CN102369277A

CN102369277A - Lung tissue model

Info

Publication number: CN102369277A
Application number: CN2010800206127A
Authority: CN
Inventors: 尤迪特·E·蓬格拉茨
Original assignee: PECS, University of
Current assignee: PECS, University of
Priority date: 2009-05-05
Filing date: 2010-05-05
Publication date: 2012-03-07
Anticipated expiration: 2030-05-05
Also published as: CA2760768A1; JP2013504303A; AU2010244121A1; HUP0900819A2; WO2010128464A1; CN102369277B; CA2760768C; HU0900819D0; SG175425A1; AU2010244121B2

Abstract

The present invention provides for an engineered tissue scaffold free three dimensional pulmonary model tissue culture which is free of any artificial scaffold. Three dimensional models of healthy lung tissue as well as disease tissues are available. The product according to the invention can be marketed e.g. in the form of tissue cultures, plates or arrays comprising such cultures or kits. The invention is applicable in medical and scientific research, for testing compounds for their effect on lung tissue, for screening, testing and/or evaluating drugs, and in certain cases in diagnostics of lung diseases.

Description

The lung tissue model

Technical field

The present invention provides the through engineering approaches that does not contain any artificial skeleton three-dimensional lung model tissue culture.The three-dimensional model of the lung tissue that can secure good health and diseased tissue.According to product of the present invention can for example be tissue culture, the form that comprises plate or the array or the test kit of said culture sells.The present invention can be applicable in medical science and the scientific research, is used for the influence of test compounds to lung tissue, screening, test and/or assessment medicine, and diagnose pulmonary disorder in some cases.

Background technology

Engineered is a field that develops rapidly in the biomedical research, and it is intended to repair, replaces or the regeneration damaged tissue.Owing to miserable II phase clinical medicine test incident (for example TGN1412) takes place recently, produces safety and the effect test of human tissue model for pharmaceutical compound so other engineered target comprises.General engineered and especially our model utilize biomorph to form, it is an instance of self-assembly.

At present carry out cell and engineered according to some viewpoints.On the one hand, it is in order to obtain pair cell biology and the more deep understanding of physiology.

Developing two main directions in the engineered research: based on the system of system that organizes skeleton and inorganization skeleton.Though mainly use biodegradable framework material to provide artificial three-dimensional structure promoting the interaction of cell based on the system of skeleton, exoskeletal system allows to instruct interaction and the permission cell of cell and cell to be grown in the model on its excretory framework material.

In US 2001/0055804A1 (" three-dimensional of human knurl fore udder disease is model (Three dimensional in vitro model of human preneoplastic breast disease) in vitro "); Disclose in vitro cell culture system of a kind of three-dimensional, it is suitable for the model of making knurl fore udder disease and supplies screening of medicaments.Said model prepares through in comprising like the substratum of other additives such as growth factor, oestrogenic hormon, reproducing knurl anterior epithelium cornea cell, endotheliocyte and the breast inoblast that common cultivation is derived from breast on the basement membrane.Therefore; In this solution, basement membrane, preferred

are as organizing skeleton.According to description, in this system, form the reticulattion of branch's envelope unit vascular system in about 3 days-7 days.

Said system relates to breast but not lung, and does not advise making the lung culture through this method.

In US 2003/0109920 (" through engineering approaches animal tissues (Engineered animal tissue) "), obtain CMEC and be placed between the two-layer human skin inoblast that is present in the three-dimensional collagen gel from lung in adult.Therefore, form sandwich structure.

Although obtain vascular endothelial cell from human lung, the artificial organ for preparing through this method is similar to human skin, and therefore, in fact it can not think the lung tissue model.

In US 2008/0112890 (" tire pneumonocyte and its purposes (Fetal pulmonary cells and uses thereof) "), teaching one kind three-dimensional tissue preparation, it is based on tire mouse epithelial cell, endotheliocyte and mesenchymal cell.The author uses the mice embryonic pneumonocyte to prepare said preparation, to obtain the lung prosthese and said type of three-dimensional tissue's preparation screened.As matrix, use hydrogel MATRIGEL ^TM, to set up suitable cell-cell interaction.From describe, find out inoblast hypertrophy when cultivating epithelial cell and inoblast altogether.

WO2008/100555 (" supplying the through engineering approaches lung tissue of high-throughput toxicity screening and drug discovery to construct (Engineered lung tissue construction for high throughput toxicity screening and drug discovery) ") relates to a kind of lung tissue model preparation, and it comprises the tire pneumonocyte and with biocompatible materials and be preferably the skeleton of organizing that fibroblast growth factor processes.The tire pneumonocyte comprises epithelial cell, endotheliocyte and mesenchymal cell.List a large amount of biocompatible materialses that are suitable for.

WO2004/046322 (" biological tissue duplicate (Replication ofbiological tissue) ") is proposed in the artificial three-dimensional tissue of preparation under the microgravity environment.This tissue is based on human breast cancer cell and can be used as the breast cancer model.In rotary bioreactor chamber, initial cultured connective tissue cell, up to forming three-dimensional ball-like structure, endotheliocyte and epithelial cell add in the culture in regular turn then.It should be noted that this teaching is theoretic, though and culturing cell and the scheme of handling culture are provided, the actual result of experiment does not disclose.

Watt Qu Si (Vertrees, RA), Wei Shenbeigeer (Zwischenberger, JB) wait people (2008) to use verified normal immortal pneumonocyte to tie up to grow in the culturing bottle conventional monolayers (ML) and rotary have grow into the dimensional culture thing in the wall container.Study these cultures and exist and the comparable situation of marker representation from differentiation between the control tissue of patient with operation sample collection.Author's purpose is the conventional monolayers and the three-dimensional cell culture of the known clone of development.

Recently, Kai Libeilvbei (Kelly B é ruB é) and her colleague are developed the three-dimensional tissue's through engineering approaches model confession safety test that human lung epithelial.It uses normal human subject bronchial epithelial cell [NHBE].Grow at microporous membrane from the primary cell that the mankind obtain, form three-dimensional (3D) cell culture.These dimensional culture things form between cell, the cell with active cilium and mucous other cell of generation justacrine and are tightly linked.These characteristics are similar in natural human respiratory epithelium tissue situation about finding very much, and think its exactly simulating human to the reaction of tissue injury.In fact this culture forms the thick cellular layer [Hughes Te Ruixi people such as (Hughes Tracy), 2007] of imitation mucomembranous surface.

As if although there is the document of a large amount of relevant lung models, prior art only discloses based on the three-dimensional model of organizing skeleton, and prior art does not disclose the simple lung tissue model that comprises epithelial cell and fibroblastic inorganization skeleton at least.

The present inventor is surprised to find that, through simple biochemical method, can build the lung tissue model system of inorganization skeleton rapidly, its aspect some all than two-dimentional system or favourable based on the system of matrix.The present invention provides a kind of model tissue that is ready for use on various tests.This model is suitable for studying in the various lung tissues cell-cell interaction with simulation normal function and disease progression.

Summary of the invention

The present invention provides a kind of through engineering approaches three-dimensional lung model tissue culture, said model tissue culture

A) do not contain any artificial organ skeleton,

B) be made up of culturing cell or have a culturing cell material, wherein said cell is in the direct cell-cell interaction with one or more other cell type cells of organization material,

C) comprise lung's epithelial cell and mesenchymal cell at least, preferred lung mesenchymal cell is preferably fibrocyte; Wherein the ratio of lung's epithelial cell and mesenchymal cell is preferably at least 1: 6 in the model tissue, and preferably at least 1: 3, and 6: 1 at the most; Preferably at the most 3: 1

D) have the form of one or more cell aggregations, the surface enrichment lung epithelial cell of aggregate wherein, or wherein lung's epithelial cell and mesenchymal cell, be preferably fibrocyte in said aggregate at least part separate and

E) wherein said epithelial cell is expressed epithelium differentiation marker thing.

A) in a preferred embodiment, said model tissue culture does not contain the artificial substratum material provides three-dimensional environment to cell.In one embodiment, said model tissue culture does not contain any artificial organ framework material, no matter is biodegradable or the biological nondegradable framework material of organizing, for example porous three-dimensional matrix; Three dimensional gel matrix.In one embodiment, said model tissue culture does not contain or does not comprise the microporous membrane upholder.

B) in a preferred embodiment, said model tissue culture also comprises extracellular matrix, its extracellular matrix protein at least a cell type that constitutes said tissue, to be preferably inoblast secreted.

C) in other preferred embodiment, lung's epithelial cell comprises at least a following cell type:

-I type pneumonocyte [type I alveolar cells (ATI)]

-II type pneumonocyte [alveolar type II cells (ATII)].

One or more following affinity tags of said II type pneumonocyte (alveolar epithelial cells) preferred expression: TTF1 transcription factor, surfactant protein A (SFPA), surfactant protein C (SFPC) and aquaporin 3 (AQP 3) with ATII characteristic.

One or more following affinity tags of said I type pneumonocyte (alveolar epithelial cells) preferred expression: TTF1 transcription factor, aquaporin 3 (AQP 3), aquaporin 4 (AQP 4) and aquaporin 5 (AQP 5) with ATI characteristic.

In various other embodiment, at least one in lung's epithelial cell and the lung's mesenchymal cell is present in the model.

Cell is preferably Amphibians, Reptilia, birds cell or mammalian cell more preferably.

Preferred birds cell is the poultry lung cells.

The preferred mammal cell is the cell of herbivore, preferred livestock animals, like cell, ox cell or the rodent cells (for example rabbit or muroid cell) of for example sheep, goat.Other preferred mammal cell is like omnivore cells such as pig cells.Extremely preferred cell is the human cell.

In other embodiments, lung's epithelial cell and/or mesenchymal cell obtain from following:

The clone of-foundation preferably obtains from commercial source,

-healthy donors

-patient donor.

In a preferred embodiment, cell is a primary cell.In a preferred embodiment, cell is not for dedifferenting cell or the cell for only partly dedifferenting.

In a further advantageous embodiment, cell is for dedifferenting cell, or cell dedifferentes before it cultivates into the three-dimensional model tissue culture.

In a preferred embodiment, lung's epithelial cell comprises stingy tract epithelial cell, preferably has a stingy tract epithelial cell of ATII characteristic.

In a further advantageous embodiment, model tissue culture of the present invention also comprises endotheliocyte.In a preferred embodiment, endotheliocyte is HMVEC or HUVEC cell.

The optional cell that can comprise other type that is selected from scavenger cell, mastocyte, smooth muscle cell in addition of model tissue culture of the present invention.

D) in a preferred embodiment; The mean diameter of aggregate or representative diameter are at least 10 μ m, 40 μ m, 60 μ m, 80 μ m, 100 μ m or 120 μ m, and the mean diameter of aggregate or representative diameter are at most 1000 μ m, 800 μ m, 600 μ m, 500 μ m, 400 μ m or 300 μ m.

The mean diameter of aggregate or the representative diameter utmost point are preferably 100-300 μ m, and in a preferred embodiment, it is about 200 μ m.

