 5.5%CO ₂Middle incubation 5-6 hour.Then Optimem/Lipofectin/DNA is positioned over and contains 1% penicillin/streptomycin, and among the 400 μ l DMEN of 1%L-glutaminate and 10%FCS, culture is at 37 ℃, 5.5%CO ₂Incubation spends the night.Second day morning, take out the micro-organs culture, with 1 * PBS washed twice, in manual glass tissue grinder, grinding in 750 μ, the 1 1X cell cultures lysis buffer (Promega).Use luciferase detection system (Promega) to detect genetically modified uciferase activity, the result reports in Figure 18.

Embodiment XVII

Gene product passes to the micro-organs culture

Carry out the micro-organs culture as the lungs of the female Lewis rat in eight ages in week of cutting as described in the embodiment XV and thymus gland and processing.The micro-organs culture is positioned in the culture hole also with the fit transfection of the plasmid dna complex of the plain enzyme of cation lipid/coding fluorescence five to six hours, simultaneously at 37 ℃ of incubations.Extract cation lipid/plasmid DNA solution out, then incubation culture two days in the substratum that adds 10% serum detects luciferase reporter gene then and expresses (expressing at light unit arbitrarily).Figure 16 has set forth this experimental result.Prove lungs, rather than thymus gland, the luciferase gene of expressing transfection in these cases as Figure 16.As expected, the light yield (23 light unit) of the lungs micro-organs culture of negative control beta-galactosidase enzymes transfection (10 μ l cation lipid enriched material) is near the machine background.

Embodiment XVIII

The hair shaft growth in vitro

Obtain newborn mice skin after the surgical operation, remove its down fatty tissue and be cut into the flap of 0.4 * 5cm, using-system knife mill or other any suitable cutting unit section of laterally being cut into 300 μ m then.Micro-organs places microplate, lacks among the DMEM of serum 5%CO ₂, 37 ℃, 1 to 14 day time of sustained oscillation.Somatomedin such as the FGF in the substratum added in some explant contact to.Changed substratum every 2 days.

When growing in MC cultivates, skin produces hair shaft can to induce new life " not have hair ".In the presence of 1ng/mlEGF, growth 3 days in the micro-organs culture 30 hour age mouse the Photomicrograph of skin show and the growth of hair shaft in the explant growth in cultivating the beginning process, to occur.

In another group experiment, observe the activation of final hair follicle.The Sencar mouse provides research hair follicle activatory useful model, because the characteristic of fine synchronous and fine description cycle stage of hair follicle.The Sencar mouse provides regeneration period activatory body inner model.Removing the clavus of final hair follicle can induce new hair to form the fine characteristic of having described of its first indication.Obtain the skin of adult sencar mouse after the surgical operation, remove the flap that it descends fatty tissue and is cut into 0.4 * 5cm, then using-system knife mill or other suitable cutting unit laterally are cut into the thin slice of 300 μ m.Micro-organs places microplate, the DMEM of serum-free, 5%CO ₂, 37 ℃, 1 to 14 day time of sustained oscillation.The hair follicle stem cells hyperplasia has proved the activation of no matter removing clavus or the final hair follicle of somatomedin processing inductive.Figure 17 shows the activation of mixing the final explant of detection as thymidine.

Example I XX

The preparation islet cells and islet

Developed the islet cells that several technology have prepared a large amount of various Mammalss source, after this they have formed the β cell mass that may transplant, with the type i diabetes of their treatment foundation.Up to now, two main shortcomings have been met with.Proved that the reproducible trusted methods that obtains preparation β cell is difficult.The second, these cells vigor in vitro and in vivo is very changeable.Part owing to first reason and part because the β cell needs the fact of the support of matrix under the pancreas islet of Normal Pancreas probably.Up to now, the process of keeping the pancreas organ in the non-body attempts all getting nowhere.Use MC culture technique described here, in the substratum of determining, the external micro-organs culture of setting up mouse, rat, cavy and pig pancreas hits pay dirk.

Select pancreas micro-organs culture to reach one month period in growth in vitro.In the culture, explant is kept organizing microarchitecture and mix with some cell subsets of marker determination as BrdU and enlivening hyperplasia of they.In addition, islet cells with insulin secretion in substratum, even in vitro culture after one month.

Implemented transplantation experiments, wherein pig micro-organs pancreas culture is implanted rat host's internal organ and parietal mesoderm.Explant keeps the period from several days to one month in vivo.Explant has had good vascularization and has incorporated into and organize among the host.

Embodiment XX

The preparation of people's psoriasis skin micro-organs culture

Use dermatome, obtain to cut thickness patient's psoriasis skin after the necrotomy.Skin is cut into the flap of 0.4 * 5cm, then with organizing knife mill laterally to be cut into 300 μ m slabs.These micro-organs explants in DMEM (serum-free), on the microplate, 37 ℃ and 5%CO ₂Under cultivated 1 to 14 day.Detect the micro-organs explant at various time points and show that the cell of explant maintains vigor, and hyperplasia takes place.

Above-cited all reference and publication are incorporated into here as a reference.

Equivalent

Those skilled in the art will recognize that, maybe can determine to use normal experiment, a lot of equivalents of special test described here and reagent.Think that this equivalent comprises within the scope of the invention and covered by following claim.

Claims

1. express the micro-organs explant of the genetic modification of at least one recombination product, this micro-organs explant comprises cell mass, the micro-organs explant keep the microarchitecture that obtains this organ and have simultaneously select to make allow enough nutrition and gaseous diffusion in this micro-organs explant cell and cellular waste from the micro-organs explant spread out make cytotoxicity and since the micro-organs explant the inadequate and refuse of nutrition accumulate the dead minimized size that occurs together, at least one recombination product of at least some cell expressings in the cell mass of described micro-organs explant.

2. the micro-organs explant of the genetic modification of claim 1, wherein said at least one recombination product is selected from recombinant protein and recombination function RNA molecule.

3. the organ that the micro-organs explant of the genetic modification of claim 2, wherein said recombinant protein are normally originated by the micro-organs explant produces.

4. the micro-organs explant of the genetic modification of claim 2, wherein said recombinant protein normally are not to be produced by the organ that the micro-organs explant is originated.

5. the micro-organs explant of the genetic modification of claim 2, wherein said recombinant protein is a labelled protein.

6. the micro-organs explant of the genetic modification of claim 2, wherein said recombinant protein is selected from Regular Insulin, amylase, proteolytic enzyme, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, rnase, deoxyribonuclease, triglyceride level enzyme, phospholipase A ₂, elastoser, amylase, thrombin, UDP glucuronyl transferase, ornithine transcarbamylase and cytopigment p450 enzyme, adenosine deaminase, serum thymic factor, Thymus humoral factor, thymopoietin, extrasin alpha ₁, tethelin, somatomedin, transforming growth factor, gastrin, secretin, cholecystokinin, Somatostatin, P material and the transcription factor of G CFS, erythropoietin, Urogastron, liver erythrogenin (liver generates plain), pHGF, interleukin, anti-somatomedin, fibroblast growth factor, 'beta ' family.

7. the micro-organs explant of the genetic modification of claim 1 can be kept at least approximately twenty four hours in cultivation.

8. the micro-organs explant of the genetic modification of claim 1 has formula 1/x+1/a＞1.5mm ^-1For the surface-area of feature to bulk index; Wherein " x " is that tissue millimeter thickness and " a " are described tissue millimeter width.

9. the micro-organs explant of the genetic modification of claim 1, wherein said organ are selected from lymphoid organ, pancreas, liver, gall-bladder, kidney, digestive organ, respiratory tract organ, reproductive organ, skin, urinary tract organ, the relevant organ of blood, thymus gland, spleen.

10. the micro-organs explant of the genetic modification of claim 1 comprises epithelium and phoirocyte, arranges with the microarchitecture of the microarchitecture that is similar to the organ that obtains this explant.

11. the micro-organs explant of the genetic modification of claim 1, wherein organ is a pancreas, and cell mass comprises youth's lattice Han Shi pancreas islet.