The mean sizes of aggregate can be assessed and calculate or estimate through any experiment and mathematics modification method.Though aggregate is spherical in shape basically, obviously be attributable to accurate spheric deviation to the diameter of a plurality of diameters of each aggregate and during looking measurement aggregate position and measuring method and confirm.For instance, minimum and maximum diameter can directly be measured in the microscope of measuring some aggregate sizes and average.

Microscope should be used to reach this purpose.

In a preferred embodiment; Most of aggregate; Preferred at least 60%, 70%, 80% or 90% aggregate has the diameter of at least 10 μ m, 40 μ m, 60 μ m, 80 μ m, 100 μ m or 120 μ m and the diameter of 1000 μ m, 800 μ m, 600 μ m, 500 μ m, 400 μ m or 300 μ m at the most; The diameter utmost point of the aggregate of above-mentioned ratio is preferably 100-300 μ m, and in a preferred embodiment, it is about 200 μ m.

In a preferred embodiment, in each aggregate or the amount of the cell that culture sample comprises in each container of test kit do

At least 10 ³Individual, preferably at least 10 ⁴Individual, more preferably at least 2 * 10 ⁴Individual, 3 * 10 ⁴Individual, 4 * 10 ⁴Individual, 5 * 10 ⁴Individual cell, and

At the most 10 ⁶Individual, more preferably at the most 5 * 10 ⁵Individual, 4 * 10 ⁵Individual, 3 * 10 ⁵Individual, 2 * 10 ⁵Individual or at the most 10 ⁵Individual cell.

In a preferred embodiment, lung's epithelial cell separates based on its capillary difference with inoblast.Preferred most of lung epithelial cell is positioned on the aggregate surface.

Preferred most of lung epithelial cell forms lung's epithelial cell lining on aggregate surface, preferred said lung epithelial cell lining part at least covers aggregate surface.

In a further advantageous embodiment, aggregate also comprises endotheliocyte.

In a preferred embodiment, than epithelial cell and inoblast, the ratio of endotheliocyte is higher than in the aggregate surface in the center of aggregate or central zone, or the ratio of endotheliocyte increases to the center of aggregate from the surface of aggregate.

In a preferred embodiment; Aggregate has laminate structure; Wherein the core of aggregate or central zone comprise the endotheliocyte of maximum rate, and the middle layer of aggregate or zone comprise the inoblast of maximum rate, and the skin of aggregate or upper layer comprise the epithelial cell of maximum layer.

E) in a preferred embodiment, the expressed epithelium differentiation marker thing of histocyte of the three-dimensional lung model tissue of through engineering approaches is at least a or more than one are selected from the affinity tag of following group:

-ATII type differentiation marker thing, preferred TTF1 transcription factor, cytokeratin 7 (KRT7), surfactant protein A (SFPA), surfactant protein C (SFPC) and aquaporin 3 (AQP 3), and/or affinity tag

-ATI type differentiation marker thing, preferably water channel protein 4 (AQP 4) and aquaporin 5 (AQP 5).

Expressed affinity tag is also looked used in the tissue culture cell type and is decided.

The content of any affinity tag can mRNA or protein content detection.Therefore, the content of affinity tag can be mRNA content and/or protein content.

The AQP3 of preferred model tissue culture of the present invention and at least one increase in the SFTPA content, promptly it raises than the two-dimentional culture of contrast.

At least a inflammation affinity tag downward modulation that is selected from IL1b, IL6 and CXCL8 of preferred model tissue culture of the present invention, promptly than the two-dimentional culture of contrast, its content reduces under dimensional culture thing condition.

The content that dedifferentes in affinity tag S100A4 and the N-cadherin at least one of preferred model tissue culture of the present invention than two-dimentional culture condition down or contrast the primary cell minimizing of the purifying in the two-dimentional tissue culture.

In various other embodiment, at least one in lung's epithelial cell and the lung's mesenchymal cell is present in the model, and is similar to the embryo lung growth,

One or more are selected from the fibroblast growth factor of FGF4, FGF8, FGF9 the epithelial cell secretion of-lung.

-lung epithelial cell is expressed on cell surface FGFR2b acceptor.

-lung mesenchymal cell, preferred fibroblasts to secrete FGF7 and FGF10, and express FGFR1c and FGFR2c acceptor.

In a preferred embodiment of tissue culture of the present invention; The expression amount of ATII type differentiation marker thing and/or ATI type differentiation marker thing is higher than measured expression amount in two-dimentional tissue culture for referencial use, the expression amount height at least 10%, at least 20% or at least 30% among the more preferably said embodiment.

The mRNA content of epithelium affinity tag and/or protein content described in the preferred said model tissue are higher than one or more or each is following with reference to culture:

The two-dimentional culture of-same cell type,

-epithelial the culture of lung only,

-only former generation inoblast, preferred human fibroblast culture.

The content of preferred at least two kinds of said affinity tags raises.

In a preferred embodiment, use stingy tract epithelial cell.

In a further advantageous embodiment, use the stingy tract epithelial cell that shows at least some ATII type characteristics.In the case, the model tissue shows at least a or more than one mRNA content and/or protein contents that are selected from the affinity tag of following ATII type differentiation marker thing group (the for example listed affinity tag of preceding text) increase.

The three-dimensional lung model tissue of through engineering approaches of the present invention is preferred to show one or more pro-inflammatory cytokines and or the expression decreased of one or more EMT affinity tags.

The mRNA content of one or more pro-inflammatory cytokines and/or protein content described in the preferred said model tissue are lower than one or more or each is following with reference to culture:

The two-dimentional culture that-same cell is formed,

-epithelial the culture of lung only, wherein said lung epithelial cell is similar to the model tissue culture and handles.

Pro-inflammatory cytokine is preferably selected from following group:

CXCL-8 pro-inflammatory cytokine, IL6, IL1a, IL1b, TNF α.

In a utmost point preferred embodiment, short inflammatory chemokine is the CXCL-8 chemoattractant.

In a preferred embodiment, the model tissue culture comprises lung's epithelial cell and lung's mesenchymal cell and does not comprise endotheliocyte, wherein

The content of-one or more following affinity tags increases with respect to the contrast that comprises non-culturing cell: E-cad, IL-1b and/or IL6,

The content of-E-cad increases with respect to the two-dimentional culture of contrast,

The content of-one or more following affinity tags reduces with respect to the two-dimentional culture of contrast: IL-1b, CXCL8, IL6.

In a preferred embodiment, the model tissue culture comprises lung's epithelial cell, lung's mesenchymal cell and endotheliocyte, wherein

The content of one or more following affinity tags reduces with respect to the contrast that comprises non-culturing cell: E-cad, N-cad,

The content of-one or more following affinity tags reduces with respect to the two-dimentional culture of contrast: N-cad, S100A4.

In a preferred embodiment, three-dimensional lung model tissue culture comprises the cell that is selected from following group in addition:

-endotheliocyte, for example with the simulation vascular system,

-scavenger cell,

-mastocyte.

In the later stage, model can use following cell type amplification:

-smooth muscle cell,

-neurocyte.

Disease model

The present invention also relates to the three-dimensional lung model tissue culture of as above defined through engineering approaches, wherein

Said epithelial cell and/or inoblast comprise the influenced cell of the pathological characters with diseased lung tissue, make that said model tissue culture is a pulmonary disorder model tissue culture.

In a preferred embodiment, disease comprises the symptom that is selected from tissue inflammation, tumour, fibrosis, damage, and the model tissue culture is regarded as inflammation model, tumor model, fibrosis model or regenerating model respectively.

The influenced cell of disease model can be the cell (patient's cell) that (but being not limited to) obtains from the patient; Clone with genius morbi, for example tumor cell line; Be exposed to the cell of the environmental activity (for example short inflammatory material) that causes genius morbi; The cell that is exposed to the mutagenic compound effect and selects to genius morbi; Or genetically modified cell, it has ill characteristic through transforming to express a kind of protein or wherein a kind of gene silencing, making.

In a preferred embodiment, cell obtains from healthy individuals, and the patient's condition that induces an illness therein.In this embodiment, for example can confirm the signal conduction or the potential drug target of tumor promotion.

In another embodiment, the tumor model tissue is from the immortality cell preparation, for example from vicious transformation or tumour cell or clone preparation.Though do not have " health " contrast in this embodiment, because the sample that contacts with the placebo medicine provides the contrast of pharmacological agent sample, so this system is applicable to drug test.

In one embodiment, tumour cell obtains from the patient, but and the efficient of test plan therapy.

Therefore, the model tissue culture can be used for setting up individualized therapy.

The preparation method

The present invention also provides the preparation method like the three-dimensional lung model tissue culture of through engineering approaches defined herein, and said method comprises following steps:

-preparation inoblast of former at least generation and the epithelial mixing suspension of lung,

-mixing suspension or its aliquots containig are placed in the container that is suitable for granulation suspension-s cell,

-granulation cell,

-at CO ₂Cultivating granulation suspension-s and maintenance under existing is enough to make cell to form the time of the three-dimensional lung model tissue that comprises cell aggregation,

-optional analysis model tissue

A) expression of peculiar one or more epithelium differentiation marker things of lung tissue, and disclosed herein suitable compared to for example with reference to culture, the expression amount increase is considered to indication and forms three-dimensional lung model tissue culture; And/or

B) expression of one or more pro-inflammatory cytokines, and disclosed herein any suitable with reference to culture compared to for example, expression amount reduce and are considered to indication and form three-dimensional lung model tissue culture.

Container is preferably non-tissue culture processing vessel.

A plurality of aliquots containigs preferably are put in a plurality of containers.

Container is preferably the for example hole of plate such as 96 orifice plates or 384 orifice plates.

Granulation was preferably carried out 1 minute to 20 minutes, preferred 2 minutes to 10 minutes under 600g at 200g.

Cell is preferably through supplying with reporter molecule, for example with the biocompatibility dyeing with report like cell characteristic disclosed herein.

Container at the bottom of container can be V-arrangement, at the bottom of the flat or U-shaped is looked its purpose that is used to reach and is decided.

When the preparation mixing suspension; The cell of one or more types is in 18 hours; In preferred 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, more preferably in 4 hours, 3 hours, 2 hours, add in the container in extremely preferred 1 hour or 0.5 hour.Used various types of cell preferably adds in the above period of defining.

In a preferred embodiment, granulation suspension-s is at CO ₂Exist and to cultivate down the period that is no more than 50 hours, 40 hours, 30 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours or 10 hours.In a preferred embodiment, granulation suspension-s is at CO ₂Exist to cultivate down and be no less than 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours period.

In one embodiment of the invention, other cell type adds in the mixing suspension of cell.In one embodiment of the invention, other cell type is at least endotheliocyte.In one embodiment of the invention, other cell type is selected from endotheliocyte, smooth muscle cell, neurocyte, granulocyte, BMDC, mastocyte, T/B lymphocyte, scavenger cell.Granulocyte, BMDC, mastocyte, T/B lymphocyte and scavenger cell can be nonactive or the immunocompetence state adds in the culture.

Optionally according to the method for the invention one or more cell types are dedifferented before being included in the preparation mixing suspension.

Optional according to the method for the invention one or more cell types of the preceding breeding of preparation mixing suspension that are included in.A large amount of samples of parallel testing if desired, for example in HTS (high flux screening) solution, this step especially needs so.

Screening method

The present invention also relates to the method for medicine to the effect screening of medicaments of lung tissue, said method comprises following steps:

-provide like the three-dimensional lung model tissue culture of through engineering approaches defined herein,

-get at least one specimen and the reference sample of said model tissue culture,

-specimen is contacted with medicine, keep specimen and reference sample simultaneously under the same conditions,

-compare with reference sample, detect any variation or the change of specimen,

If wherein detect any variation or the change of specimen, think to indicate the effect of medicine so.

In some variant of said method, only observe the direction that changes or change, and do not calculate physiological values.In some variant, based on working curve, define predetermined threshold value, and value compares therewith.

In a preferred embodiment, the model tissue culture is the undesirable action of healthy lung tissue model and testing drug, wherein the deleterious variation of specimen cell or change is considered to the poisonous or undesirable action of said medicine.

In a preferred embodiment, the model tissue culture is the pulmonary disorder model tissue culture that comprises the influenced cell with pathological characters, and the beneficial effect of testing drug, wherein

-provide the analysis of measuring or assess said pathological characters to measure to said model tissue culture to obtain disease,

-reference sample (healthy reference sample) of healthy lung tissue and/or the reference sample (ill reference sample) of diseased lung tissue be provided,

-in healthy reference sample and/or ill reference sample, and in said at least a specimen, contact front and back with medicine at it, measure or the assessment pathological characters, wherein

Disease in the specimen is measured shift to disease in the healthy reference sample measure with or be considered to the beneficial effect of said medicine away from any variation or change in the specimen that disease is measured in the ill reference sample.In other words, compare with ill reference sample, it more is similar to the state of healthy reference sample.

In a preferred embodiment, use the primary cell that obtains from the patient.In a preferred embodiment; From set patient's primary cell do not dedifferente or only part dedifferente, and obtaining to be used for preparing in back 5 days, 4 days, 3 days, 2 days or 1 day or in 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour the mixing suspension of cell from said patient.In the model tissue culture of processing with primary cell, can study the characteristic of the set disease of patient patient's condition and can test medicine and/or scheme.

Test kit

The present invention also relates to the three-dimensional lung model of a kind of through engineering approaches and organize test kit, it comprises the test panel with vessel array, and wherein at least two containers contain

The sample like the three-dimensional lung model tissue culture of defined through engineering approaches in arbitrary technical scheme in the prior art scheme of-one or more types, each sample is put into the container out of the ordinary of said plate,

The appropriate culture medium of the cell of-confession culture model tissue culture.

A container subclass preferably comprises one or more control samples.Control sample can be the pure growth of some cell type, for example only epithelial cell and fibroblastic culture and/or two dimension (2D) culture.In disease model, contrast can be the culture of healthy cell.

The three-dimensional lung model of through engineering approaches organizes test kit preferably to have one or more feature:

-plate is 96 orifice plates.

-plate be V-arrangement base plate or flat underside or comprise V-arrangement at the bottom of with the plate of flat-bottom hole.The U-shaped base plate also can be used.

The amount of the cell that culture samples comprises in-each container does

At the most 10 ⁶Individual, preferably at the most 5 * 10 ⁵Individual, 4 * 10 ⁵Individual, 3 * 10 ⁵Individual, 2 * 10 ⁵Individual or at the most 10 ⁵Individual cell.

-container distinctly or together seals, and contains enrichment CO ₂Environment that is suitable for the lung tissue culture or atmosphere.

-enrichment CO ₂Environment or atmosphere comprise

At least 2%, 3%, 4%CO ₂Environment,

At the most 10%, 9%, 8% or 7%CO ₂Environment,

Extremely preferably about 5%CO ₂

In a preferred embodiment, sample comprises specimen and corresponding control sample.

In a preferred embodiment, specimen is present on the V-arrangement base plate or is present in the V-arrangement bottom outlet on the plate, and control sample is present on the flat underside or is present in the flat-bottom hole on the plate.

Definition

In context of the present invention, the implication of " artificial organ skeleton " is a kind of solid support material, and it has the structure three-dimensional structures arranged that is designed for and is applicable to cell in cell attachment and/or aid in tissue or the cell culture especially.Said artificial organ skeleton preferably before cultured tissue or cell, make and before cultivating said tissue or cell or during this period with tissue or cells contacting.Therefore, the artificial organ skeleton is generally through influencing at least a portion cell interaction (for example cell-cell interaction) or cellular environment self, promotes the cell growth supporting structure or the material of for example structure such as tissue or cell culture three-dimensional structure formation.Therefore, if organize skeleton to remove from tissue or cell culture, tissue or cell culture will disintegrate or become chaotic so.Yet; It will be understood by one of ordinary skill in the art that; If the artificial organ skeleton processes with Biodegradable material and it is degraded gradually, make cell-cell interaction form, do not think that so this organizes in skeleton and this method tissue or cell culture maybe not can to disintegrate or becomes confusion for removing.

In a pattern, the artificial organ skeleton is a three dimensional matrix, is preferably three dimensional gel matrix or porous three-dimensional matrix, and said matrix optimization has between the residing microvoid of cell or the hole.In a pattern, the artificial organ skeleton is preferably the porous-film upholder from as cell attachment upholder from the teeth outwards.In this pattern of skeleton, it has the structure that is designed for and is applicable to cell attachment especially, and for example porous or bending or spination or flute profile are surperficial, and cell attachment is in this surface, thus the formation of promotion three-dimensional structure.

The artificial organ skeleton is preferred

-have a three-dimensional structure that defines,

-be porous, preferred heights porous material or matrix,

-be porous-film,

-be porous three-dimensional matrix,

-process with biocompatible materials, and/or

-process with polymkeric substance.

" artificial organ skeleton " chosen wantonly and is the matrix based on polysaccharide, and for example it is based on cellulosic matrix, for example methylcellulose gum matrix.

" artificial organ skeleton " is optional to have the bead structure, and for example it is this bead of Saden (cytodex bead).

In this article; The tissue culture that " the three-dimensional histoculture thing that does not contain any artificial organ skeleton " is interpreted as having three-dimensional structure, the three-dimensional structure of wherein said tissue culture are to form or facilitate and do not obtain the artificial organ skeleton and help through intrinsic cell-cell interaction.

Therefore, the three-dimensional histoculture thing that does not contain any artificial organ skeleton is not disintegrated under the situation that lacks the artificial organ skeleton or is become confusion, but keeps its three-dimensional structure.Even the said three-dimensional histoculture thing that does not contain any artificial organ skeleton is cultivated on the solid support material and is formed; The formation of three-dimensional structure do not obtain yet this solid support material help and be not owing to cell attachment in this solid support material, and it can separate under the situation of not destroying three-dimensional structure.

" cellular segregation " as used herein is meant the spatial separation of at least two types of cells of tissue or cell culture; Thereby after this spatial separation (promptly separating); The ratio that can define or find two types of cells be different from separate before in culture the same area in ratio and other zone of culture of same type cell the culture of the ratio of same type cell regional, for example (part) volume or surface.The surface tension significant difference of preferred at least two types of cells promotes it in vitro to separate.

For example a certain cell type of a certain volume of culture or zones such as partial volume or surface or part surface " enrichment " ratio that is interpreted as a certain cell type in this zone is higher than the phenomenon in the reference zone (for example other zone of said culture).Usually, the zone of culture " enrichment " is the result of cellular segregation.

" inflammation " is for example to infect the adaptation reaction that causes with noxious stimulus such as tissue injury and situation.

A large amount of cytokines are referred to as " pro-inflammatory cytokine " because of quickening inflammation, and it also directly or through it brings out cell adhesion molecule or other cytokine synthetic capacity adjustment inflammatory response in some cell type.The main pro-inflammatory cytokine of being responsible for early response is IL1-α, IL1-β, IL6 and TNF-α.Other short inflammatory mediators comprises that IFN-γ, CNTF, TGF-β, IL12, IL17, IL18, IL8 (CXCL8) and number of chemical lure other chemokine and the various neuregulins of inflammatory cells.The actual result of inflammatory response is to confirm through the equilibrium between pro-inflammatory cytokine and the anti-inflammatory cytokine (for example IL4, IL10 and IL13, IL16, IFN-α, TGF-β, IL1ra, G-CSF, to the soluble receptors of TNF or IL6).Various Casproteases carry out in being called the large-scale polyprotein mixture of inflammation body the activation of IL1-β.

LIF, GM-CSF, IL11 and OSM prepare disease model of the present invention for other cytokine and its that influence inflammatory process possibly be applicable to.

On the contrary, regulate inflammatory process, it is suppressed or alleviates as " anti-inflammatory cytokines " such as IL10.

" mean diameter " of three-dimensional tissue is regarded as the arithmetical av of some observed values of the three-dimensional tissue's diameter that produces through aforesaid method.

" representative diameter " (median diameter) is the diameter that the set throw out sample of mark (sediment sample) is divided into two equal weight parts, and a weight part contains greater than all aggregates of this diameter and another weight part and contains all aggregates less than this diameter.

Container " array " is interpreted as arranging a plurality of have same size, shape and container of material.Layout can be for example a succession of container, or the two-dimentional matrix of container.

Virus is the obligate intracellular pathogens of cells infected, usually particular cell types is had huge specificity.In " recombinant viral vector ", needed genetically deficient of the duplicate stage of viral life cycle and genes involved add in the viral genome.Recombinant viral vector transducible its usually with the cell type that infects.For producing said recombinant viral vector, non-essential gene provides through transduction (in trans), is incorporated in the genome of package cell line or on the plasmid.Research and develop a large amount of viruses, mainly paid close attention to 4 types; Retrovirus (comprising slow virus), adenovirus, adeno-associated virus and herpes simplex virus type 1.

" cancer " is that one group of cell shows uncontrolled growth, invasion and attack (invading and destroy adjacent tissue) and shows the one type of disease that shifts (being diffused into other position in the body through lymph or blood) sometimes.Three kinds of pernicious characteristics of this of cancer distinguish itself and innocent tumour, and the innocent tumour self-limiting is not attacked or shifted.95% lung tumor is a bronchogenic carcinoma; Be carcinoid of bronchus, mesenchyme also, mix vegetation.

" fibrosis " be in the organ or tissue as repairing or the excess fibre reticular tissue of reaction process forms or occurs, form on the contrary with fibrous tissue as organ or tissue's normal components.Lung fibrosis is serious chronic disease, it is characterized by elasticity and disappears, and matter myofibroblast replacement between the pulmonary epithelial cells warp, and extracellular matrix protein deposition in the interstitial lung, cause lung's structure to be reinvented.