12. the micro-organs explant of the genetic modification of claim 1, wherein organ is a skin, and explant comprises at least one hair follicle and body of gland.

13. the micro-organs explant of the genetic modification of claim 1, wherein organ is an affected skin, and explant comprises hyperplasia or new outgrowth cell mass from affected skin.

14. the micro-organs explant of the genetic modification of claim 1, wherein explant can maintain in the minimal medium.

15. the micro-organs explant of the genetic modification of claim 1, wherein the microarchitecture of the reservation of explant comprises one or more cell-cells and the cell-matrix between two or more tissues of the organ that is positioned to separate this explant.

16. the micro-organs explant of the genetic modification of claim 1, wherein at least a portion cell mass is carried the recombinant virus infection of the recombination of the described recombination product of coding.

17. the micro-organs explant of the genetic modification of claim 16, wherein said recombinant virus are selected from recombinant hepatitis virus, recombinant adenovirus, recombinant adeno-associated virus, recombinant papillomavirus, recombinant herpesvirus, recombinant slow virus, recombinant retrovirus, recombined cytomegalovirus and reorganization simian virus.

18. the micro-organs explant of the genetic modification of claim 1, the method for transformation of the transfection of the wherein transfection of at least a portion cell mass by being selected from calcium phosphate mediation, the mediation of DEAE-dextran, electroporation, liposome-mediated transfection, direct injection and receptor-mediated absorption has transformed exogenous nucleic acid sequences.

19. a conditioned medium, its micro-organs explant with the genetic modification of claim 1 are condition and comprise described recombination product.

20. comprise the pharmaceutical preparation of micro-organs explant of the genetic modification of claim 1.

21. a method for preparing the micro-organs explant of expressing at least one recombinant protein product, the method comprising the steps of:

(a) separate a part of organ comprise cell mass from animal, the part organ kept the microarchitecture that this organ originates and have simultaneously select to make allow enough nutrition and gaseous diffusion in this micro-organs explant cell and cellular waste from the micro-organs explant spread out make cytotoxicity and since this part organ the inadequate and refuse of nutrition accumulate the dead minimized size that occurs together; With

(b) come at least some cells in the cell mass of the described organ of genetic modification part to express at least one recombination product with recombination.

22. the method for claim 21, wherein said at least one recombination product is selected from recombinant protein and recombination function RNA molecule.

23. the organ that the method for claim 22, wherein said recombinant protein are normally originated by the micro-organs explant produces.

24. the method for claim 22, wherein said recombinant protein normally are not to be produced by the organ that the micro-organs explant is originated.

25. the method for claim 22, wherein said recombinant protein is a labelled protein.

26. the method for claim 22, wherein said recombinant protein are selected from Regular Insulin, amylase, proteolytic enzyme, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, rnase, deoxyribonuclease, triglyceride level enzyme, phospholipase A ₂, elastoser, amylase, thrombin, UDP glucuronyl transferase, ornithine transcarbamylase, cytopigment p450 enzyme, adenosine deaminase, serum thymic factor, Thymus humoral factor, thymopoietin, extrasin alpha ₁, tethelin, somatomedin, transforming growth factor, gastrin, secretin, cholecystokinin, Somatostatin, P material and the transcription factor of G CFS, erythropoietin, Urogastron, liver erythrogenin (liver generates plain), pHGF, interleukin, anti-somatomedin, fibroblast growth factor, 'beta ' family.

27. the method for claim 21, the micro-organs graft of wherein said genetic modification can be kept at least approximately twenty four hours in cultivation.

28. the method for claim 21, the micro-organs graft of wherein said genetic modification has formula 1/x+1/a＞1.5mm ^-1For the surface-area of feature to bulk index; Wherein " x " is that tissue millimeter thickness and " a " are described tissue millimeter width.

29. the method for claim 21, wherein said organ are selected from lymphoid organ, pancreas, liver, gall-bladder, kidney, digestive organ, respiratory tract organ, reproductive organ, skin, urinary tract organ, the relevant organ of blood, thymus gland, spleen.