Description of drawings

Fig. 1. the structure of three-dimensional two kinds of cell types trace culture.SAEC and NHLF cell dye with the vital dye of CFSE or DiI respectively.Cultivated at 24 hours the back granulation pure or with the cell colony of various ratio mixed and form aggregate, then transfer to and supply imaging in the 24 porocyte culture plates.Top row: differ micro-imaging; Middle line: fluorescence microscopy image; End row: confocal image.SAEC: green channel; NHLF: red channel.

The culture based on matrigel (Matrigel) of Fig. 2 .50%SAEC and 50%NHLF mixture.

Fig. 3. contain the granulation microcomponent culture of different ratio SAEC and NHLF.Owing to the physiology fluorescent marker, the SAEC cell is green or light gray on the black and white copy, and the NHLF cell is redness or lead.

The mRNA content of TTF-1 in the human lung of Fig. 4 a. three-dimensional microcomponent.The TTF1 transcription factor is the signature thing of alveolar epithelial cells.Do not express though three-dimensional fibroblast cell cultures shows TTF1, TTF1 is present in the D S AEC monoculture and in two-dimentional SAEC/NHLF coculture to be increased, and shows fibroblastic beneficial effect.TT1 reaches high expression level amount in D S AEC/NHLF tissue.

The mRNA content of AQP-3 water translocator in the human lung of Fig. 4 b. three-dimensional microcomponent.AQ3 is an ATII epithelial cell type mark thing in the lung.Though showing AQ3, three-dimensional fibroblast cell cultures do not express; AQ3 is present in the D S AEC monoculture and in two-dimentional SAEC/NHLF coculture to be increased; Show fibroblastic beneficial effect, but in D S AEC/NHLF tissue culture, still observe the most high-load AQ3.

The changes in gene expression of Fig. 5 .SAEC differentiation marker thing.Figure A: the relative mRNA content of AQP3 water translocator in the human lung of the two and three dimensions microcomponent.In the cell mixing culture relatively the AQP3 expression amount increase with respect to SAEC culture only, and do not detect difference between two dimension and the three-dimensional cultivation condition.Figure B: compared to SAEC culture only, in the cell mixing culture relatively the KRT7mRNA expression amount increase.(independent experiment: n=3) figure C: the RT-PCR that SFTPA1 and beta-actin are expressed in the two and three dimensions culture analyzes.Only in the SAEC+NHLF coculture, detecting SFTPA1 expresses.The expression of SFTPA1 is consistent in the dimensional culture thing is higher than in the two-dimentional culture.The representative image that shows 3 independent experiments.Scheme D: after cultivating 72 hours altogether with NHLF cell two dimension or three-dimensional, the changes in gene expression of check FACS sorting SAEC.Compared to two-dimentional coculture, three-dimensional cultivate altogether among the SAEC of repurity the content of differentiation marker thing AQP3 and TTF-1 significantly raise.Shown in data be the MV of two independent experiments.(the former generation pneumonocyte that is used for the purifying of all experiments is to be derived from donor at random).

Fig. 6. the EMT affinity tag in the three-dimensional lung tissue model.Figure A: the relative mRNA content of S100A4 in the two and three dimensions coculture.Compared to SAEC culture only, fibroblastic existence significantly reduces the content of S100A4 in the SAEC-NHLF coculture, and two dimension or three-dimensional cultivation condition significantly do not change S100A4 and express.The relative mRNA content of figure B:E-cadherin (E-cad) increases in the presence of NHLF in the dimensional culture thing.Figure C: the relative mRNA content of N-cadherin (N-cad) in the human lung of the two and three dimensions microcomponent.Scheme D: after cultivating 72 hours altogether with NHLF cell two dimension or three-dimensional, the changes in gene expression of check FACS sorting SAEC.Compared to two-dimentional coculture, the content of EMT affinity tag S100A4 and E-cad increases in three-dimensional two kinds of co-culture of cells things in the pulmonary epithelial cells of sorting, and the expression amount of N-cad in the three-dimensional hybrid culture is much lower.Former generation pneumonocyte of the purifying that is used for testing is to be derived from donor at random.Shown in data be the MV of three (figure A-figure C) or two (figure D) independent experiments.The former generation pneumonocyte that is used for the purifying of all experiments is to be derived from donor at random.

Fig. 7. the secretion of inflammatory cytokine and chemokine in the human former generation pneumonocyte culture.Figure A: in cell culture supernatant, almost do not detect the CXCL-8 secretion of two and three dimensions NHLF monoculture.D S AEC culture still produces CXCL8, but degree is lower than two-dimentional SAEC culture.The two dimension coculture does not significantly change the expression of CXCL-8, shows that fibroblastic existence can not influence cytokine expression.The CXCL-8 expression amount significantly reduces in the system of three-dimensional tissue in pure SAEC and SAEC-NHLF coculture.Figure A and B: the expression amount that is respectively IL-1b and IL-6mRNA in human former generation pneumonocyte culture.Compared to two-dimentional culture, the inflammatory mRNA content of inflammatory cytokine IL-1b and IL-6 is consistent lower in the dimensional culture thing.In pure fibroblast cell cultures, do not detect IL-1b mRNA and express, and IL-6 content is much lower and it is also not too remarkable to express variation.(in addition referring to table 2) figure D: be similar to the cell mixing culture samples, in two-dimentional culture, inflammatory cytokine IL-1b and IL-6 content are also significantly reducing from the SAEC of dimensional culture thing purifying.Shown in data be the MV of three (figure A-figure C) or two (figure D) independent experiments.The former generation pneumonocyte that is used for the purifying of all experiments is to be derived from donor at random.

Fig. 8. the structure of three kinds of cell types trace of the three-dimensional of forming by SAEC, NHLF and HMVEC culture.SAEC, NHLF and HMVEC with vital dye CFSE, DiI or DiD dyeing, then assemble respectively.After cultivating in 24 hours, the two or three cell type trace culture of spontaneous rearrangement is carefully transferred to and is supplied imaging in the 24 porocyte culture plates.Figure A: two kinds of cell type cultures; Figure B: three kinds of cell type cultures.Top row: differ micro-imaging; Middle line: fluorescence microscopy image; End row: confocal image.SAEC: green channel; NHLF: red channel; HMVEC: blue channel.

Fig. 9. the changes in gene expression of three kinds of cell type cultures.Figure A: compared to two-dimentional culture, the expression amount of AQP3 and KRT7 increases in the dimensional culture thing, and the expression amount of S100A4 and N-cad reduces.The comparison that the expression of molecular marked compound changes in figure two kinds of cell type cultures of B: D S AEC-NHLF and three kinds of cell type cultures of SAEC-NHLF-HMVEC.AQP3 and E-cad mRNA content increase, and S100A4 and N-cad reduce, and show in three kinds of cell type models and keep tissue differentiation.The former generation pneumonocyte that is used for the purifying of all experiments is to be derived from donor at random.

The preparation flow figure of the subsequent use lung tissue test kit of test that transmits in Figure 10 .96 orifice plate.

Figure 11: adenoviral gene is delivered among the SAEC in two kinds of cell type models.

Figure A:SAEC expresses owing to GFP and in three-dimensional tissue's mold surface, presents green.Inoblast is dyed redness with the physiology dyestuff in advance before gathering.Figure B:RT-PCR reaction confirms that the GFP gene effectively is delivered in the model.Can in the model culture of adenovirus transduction, detect GFP.

Embodiment

The present inventor sets up the three-dimensional lung model tissue culture of a kind of simple through engineering approaches, and it is suitable for makes the lung tissue model and be ready for use in the various testing method.

During producing said model, consider some aspects of tissue signature, comprise the principal character of lung tissue type, the interaction of embryo lung cell type between the growth period and engineered technical progress.

Based on the system of skeleton and exoskeletal as described herein test of system.The analysis of lung tissue specific marker thing is showed that all of a sudden three-dimensional exoskeletal system shows and the surprising similarity of natural lung tissue.

In the prior art, the inoblast hypertrophy is often run into (US2008/0112890) during attempting in the model tissue, utilizing these cell types.According to the result that this paper appeared, can suppose the former of said hypertrophy because lack the formation of aggregate.The cell that leaves from aggregate possibly be attached to vessel surface, begins breeding, up to realizing that through it contact suppresses.

So far the lung tissue model of development use special framework material and keep to cultivate chronically [Nichols (Nichols, J.E.) and Ke Dila (Cortiella J.) 2008].By contrast, the model that this paper appeared is handled easily, uses simple experimental apparatus and relatively short incubation time.In addition, need not special laboratory equipment.System of the present invention is applicable to the human cell, comprises human primary cell and unconverted human cell.

Should be appreciated that in some system, for example skeleton such as matrix is biodegradable.Yet, if because and skeleton influence or define the structure or the shape of tissue culture, so even and so these systems after the skeleton degraded, should not be considered to exoskeletal system yet.In addition, in vitro in the system, in the present invention, general inexpectancy degradable membrane can dissolve.

In addition, the dissolving of biodegradable framework needs for a long time, is longer than the preparation and the duration of service of tissue culture of the present invention.

Not for the cell that dedifferentes cell also can be applicable among the present invention, yet cell number should be very little.Therefore, this embodiment is applicable to that mainly small amounts of cells enough is used for scenario test, for example tests under the enough responsive situation.In testing fast, can begin to prepare model tissue culture of the present invention from the noble cells of purifying.Said cell can make from individuality is fresh.The method of this pattern is particularly useful for the patient-specific test of medicine for example or compound, or the effect of promoting agent remains the situation of in specified disease background (for example to potential manufacturers), testing.

Primary cell also can obtain from commercial source.The for example for a short time industry park Long Shaweierweiye ltd (Lonza Verviers, S.p.r.l.Parc Industriel de Petit-Rechain) still of reining in, Belgium (Belgium) Wei Erweiye B-4800; Biological center ltd (Biocenter Ltd.); Valley, Sai Gede (Szeged) tower Metz (Temesv á ri krt.) 62H-6726; Hero company (Invitrogen Corporation) (part of Life Technologies, Inc. (Life Technologies Corporation) (model Alan road, Carlsbad, the U.S. (USA) California (California) (Carlsbad) (Van Allen Way) 5791,92008)).

A large amount of if desired samples, cell remains before the preparation model is organized culture samples, to be bred so.During this process, possibly occur dedifferenting.This is the situation in screening (for example HTS) application.In the patient-specific test, more a spot of sample is promptly enough.

When cell differentiation phase in aggregate, breeding is slowed down or is stopped, and after this aggregate does not significantly increase.

If small amount of sample is promptly enough, need not so to breed in advance.Usually, this situation possibly occur in in patient's sample test of several drugs or remain to be observed under the situation of some phenomenon such as signal conductive process for example.Yet, in screening process, need a large amount of samples and should be before preparation model tissue culture propagated cell.

In a preferred embodiment of the invention, use the adult to dedifferente epithelial cell.Under in the field and do not know that the simple adult who cultivates altogether dedifferentes epithelial cell under inoblast and can realize breaking up, and this situation is under three-dimensional condition, especially is being suitable for forming further increase under the condition of three-dimensional aggregate.Therefore, in a preferred embodiment, the preparation of model tissue culture begins from dedifferenting cell.

Remain on primary cell in the culture such as two-dimentional tissue culture for example and will show that some dedifferentes affinity tag.Therefore, said cell also can be applicable among the present invention.Dedifferente affinity tag and comprise S100A4, N-cadherin and inflammation affinity tag.Thereby can use more a large amount of cells.

After adding the structural constituent and/or the factor, multipotency or undifferentiated cell can be broken up.

Cell interaction in the lung tissue

Cell dedifferentes and differentiation again

Stem cell and tissue specificity progenitor cell can carry out the orientation step of tissue specificity differentiation, and therefore representative produces the ideal source of organ specific tissue cultivated material.Regrettably, these two kinds of cell types use with finite population in differentiated tissue only.

Because organize models need regularly set up according to experiment and/or test request, so the tissue specificity noble cells is represented better starting material source, this is for no other reason than that they are more.The phenomenon that model system of the present invention at least partly utilizes noble cells under two-dimentional culture condition, can dedifferente and use suitable culture condition can force it to break up again.

Cell interaction

As if lung development and epithelial damage reparation come close coordination through the stimulated gene of the function of stem cell and adult's progenitor cell and the fine balance between the suppressor gene in the common adjusting lung.For instance, the conduction of FGF receptor tyrosine kinase signal is indispensable concerning respiratory organs forms, and oppositely regulates (opening (Zhang), Si Tapeng Bake people such as (Stappenbeck), 2005) through inducibility FGF path suppressor factor family.In addition, the FGF signal conducts for forming new alveolar, protecting alveolar epithelial cells to avoid supposing the migration of alveolar stem/progenitor cells and breeding required during damage and the lung reparation.Otherwise the TGF beta receptor serine-threonine kinase signal conduction through Smad 2,3 and 4 suppresses the lung form and forms, and the back alveolar that can suppress to be born grows, and the excessive TGF signal conduction through Smad3 then can cause interstitial fibrosis.

Therefore, need have interactional more complicated interactive system between mesenchymal cell and the epithelial cell.The present inventor supposes that basic lung model can produce through alveolar epithelial cells and the fibroblastic mixture that uses purifying.

Therefore, only use two kinds of cell types at first: former generation human fibroblast (NHLF) and stingy tract epithelial cell (SAEC with ATII characteristic), the both can buy (Long Sha company (Lonza)).Be surprised to find that these two kinds of cell types enough provide the essential factor that forms three-dimensional lung tissue model.It will be understood by one of ordinary skill in the art that the cell type of for example listing among this paper can further develop this model through adding other cell type.

The separation of cell type (sorting) in the mixed culture

Microscopy proves, spontaneous tissue reorganization-" sorting "-occur in the three-dimensional lung primary cell culture.The present inventor uses simple centrifugal method to make cell aggregation in this article, is similar to the method for preparing tire thymus gland organ cultures.The evidence that [extra large ear (Hare K.J.) waits people (1999)] this paper provides proves, former generation lung's epithelial cell cultivate than more help in two dimension or the three-dimensional in vitro monoculture SAEC altogether with fibroblastic three-dimensional and keep and more the state that breaks up more.Comprise that NHLF not only promotes epithelial cell differentiation, and (Fig. 8 is culture a), the adhesion of three-dimensional microcomponent and firm in structure much fine and close compared to only SAEC (Fig. 3) or SAEC-HMVEC.

Two kinds of cell type cocultures of the present invention system that is made up of stingy tract epithelial cell of the mankind (SAEC) and normal human subject lung fibroblast (NHLF) need not to exist the outside ECM that adds to form and keep three-dimensional structure (Fig. 3).The SAEC of the cell mixture of unicellular type of granulation and various ratios and NHLF cell suspending liquid show, set up microcomponent of lung and need exist inoblast to keep the three-dimensional structure of form compact and stable (Fig. 3).The morphological examination of three-dimensional microcomponent shows that the separation of two kinds of cell types is characteristics of three-dimensional microcomponent in the mixed culture, and inoblast forms the internal core part, and epithelial cell covers outer (Fig. 3).Separation or " sorting " phenomenon are based on the difference adhesion energy feature of cell type and had before described to former generation mankind lung tissue.Spontaneous cell " sorting " is based on the difference of bounding force between the different cell types: the most bonding core or the central zone of microcomponent of lung are formed by NHLF colony, and NHLF colony quilt not too adherent SAEC centers on.Especially pay close attention to the sepn process in the human lung tissue of former generation differentiation, even because the ability that it initiatively probes into himself microenvironment is also kept for the adult humans cell of differentiation in the basis of this idea.Cell in the three-dimensional coculture can with adjacent cell switch, tissue is reorganized.This process also needs the reorganization of extracellular matrix.Study of model to various SAEC/NHLF ratios in the three-dimensional microcomponent model shows that ratio was enough to make epithelial cell to cover fibroblastic internal core in 1: 1, therefore uses this model of further analysis is set at 1: 1.Yet model can use by other epithelial cell-mesenchymal cell ratio.The ratio that is enough to cover fully is visual cell's type and changing to a certain extent.

Do not accept opinion constraint, the present inventor thinks that the separation of cell is different owing to the binding property of cell type in the model of the present invention, and wherein its capillary difference plays an important role.

Any cell all can initiatively be probed into the microenvironment of himself, and it can or reorganize near the extracellular matrix it with adjacent cell switch.Known back one process relates to the enzymic activity of mechanical haulage power and matrix metalloproteinase (MMP).Based on different adhesion energy features, some cell composition of experimentally known cell mixing type can be aggregate form and separate.

In the isolated cells aggregate of hanging drop culture, the adherent colony zone of plying in the centre is centered on by adherent colony not too.Measuring of tissue adhesion property is the surface tension of cell.Therefore, as detectable amount experimentally, the measurable sorting level of surface tension.Therefore, early stage trial can predict whether comprise new cell type (Nei Yagu (Neagu), 2006) to a certain extent through this sorting level in the affiliated field.

Yet, and do not know the surface tension factor of the particular cell types of human lung.Prior art is not mentioned the phenomenon of organizing sorting fully and is occurred in other culture or only occur in the hanging drop culture.The present inventor confirms through experiment, shows all of a sudden first that in this article epithelial cell separates in the mixing alveolar epithelial cells and fibroblast cell cultures that occurs in granulation with fibroblastic.

The size of little aggregate and structure

The present inventor is surprised to find that, even therefore the very little aggregate display organization characteristic that also has structure is suitable for studying interaction and test compounds or environmental influence.

Little aggregate has some advantages, for example, need not special reaction vessel, and the ratio of its size and different cell types can reproduce, and interacts thereby be easier to control.In little aggregate, possibly organize the internal portion of aggregate downright bad hardly.In addition, can realize that surprising uniform grain sizes distributes, this makes it be very suitable for parallel testing.

Therefore, preferably, according to the present invention, the size of aggregate should remain little, as long as present tissue signature, thereby can check interaction to get final product.

If aggregate is too small, so as correct form disclosed herein may not be shaped and aggregate type of having tissue signature not.If aggregate is excessive, its size departure basically so.

In addition, necrosis possibly occur in aggregate inside, and reason is that incubation time is long and the aggregate perfusion is less.

Lacking special additive, for example receive only under the situation of cell culture medium, the growth of aggregate suppresses to control through the contact of cell.

Yet size depends on the cell number in the aggregate.The size and the cell number that it will be understood by one of ordinary skill in the art that aggregate can change in the boundary that this paper provides, as long as meet above-mentioned requirements.

Have been found that and add the size that endotheliocyte does not significantly change aggregate.

Be applicable to cell type of the present invention

Inoblast

Inoblast is the most omnipotent cell in the phoirocyte family, and it is actually the most ubiquitous cell type.Inoblast is the important structure composition of tissue integrity.Its participation comprises the lung almost reparation and the regenerative process of each human tissue and organ.Its major function is that normal repair for event such as secretion usual practice such as epithelial cell migration provides the extracellular matrix (ECM) of organizing skeleton albumen.

Inoblast or its different subgroups are carried out the organizing specific sexual function as immunity regulatory cell, and secretion can cause immunoreactive chemokine and cytokine through attracting inflammatory cells and immunocyte.Inoblast from the different anatomic position shows a series of common phenotype attributes.Yet the inoblast of different anatomic position shows different phenotypes.The feature representation of fibroblast growth factor and acceptor also is the fibroblastic characteristic of lung [Mo Lelusi (De Moerlooze), Edward Spencer Dien people (2000) such as (Spencer-Dene)].

The present inventor finds, can be dependent on inoblast physiology, set up the nothing manual work and organize the tissue system of skeleton to simulate some aspects of far-end lung tissue, and may not be for setting up the only way of three-dimensional lung culture based on the model of artificial substratum.Do not accept the opinion constraint, the present inventor supposes that the fact of fibroblasts to secrete ECM in the lung helps to realize this result.

Lung's epithelial cell (pneumonocyte)

Pneumonocyte (lung or alveolar epithelial cells or AEC) is the epithelial cell of lining in normal ABM (being the surrounding gas switched area in the lung far-end air flue).Pneumonocyte or AEC can be divided into I type and II type pneumonocyte again.

The signature thing of two kinds of alveolar epithelial cells types is followed the trail of easily, and can for example use RT-PCR reaction or immunohistochemistry monitoring at experimental session.

1 type pneumonocyte

1 type pneumonocyte [alveolar 1 type pneumonocyte, 1 type i alveolar cell, alveolar 1 type cell (abbreviation ATI cell) are also referred to as little alveolar cell, squamous alveolar cell, membrane lung cell or 1 type alveolar epithelial cells] is the compound branched cells with the cell scutum on gaseous interchange surface in a plurality of expression alveolars.These cells have metabolic activity and have to multiple material, comprise the cell surface receptor of extracellular matrix (ECM) albumen, growth factor and cytokine.About 95% alveolar surface is covered by I type pneumonocyte.

2 type pneumonocytes

2 type pneumonocytes (alveolar 2 type pneumonocytes, alveolar 2 type cells; Abbreviation ATII cell, T2P) be the cube epithelial cell; Be also referred to as 2 type alveolar epithelial cellss (abbreviation AEC is also referred to as the EPII cell), 2 type particulate state pneumonocytes, 2 type cells, 2 type i alveolar cells, septal cell or bulla cell (great alveolar cell), big type i alveolar cell (large alveolar cell) or particulate state pneumonocyte.These cells are produced by immature epithelial cell progenitor cell.Alveolar 2 type pneumonocytes are considered to the progenitor cell of alveolar epithelial cells.It can self and is divided into squamous 1 type pneumonocyte.II type cell is a cuboid cell, and it only accounts for 4% of alveolar surface-area, but constitutes the 10%-15% (carat ripple people such as (Crapo), 1982) of 60% and all pneumonocytes of alveolar epithelial cells.