30. the method for claim 21, the micro-organs graft of wherein said genetic modification comprises epithelium and phoirocyte, arranges with the microarchitecture of the microarchitecture that is similar to the organ that obtains this explant.

31. the method for claim 21, wherein organ is a pancreas, and cell mass comprises youth's lattice Han Shi pancreas islet.

32. the method for claim 21, wherein organ is a skin, and explant comprises at least one hair follicle and body of gland.

33. the method for claim 21, wherein organ is an affected skin, and explant comprises hyperplasia or new outgrowth cell mass from affected skin.

34. the method for claim 21, the micro-organs graft of wherein said genetic modification can maintain in the minimal medium.

35. the method for claim 21, wherein the microarchitecture that keeps of the micro-organs graft of genetic modification comprises one or more cell-cells and the cell-matrix between two or more tissues of the organ that is positioned to separate this explant.

36. the method for claim 21, wherein at least a portion cell mass is carried the recombinant virus infection of the recombination of the described recombination product of coding.

37. the method for claim 36, wherein said recombinant virus are selected from recombinant hepatitis virus, recombinant adenovirus, recombinant adeno-associated virus, recombinant papillomavirus, recombinant herpesvirus, recombinant slow virus, recombinant retrovirus, recombined cytomegalovirus and reorganization simian virus.

38. the method for claim 21, the method for transformation of the transfection of the wherein transfection of at least a portion cell mass by being selected from calcium phosphate mediation, the mediation of DEAE-dextran, electroporation, liposome-mediated transfection, direct injection and receptor-mediated absorption has transformed exogenous nucleic acid sequences.

39. a gene product passes to the method for acceptor, the method comprising the steps of:

(a) provide the micro-organs explant of expressing at least one recombination product, this micro-organs explant comprises cell mass, the micro-organs explant kept the microarchitecture that this organ originates and have simultaneously select to make allow enough nutrition and gaseous diffusion in this micro-organs explant cell and cellular waste from the micro-organs explant spread out make cytotoxicity and since the micro-organs explant the inadequate and refuse of nutrition accumulate the dead minimized size that occurs together, at least one recombination product of at least some cell expressings in the cell mass of described micro-organs explant; With

(b) this micro-organs explant is implanted in the acceptor.

40. the method for claim 39, wherein said micro-organs explant comes autoreceptor.

41. the method for claim 39, wherein said micro-organs explant is from the donor experimenter.

42. the method for claim 39, wherein said micro-organs explant is from the people.

43. the method for claim 39, wherein said micro-organs explant is from the non-human animal.

44. the method for claim 39, wherein acceptor is the people.

45. the method for claim 39, wherein acceptor is inhuman animal.

46. the method for claim 39, wherein said at least one recombination product is selected from recombinant protein and recombination function RNA molecule.

47. the organ that the method for claim 46, wherein said recombinant protein are normally originated by the micro-organs explant produces.

48. the method for claim 46, wherein said recombinant protein normally are not to be produced by the organ that the micro-organs explant is originated.

49. the method for claim 46, wherein said recombinant protein is a labelled protein.

50. the method for claim 49, wherein said recombinant protein are selected from Regular Insulin, amylase, proteolytic enzyme, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, rnase, deoxyribonuclease, triglyceride level enzyme, phospholipase A ₂, elastoser, amylase, thrombin, UDP glucuronyl transferase, ornithine transcarbamylase, cytopigment p450 enzyme, adenosine deaminase, serum thymic factor, Thymus humoral factor, thymopoietin, extrasin alpha ₁, tethelin, somatomedin, transforming growth factor, gastrin, secretin, cholecystokinin, Somatostatin, P material and the transcription factor of G CFS, erythropoietin, Urogastron, liver erythrogenin (liver generates plain), pHGF, interleukin, anti-somatomedin, fibroblast growth factor, 'beta ' family.

51. the method for claim 39, the micro-organs graft of wherein said genetic modification can be kept at least approximately twenty four hours in cultivation.