3 type alveolar epithelial cellss

The difference of 3 type alveolar epithelial cellss and flat 1 type cell and cube 2 type cells is that there is microvillus top bundle in it and lacks the slice-type secretory granules.These cells are also referred to as brush cell of alveolus.

Endotheliocyte

Endotheliocyte is for being single flakey epithelium layer, the rectangle cell of lining in all vessel lumen.It derives from blood vessel parent cell and angioplast.

Scavenger cell

Scavenger cell is to derive from the monocytic cell of bone marrow derived (bone marrow derived scavenger cell), and it is finally moved in the tissue.Scavenger cell differentiation of monoenergetic and dual intensity progenitor cell from marrow receives the various kinds of cell FACTOR CONTROL.Other differentiation occurs in the tissue and gained scavenger cell colony is called resident macrophage.

Mastocyte

Mastocyte produces (flagpole-waving (Nakahata) and thorough (Toru) 2002 by multipotency CD34 (+) precursor cell in the marrow; Jane Austen (Austen) and Bo Yisi (Boyce), 2001).Immature mastocyte is taked its exemplary particles form in the tissue of moving into the time.These cells are also expressed Fc-ε R1 and are stopped to express CD34 and Fc-γ R2.Most of mastocyte only produces tryptase (being called MCT) or only produces chymase in lung and the intestines mucosa.Mastocyte plays an important role through discharging effective amboceptor in type.

Smooth muscle cell

Smooth muscle cell is highly specialized multi-functional collapsible cell, and its of short duration (reversible contraction) or long-term (owing to fibrosis and muscle hypertrophy) regulated hollow organ's tube chamber.Smooth muscle cell plays an important role in blood vessel takes place, and makes the vessel wall shaping and keep vascular tone.

Observation under other cell

Add endotheliocyte and can produce the stable aggregate that comprises noble cells.Such as based on the affinity tag of expressing discovery, if endotheliocyte is included in the model tissue culture, degree of differentiation can not reduce so.As if these aggregates are kept a kind of laminate structure, and wherein endotheliocyte is positioned at inside.

Embodiment

Preparation three-dimensional model tissue culture

In the method for the invention, use lung's epithelial cell and mesenchymal cell at least, be preferably fibrocyte.Cell is distinctly cultivated, and the culture to obtain to live then mixes with adequate rate, and at CO ₂Exist down as the felicity condition cultivation altogether down understood based on the present invention and affiliated field method.Through setting cells ratio and selection condition, can avoid a kind of cell type with respect to another kind of cell type hypertrophy.

In a preferred embodiment, said cell obtains as primary cell from the human individual, and dedifferentes or use immediately.Dedifferente and for example to carry out through currently known methods (go down to posterity, remove other cell type, add growth factor).If cell can converge, think that so it dedifferentes.

Granulation cultured cells mixture altogether is to set up cell-cells contacting and make the important step that has suitable distance between the cell.The convenient manner of granulation cell be use centrifugal.The teaching that the those skilled in the art provides based on this paper can be selected suitable granulating method.

In model of the present invention, in principle, can use the listed cell of any preceding text, obtain lung model tissue near natural lung tissue.Applied each cell type must be applicable to obtain as the condition of three-dimensional model tissue disclosed herein under grow and can combine with other cell type of model.

These factors should be tested in preliminary experiment.Initial other cell that should use less relatively ratio, then the ratio of other cell type can increase, and is similar in vivo ratio up to ratio usually.

Other cell type that can be included in the model in a preferred embodiment, is for example endotheliocyte and smooth muscle cell.

Disease model

Based on above model and use range gene transmission method and variable target gene, the gene that above system is suitable for studying during the pulmonary disorder easily changes, thereby can differentiate the therapy that new medicine target is new with development:

Wherein disease comprises inflammation, and influenced cell, preferably epithelial cell expression inflammatory cytokine (surpassing normal contents), and model are the inflammation model,

Wherein disease is a tumour, cell for transform, for example vicious transformation or immortality cell, and model is tumor model,

Wherein disease comprises fibrosis, and model is the fibrosis model,

Wherein disease comprises tissue injury, and model is a regenerating model.

Disease model can be used for drug test.

Cell from patient's acquisition

In one embodiment, lung cells obtains from the patient, and cultivates according to the present invention.In this embodiment, preferably do not dedifferente or only partly dedifferente.After this, with rapid preparation method, form the three-dimensional model tissue culture, and test the medicine that proposal is used to treat said patient, but or the regimen of test plan.The advantage of this embodiment is especially for preparing the pure and parallel sample cultivation thing with homogeneous composition and size.Said sample does not contain any pathogenic agent and purifying on demand yet.

Model from the healthy cell preparation

In a preferred embodiment, disease model begins to prepare from healthy cell, adds the factor that realizes genius morbi (symptom) in the cell subsequently.

For instance, tumor model is prepared by healthy cell, and adds the factor that realizes vicious transformation, and/or expresses the gene that causes vicious transformation therein.For example observing in the lung tumors tissue Wnt Protein content such as Wnt5 increases.Therefore, the expection tumor model can prepare in cell mixture of the present invention or culture like tumorigenesis factors such as EGF (epidermal growth factor), IGF (rhIGF-1), Regular Insulin, the Wnt factor (for example Wnt5) or its mixtures through adding.

In an alternative method of this method, tumour cell adds in the substratum, exist according to model culture of the present invention in the said substratum, but it separates through semi-permeable membranes.Thereby the factor that tumour cell produces is brought out the tumour (pernicious) of culturing cell of the present invention and is changed.

With lung tumor cell is the lung tumor model of processing

The lung tumor model can be by the preparation of lung tumor cell system.Said clone is easily at American Type Culture Collection (American Type Culture Collection) (ATCC; Rockville city, the Maryland State (Rockville, MD)) obtains when searching tumor cell line.

Advisably, seek the experiment of the ratio of used cell type in the condition that is suitable for culturing cell and the optimizing cell mixture.

The inflammation model

For the inflammation model, preferably during the preparation process, monocyte and/or scavenger cell can add in the model culture of the present invention.

In this model, should be with LPS or WNT5A pre-treatment.

The production of cytokines of activatory scavenger cell and like the tissue culture and give the inflammation model capability of exerting an influence of other factors such as Wnt5.

If desire check inflammation model system can be provided in the suitable chamber through film and the isolating neutrophil leucocyte of lung's aggregate so.In the case, also can measure the migration of neutrophil leucocyte and the generation of MMP.

In an alternate embodiment, disease model lung cells system cultivates according to the present invention.In this embodiment, but testing drug to the efficient of said disease.

In said disease model, should avoid the use of the over-expresses gene, more precisely say, should use inducible promoter.

Inflammation model from natural three-dimensional lung cells aggregate

Be simulation inflammation symptom, natural three-dimensional lung cells aggregate can be with the various mass treatment that cause inflammatory response.

Said material is for for example:

The chemical substance that sets up an acute inflammation, for example vasoactive amines, eicosanoid etc.

Short inflammatory polypeptide, for example growth factor, lytic enzyme etc.

Active oxygen,

Pro-inflammatory cytokine, for example IFN-γ and other cytokine,

Bacterial cell wall extracts.

The inflammation symptom is tested through method (for example PCR in real time) or expression analysis (for example applying gene chip) the detection cytokine-expressing of for example using biochemical analysis, immune analysis (for example ELISA), PCR-based.

The genetic modification of primary cell

Epithelial cell and mesenchymal cell can use recombinant virus transmission carrier (recombinant adenovirus and recombined lentivirus vector) to carry out genetic modification, and these gene transmission methods do not damage the ability of cell aggregation.But inflammation, tumour, fibrosis and regenerated characterizing gene composition or inducibility over-expresses or silence, and can in three-dimensional microenvironment, research organization's form, cell response, gene and protein expression change.

For instance, one or more genes that known promotion tumour forms can be introduced lung cells system, and for example alveolar I type or II type clone in the preferred II type clone, or are introduced in the fibroblast.This genoid can for example be for example oncogene such as ras gene or for example COX-2 gene etc. represent gene or one group of gene of tumour expression pattern.Separately ras genetic expression be not enough to transformant, preferred immortality cell, but propagation possibly increase [king (and Wang, XQ), Lee (Li H) waits people (2009)], the genius morbi that this result can supply a model.

Use genetically modified and EF composition in the clone change primary cell aggregate of sublethal exposure

In this embodiment, all cells type all infects, so the same normal in genetic expression and any set three-dimensional lung tissue model.Yet; The cell of aggregate is formed cell (5-10% of the whole cell numbers of the aggregate)-inoblast (WI-38) or the alveolar epithelial cells (A549) that contain through sublethal exposure, and these cells are genetically modified and produce the EF (Wnt-s, bone morphogenetic protein (BMP)-s, inflammatory and pro-inflammatory cytokine, growth factor etc.) that changes aggregate inner cell microenvironment.Sublethal exposure can reduce cell proliferation and prevent that a kind of cell type is with respect to other cell type hypertrophy.

Product

The present invention also provides a kind of test kit that comprises a plurality of samples of three-dimensional model tissue culture.

The three-dimensional model tissue can be health tissues model or disease model (disease model test kit).

Plate should comprise the array in container or hole, and wherein a plurality of containers contain the sample of the three-dimensional lung model tissue culture of one or more type through engineering approaches in appropriate culture medium.

That container can be is for example flat, container at the bottom of the U-shaped or at the bottom of the preferred V-arrangement, on the plate that allows a plurality of samples of parallel testing.

Container is preferably the container of handling without tissue culture, adheres to wall of container to avoid cell.

In a preferred embodiment, each container comprises single aggregate.In a preferred embodiment, the culture samples in each container comprises the cell like the amount that is defined in the summary of the invention.

Preferably, container distinctly or together seals, and the enrichment CO that contains just like defined in the summary of the invention ₂Environment that is suitable for the lung tissue culture or atmosphere.

Disease model needs equivalent environment usually.

Cell is preferably with being suitable for reporting the biocompatibility dyeing of one or more following cell characteristics: cell state, the for example apoptosis or the moribund condition of phase (cell phase), cell viability, cell during cell fission; Cell type; Cell position; Vicious transformation; Inflammation.

Contrast

As control sample, test kit contains only epithelial cell and fibroblastic culture.Onboard, preferred at least 3-3 holes contrasts of existence (being respectively epithelial cell and inoblast).

As another contrast of two-dimentional lung tissue preferably in order to differentiate or assessment three-dimensional tissue specific characteristics.

Therefore, when needing, can comprise that two-dimentional control board (preferred flat adhesion tissue culturing plate) follows three-dimensional tissue.Perhaps, plate also can contain the hole as the two-dimentional lung tissue of contrast, and said tissue is preferably in flat-bottom hole.

Therefore, in one embodiment of the invention, use to contain V-arrangement bottom outlet that is useful on three-dimensional tissue and the plate that is used for the flat of two dimensional tissues or U-shaped bottom outlet.

Instance

Instance 1-materials and methods

Former generation SAEC, NHLF and the HMVEC of lung cell are buied from Long Sha company (Lonza).The all cells type all is that the lung from a plurality of donors at random at different sexes and age separates.Such as manufacturers suggestion, use SAGM, FGM or EGM-2 substratum to carry out the initial amplification of SAEC, NHLF or the HMVEC of lung respectively.All types of cell cultures are all containing 5%CO ₂Atmosphere in cultivate down in 37 ℃.Cultivate for two and three dimensions, pure or mixed cell population is cultivated in the 50%-50% mixture of SAGM (stingy road growth medium, Long Sha company (Lonza)) and complete DMEM.For two kinds of cells and three kinds of cell cultures of containing the HMVEC cell, the suitable growth factor fill-in of HMVEC cell adds in the 50%-50% mixture of SAGM and DMEM.The compsn of cell culture medium is according to the specification sheets preparation of manufacturers.Cultivate for two and three dimensions, cell also is assigned to respectively on flat 6 orifice plates or the 96 hole V-arrangement base plates (Sa Ershitaite (Sarstedt)) with indicated ratio mixed.The V-arrangement base plate was at behind the cell inoculation under room temperature with 600 * g centrifugal immediately 10 minutes.

SAEC and NHLF with following fluorescin dyeing: Dil of science [Huo Nige (and Honig, M.G.) with stop nurse (R.I.Hume) (1989)] with CFSE [king (Wang, X.Q.), section people (2005) such as (X.M.Duan)], it can follow the tracks of the motion of cell in the culturing process.Have or do not have the cell of matrigel inhale to move at the bottom of the V-arrangement of handling without tissue culture in 96 orifice plates, and at CO ₂Cultivated 1 hour down in 37 ℃ in the incubator.After the cultivation, cell is at room temperature with 2000rpm granulation 5 minutes, and then the gained cell mass is cultivated the (5%CO that spends the night ₂, 37 ℃).

1972, Gerard people (1973) such as (D.J.Giard) cultivated through the cancerous lung tissue from 58 years old man being carried out explant, initiative A549 clone.The A549 cell is the human alveolar substrate of a gland cancer epithelial cell.The A549 cell is included into epithelial squamous branch.In above substratum with 2 * 10 ⁴The cell of the concentration of individual cells/square cm inoculation will be in 5 days 100% converges.

The A549 cell can be from American Type Culture Collection (ATCC; Rockville city, the Maryland State (Rockville, MD)) obtains as CCL-185, and can be grown in and have 10% foetal calf serum (FCS; Ji Bu company (GIBCO BRL)) Durham F-12 substratum (Ham ' s F-12medium) (grow in Glan Tokushima city, New York (Grand Island, NY)) or according to supplier's suggestion by Ji Bu company (GIBCO BRL).

1962, from the lung tissue development WI-38 cell system of the TA fetus of taking from about 3 months gestational ages.The cell that the tryptic digestion lung tissue is discharged is used for primary culture.Cellular form is similar to inoblast.Karyotype is 46, XX; The normal diploid women.The maximum life that obtains this culture from Rui Pusituoku (Repository) is 50 population doublings number of times.Reclaim the back and obtain 86% thymidine labeling index.G6PD is the Type B isozyme.This culture of WI-38 is from the 9th generation frozen cell amplification available from the person of presenting.

Can be from American Type Culture Collection (ATCC; Rockville city, the Maryland State (Rockville, MD)) http://www.atcc.org/ATCCAdvancedCatalogSearch/tabid/112/Default .aspx grows according to supplier's suggestion as the WI-38 cell that CCL-75 obtains.

CL13 or CL30 cell (Lip river, Ward people such as (Wardlaw), 2002) cultivation in containing the Dulbecco improvement Iger/F12 substratum of 5% foetal calf serum and 25% mcg/ml qingfengmeisu qiong (gentamicin) (Dulbecco ' s modified Eagles/F12medium).

The human cell preferably maintains contains CO on demand under 37 ℃ ₂Damp atmosphere in.

Fluorescence and confocal microscopy

Before two and three dimensions was cultivated, SAEC, NHLF and HMVEC used fluorescin dyestuff CFSE of science, DiI and DiD (obtaining from molecular probe company (Molecular Probes)) dyeing respectively.Cell is washed twice in PBS, and the concentration with 0.5 μ g/ml was cultivated 10 minutes with CFSE, DiI or DiD under 37 ℃.Through removing excessive dyestuff with the DMEM+10%FCS washed cell.The two and three dimensions culture uses fluorescently-labeled cell, like previous indicated the preparation.After cultivation is spent the night, remove the three-dimensional cell culture from the V-arrangement base plate carefully, and transfer to the bottom in the plate of deckglass (horse Tyke (MatTek)).Through fluorescent microscopy (Olympus (Olympus) IX-81 microscope) or confocal microscopy (Olympus (Olympus) FV1000 confocal imaging system) research lung tissue trace culture.

Cell sorting

According to the specification sheets (molecular probe company (Molecular probes)) of manufacturers, SAEC and NHLF are with CFSE and DiI dyeing.

Cell mixing was also cultivated 72 hours in the two and three dimensions system.Staining cell is through slight trypsin treatment, and then PBS+EDTA handles and dissociates.Dissociated cell uses BD FACSVantage cell sorter to be sorted into to have the pipe (beautiful day Ni biotechnology (Miltenyi Biotech)) of the lysis buffer that is used for preparing mRNA.

Synthetic and the quantitative RT-PCR of cDNA

Total RNA carries out on the post dnase-from the two and three dimensions cell culture through use Niu Sipan (NucleoSpin) RNAII test kit (Mai Xierui-Na Jier (Machery-Nagel)) to digest and prepare.Messenger RNA(mRNA) is SAEC sample use μ MACS mRNA separation system (beautiful day Ni biotechnology (Miltenyi the Biotech)) preparation from sorting.CDNA uses MMuLV reversed transcriptive enzyme test kit (Sai Mo flies science and technology (Thermo Scientific)) preparation from the RNA sample.The real-time quantitative PCR inspection is to use female mixture (A Jiying (ABGene)) of ABsolute QPCR SYBR Green Low ROX and applying biological system 7500 heat circulating systems (Applied Biosystems 7500thermal cycler system) to carry out.Primer is listed in the table 1.

Recombinant adenoviral vector

Fully genes involved or only the GFP sequence use forward (5 ') through the PCR reaction: 5 '--3 ', reverse (3 '): 5 '--3 ' primer sequence amplification, and being cloned in the shuttle vectors, then be cloned in the adenovirus carrier through homologous recombination.(American Type Culture Collection produces in the Maryland State Rockville city (Rockville, MD)) adenovirus to 293 package cell lines through using fat to dye amine 2000 (Lipofectamine 2000) (hero (Invitrogen)) transfection linearization plasmid DNA.The gained bacterial plaque enlarges, and uses adenovirus purification kit (BD bio-science (BD Biosciences) purifying and concentrated adenovirus.

Epithelial adenovirus infection

The adenovirus that contains GFP or genes involved-GFP adds among the SAEC in two dimension or the three-dimensional.At 37 ℃ following 1 * 10 ⁶Individual cell resuspending is in 250 μ l cell culture mediums and 50 μ l viruses and kept 90 minutes.

Table 1

The CXCL-8 analytical method

CXCL-8 (IL-8) content of two and three dimensions cell culture supernatant liquid is measured with Quinn (Quantikine) CXCL-8/IL-8ELISA test kit (peace enlightening biological (R&D Systems)).The sandwich ELISA analytical method is carried out according to the specification sheets of manufacturers.Briefly, the cell culture supernatant liquid sample and the CXCL-8 standard substance of equal dilution are assigned on the hole that scribbles anti-CXCL-8 monoclonal antibody in advance.After at room temperature cultivating 2 hours, use the lavation buffer solution wash plate that provided 4 times.Then add the anti-CXCL-8 antibody of HRPO bonded polyclone, kept 1 hour.Behind the last washing step, tmb substrate solution adds in the hand-hole.Optical density (OD) is measured under 450nm and 570nm with iEMS reader MF (thermoelectric thunder vigorous (Thermo Labsystems)), and uses A Sente software (Ascent software) analytical data.

Data are quantitative

Quantitatively δ Ct (dCt) and relative quantity (RQ) method of real-time RT-PCR data through being proposed like Applied Biosystems, Inc. (Applied Biosystems) uses 7500 system's SDS softwares to analyze.All samples is all set up in duplicate.Briefly, use the automatic threshold level of measuring through 7500 SDS of system softwares (threshold level), measure the Ct value of each sample.δ Ct (dCt) value is confirmed according to following formula: dCt (target gene)=Ct (target gene)-Ct (house-keeping gene).The variation of genetic expression is shown as the RQ value, and said value is used computes:

RQ=2 ^-ddCt, wherein the ddCt value is pressed ddCt=dCt (sample)-dCt (reference sample) calculating.

Through comparing OD and the typical curve that calculates by 7 kinds of different concns in 31.2-2000pg/ml CXCL-8 scope, confirm the CXCL-8 content of cell culture supernatant liquid.Sample distributes duplicate and MV is used for further data analysis.

Instance 2-develops the experiment of three-dimensional lung tissue model

The hanging drop model

Be the simulating human lung structure, since the three-dimensional cell aggregate of 100000 cells, these cells contain roughly different inoblasts (NHLF) and stingy tract epithelial cell colony (SAEC), the intermingling at random of equivalent.In one day that cultivates with the hanging drop analytical method, cell produces sparse weave construction.Fig. 1 shows the hanging drop culture of 50%SAEC and 50%NHLF mixture.

Yet this formation is unstable, and can not be under the situation that weave construction is not caused the infringement that can't save with the microcomponent that produces from initial incubation jump if not to another test panel.

The granulation model that contains matrigel

For improving the stability of blended lung microcomponent, the SAEC and the NHLF of 1: 1 ratio of granulation also grows it in the presence of matrigel.Many three-dimensional lungs use matrigel to set up with other organize models can make various cell type growths and three-dimensional structure interact with each other.SAEC and NHLF dye with CFSE (king (Wang), section people such as (Duan), 2005) with the fluorescin that can follow the tracks of cell movement in culturing process dyestuff Dil of science (Huo Nige (Honig) with stop nurse (Hume), 1989).Cell and matrigel are inhaled and are moved into without in 96 orifice plates at the bottom of the V-arrangement of tissue culture processing, and place 37 ℃ of following CO ₂In the incubator 1 hour.After the cultivation, cell is at room temperature with 2000rpm granulation 5 minutes, and then the gained cell mass is cultivated the (5%CO that spends the night ₂, 37 ℃).

Fig. 2 shows the culture based on matrigel of 50%SAEC and 50%NHLF mixture.Clearly, although there is matrigel, SAEC and NHLF can not form stable three-dimensional structure.In addition, as if mainly contain epithelial pellet shapes weave construction exiting tissue/matrigel block.