52. the method for claim 39, wherein the micro-organs graft of wherein said genetic modification has formula 1/x+1/a＞1.5mm ^-1For the surface-area of feature to bulk index; Wherein " x " is that tissue millimeter thickness and " a " are described tissue millimeter width.

53. the method for claim 39, wherein said organ are selected from lymphoid organ, pancreas, liver, gall-bladder, kidney, digestive organ, respiratory tract organ, reproductive organ, skin, urinary tract organ, the relevant organ of blood, thymus gland, spleen.

54. the method for claim 39, the micro-organs graft of wherein said genetic modification comprises epithelium and phoirocyte, arranges with the microarchitecture of the microarchitecture that is similar to the organ that obtains this explant.

55. the method for claim 39, wherein organ is a pancreas, and cell mass comprises youth's lattice Han Shi pancreas islet.

56. the method for claim 39, wherein organ is a skin, and explant comprises at least one hair follicle and body of gland.

57. the method for claim 39, wherein organ is an affected skin, and explant comprises hyperplasia or new outgrowth cell mass from affected skin.

58. the method for claim 39, the micro-organs graft of wherein said genetic modification can maintain in the minimal medium.

59. the method for claim 39, wherein the microarchitecture that keeps of the micro-organs graft of genetic modification comprises one or more cell-cells and the cell-matrix between two or more tissues of the organ that is positioned to separate this explant.

60. the method for claim 39, wherein at least a portion cell mass is carried the recombinant virus infection of the recombination of the described recombination product of coding.

61. the method for claim 60, wherein said recombinant virus are selected from recombinant hepatitis virus, recombinant adenovirus, virus related to rocombinant adenovirus, recombinant papillomavirus, recombinant herpesvirus, recombinant slow virus, recombinant retrovirus, recombined cytomegalovirus and reorganization simian virus.

62. the method for claim 39, the method for transformation of the transfection of the wherein transfection of at least a portion cell mass by being selected from calcium phosphate mediation, the mediation of DEAE-dextran, electroporation, liposome-mediated transfection, direct injection and receptor-mediated absorption has transformed exogenous nucleic acid sequences.

63. the method for claim 39 further comprises the step of the micro-organs culture that wraps up described genetic modification before in described step (c).

64. the method for claim 39, wherein step (a) is finished by following:

(i) separate a part of organ comprise cell mass from animal, the part organ kept the microarchitecture that this organ originates and have simultaneously select to make allow enough nutrition and gaseous diffusion in this micro-organs explant cell and cellular waste from the micro-organs explant spread out make cytotoxicity and since the micro-organs explant the inadequate and refuse of nutrition accumulate the dead minimized size that occurs together; With

(ii) come at least some cells in the cell mass of the described part organ of genetic modification to express at least one recombination product with recombination.

65. the method for claim 39, wherein said step (a) is finished by obtain described micro-organs explant from the organ of the transgenic animal of expressing described recombination product.

66. method for preparing the micro-organs explant of expressing at least one recombination product, the method comprising the steps of: separate a part of organ that comprises cell mass from transgenic animal, the part organ kept the microarchitecture that this organ originates and have simultaneously select to make allow enough nutrition and gaseous diffusion in this micro-organs explant cell and cellular waste from the micro-organs explant spread out make cytotoxicity and since the part organ the inadequate and refuse of nutrition accumulate the dead minimized size that occurs together, at least one recombination product of at least some cell expressings in the cell mass of described part organ.

67. medical apparatus, the poly-unit that comprises the micro-organs explant that wraps up the genetic modification of expressing at least one recombination product, this micro-organs explant comprises cell mass, the micro-organs explant kept the microarchitecture that this organ originates and have simultaneously select to make allow enough nutrition and gaseous diffusion in this micro-organs explant cell and cellular waste from the micro-organs explant spread out make cytotoxicity and since the micro-organs explant the inadequate and refuse of nutrition accumulate the dead minimized size that occurs together, at least one recombination product of at least some cell expressings in the cell mass of described micro-organs explant.