Instance 3-does not have the granulation model that skeleton is organized in manual work

As the three-dimensional cultivation condition of simulating human lung next step, under the situation of no matrigel, divide the epithelial cell (SAEC) of two stage granulation equal number and the random mixture of inoblast (NHLF).Cell is inhaled and is moved into without in 96 orifice plates at the bottom of the V-arrangement of tissue culture processing, and places 37 ℃ of following CO ₂In the incubator 1 hour.After the cultivation, cell is at room temperature with 2000rpm granulation 5 minutes, and then the gained cell mass is cultivated the (5%CO that spends the night ₂, 37 ℃).Cell before the mixed culture uses physiology fluorescent dye Dil (1mg/ml stoste among the DMSO) and CFSE (1mg/ml stoste among the DMSO) the diluent dyeing in 1: 1000 in PBS (phosphate buffered saline, pH 7.2).Under these culture condition, cell forms stable aggregate, and wherein the most sticking inoblast (redness or lead) is centered on (Fig. 3) by not too sticking epithelial cell cell type (green or light gray).The aggregate diameter is about 200 μ m.

In another experiment, in an organized way change the ratio of SAEC and NHLF cell, and use following SAEC respectively: the NHLF ratio prepares culture: 0%/100%; 25%/75%, 50%/50%, 75%/25%, 100%/0%, others as stated.Fig. 3 demonstration contains the SAEC of different ratios and the granulation microcomponent culture of NHLF.As scheme proof; When the epithelial cell of using equivalent and inoblast, form significantly three-dimensional structure aggregate with superficial epithelium cell lining, aggregate has clearly epithelial cell lining under 25%/75% ratio; But smaller and form is not too convincing; And when the SAEC cell is excessive, promptly when cell epithelial cell and fibroblastic ratio are 75%/25%, form much smooth epithelial cell lining.Pure growth do not form aggregate (epithelial cell) or the aggregate size of three-dimensional structure little many (inoblasts).

The sign of the three-dimensional lung tissue model of two kinds of cell types of instance 4-inorganization skeleton

The differentiation marker thing

The characterization of molecules of model is to use the PCR in real time analysis, based on epithelial cell differentiation marker thing.MRNA is from the cell aggregation purifying and produce cDNA.Use TTF1 (Fig. 4 a), AQ3 (Fig. 4 b) and AQ5 Auele Specific Primer, with respect to beta-actin analytical results as internal contrast.

Fig. 4 a. indicates the mRNA content of TTF-1 in the three-dimensional human lung microcomponent.The TTF1 transcription factor is the signature thing of alveolar epithelial cells.Do not express though three-dimensional fibroblast cell cultures shows TTF1, TTF1 is present in the D S AEC monoculture and in two-dimentional SAEC/NHLF coculture to be increased, and shows fibroblastic beneficial effect.TT1 reaches high expression level amount in D S AEC/NHLF tissue.

Fig. 4 b. shows the mRNA content of AQP-3 water translocator in the three-dimensional human lung microcomponent.AQ3 is an ATII epithelial cell type mark thing in the lung.Though showing AQ3, three-dimensional fibroblast cell cultures do not express; AQ3 is present in the D S AEC monoculture and in two-dimentional SAEC/NHLF coculture to be increased; Show fibroblastic beneficial effect, but in D S AEC/NHLF tissue culture, still observe the most high-load AQ3.

Therefore, above affinity tag shows the increase of ATII type induced differentiation property, and this increase can be expressed not increase further and confirm by ATI phenotypic marker thing.

The differentiated cell types of the purifying that is used for testing obtains from commercial source.

Though these cell types derive from differentiated tissue, in case its purifying and remaining under the two-dimentional culture condition then increases indicatedly like S100A4 content, cell almost shows immediately and dedifferentes sign (Fig. 6 and table 2).In case SAEC and NHLF cultivate altogether, then S100A4 and N-cadherin content significantly reduce, and E-cadherin content increases." cadherin conversion " [this Burger (Zeisberg M) and Nelson (E.G.Neilson) (2009) now] be remarkable (Fig. 6) in three-dimensional cultivation condition than in two-dimentional culture condition, shows that three-dimensional structure also need reduce SAEC and dedifferente except there being NHLF.

These change also is that epithelial cell-mesenchymal cell changes the characteristic of (EMT), characterizes epithelial cell and dedifferentes process.

The expression of pro-inflammatory cytokine

Like pulmonary infection or alveolar epithelial cells damage (epithelial cell damage layer continuously) institute's initiations, pro-inflammatory cytokine is comprised the inflammatory cells of neutrophil leucocyte with attraction by the alveolar epithelium generation.Be the expression of test CXCL-8 pro-inflammatory cytokine, by the cell conditioned medium liquid test CXCL-8 protein content of two-dimentional monoculture and coculture and three-dimensional tissue's coculture (setting is found among Fig. 3).Use commercially available ELISA test kit (peace enlightening laboratory (R&DLaboratories)); Obviously compared to the two-dimentional tissue culture of routine; The CXCL-8 expression amount significantly reduces (Fig. 7 .a) in the system of three-dimensional tissue; And under the highest situation of epithelial cell ratio, detect minimum content, show that the interruption through epithelium layer causes the CXCL-8 expression.

On Fig. 7 .a graphic, show the CXCL-8 secretion in the human in vitro three-dimensional lung model.Find that fibroblastic three-dimensional monoculture do not secrete CXCL-8, and D S AEC still produce cytokine (but than the degree of two-dimentional SAEC culture low-data not shown).The two dimension coculture does not significantly change the expression of CXCL-8, shows that fibroblastic existence can't influence cytokine-expressing.The expression amount of CXCL-8 is significantly to reduce in 75%/25% the system of three-dimensional tissue at epithelial cell-inoblast ratio; Epithelial cell covers the inoblast ball basically fully in said system, shows that the CXCL-8 expression can cause through the interruption of alveolar epithelial cells layer.Said CXCL-8 expresses is reduced in 50%/50% and 25%/75% ratio and has a few not too remarkable down.

Inflammatory cytokine IL-1 β and IL-6mRNA content also use quantitative real-time RT-PCR analysis to study.When the mixture to SAEC-NHLF mRNA carried out PCR, in the dimensional culture thing, compared to two-dimentional culture out of the ordinary, IL-1 β and IL-6mRNA content were significantly reduced (being respectively Fig. 7 B and 7C).In case the SAEC cell sub-elects from two and three dimensions NHLF coculture, among the SAEC that then under three-dimensional condition, cultivates the expression decreased of IL-1 β and IL-6 remarkable many (Fig. 7 D).

Instance 5-has three kinds of cell type models of epithelial cell, endotheliocyte and inoblast component

Be further to improve the complicacy of lung tissue culture model, human pulmonary CMECs of former generation (HMVEC) add in SAEC and the NHLF cell, and are similar to SAEC and NHLF coculture and set up two and three dimensions and organize micro-culture.

Show through the morphological examination of fluorescence and confocal microscopy microcomponent, HMVEC can be successfully under three-dimensional condition with SAEC and the common cultivation of NHLF cell (Fig. 8 .a).Enjoyably, with the coculture of SAEC or NHLF in, HMVEC forms the interior solid core of micro-culture.When three kinds of cell types (1: 1: 1) were cultivated under three-dimensional condition together, it sticked together and forms the three-dimensional structure (Fig. 8 .b) of obvious form compact and stable.

The characterization of molecules of three kinds of cell cultures shows that compared to measured under two-dimentional culture condition, the expression amount of AQP3 and KRT7 obviously increases (Fig. 9 .a and table 2) in the dimensional culture thing.When cell remained in the two-dimentional culture, the content of EMT affinity tag S100A4 and N-cad increased, but stable in the dimensional culture thing.The slight minimizing of E-cad is less than detected minimizing under the two-dimentional culture condition (Fig. 9 .a and table 2) in the dimensional culture thing.Yet, when the mixing mRNA to three kinds of cell types carries out quantitative RT-PCR, need further to analyze three kinds of cell type dimensional culture things, to find to help to carry out the optimum proportion of three kinds of cell types of histological structure from the tissue element of purifying.

Table 2 shows the changes in gene expression of human former generation pneumonocyte culture.Number is the RQ value, according to formula RQ=2 ^-ddCtCalculate, wherein the ddCt value is pressed ddCt=dCt (sample)-dCt (reference sample) calculating.SAEC except sorting ^*, wherein data appear with the dCt value, calculate as follows: dCt=Ct (target gene)-Ct (house-keeping gene).The cell that a part is collected before setting up various cultures is all the time as reference sample.δ Ct (dCt) value is calculated as follows: dCt (target gene)=Ct (target gene)-Ct (house-keeping gene).The 18S ribosome-RNA(rRNA) is as house-keeping gene, except the SAEC of sorting ^*The situation of sample, wherein Actin muscle is as house-keeping gene.

Abbreviation: N.A.: data are unavailable, the expression amount undetermined.N.D.: do not detect specific PCR product with real-time QPCR.N.D. ^*: do not detect specific PCR product with conventional PCR.N.C.: use real-time QPCR, the particular expression amount does not measure thereupon in parallel hole or the sample.Low ^*: detect relatively low expression amount through conventional PCR.High ^*: detect higher relatively expression amount with conventional PCR.

The cell marker of emiocytosis and the result of factor general introduction

Here, the proof of producing evidence, former generation lung's epithelial cell cultivate than more help in two dimension or the three-dimensional in vitro monoculture SAEC altogether with fibroblastic three-dimensional and keep and more the state that breaks up more.Comprise that NHLF not only promotes epithelial cell differentiation, and compared to SAEC (Fig. 3) or SAEC-HMVEC (Fig. 8 .a) culture only, the adhesion of three-dimensional microcomponent and firm in structure much fine and close.

The TTF1 transcription factor be during the fetal development with birth back ATII cell in the characteristic universal marker of alveolar epithelial cells.Cytokeratin is the component of the intermediate filament of cytoskeleton, and its expression pattern is important in cell lineage is differentiated.In experiment, pulmonary epithelial cells affinity tag TTF-1 and cytokeratin 7KRT7 are presented at expression amount rising in the three-dimensional coculture.

II type pneumonocyte promote water through epithelium motion (through aquaporin (AQP) family member).ATII affinity tag aquaporin 3 is presented at inoblast and has content rising (Fig. 5) down.The secretion surfactant protein is the specific characteristic of ATII pulmonary epithelial cells.[Du Busi (Dobbs, L.G.) (1989), Ai Erken (Alcorn; People (1997) such as J.L.)] in experiment; SAEC cell in the monoculture fails to express surfactant protein, and surfactant protein A1mRNA thereby in three-dimensional, express, and amount is less than (Fig. 5 .c) in the two-dimentional coculture.Yet, do not detect surfactant protein B and C (data not shown) in the three-dimensional coculture system thereupon.Also check the expression of ATI affinity tag aquaporin 4 in two kinds of cell cultures and the three kinds of cell cultures and 5, but the expression of these molecules do not detect thereupon, estimation possibly be because cell used herein (SAEC) is the ATII type.In addition, the ATI differentiation needs the plenty of time (data not shown).During a little long incubation time, if use the cell with ATI type characteristic, so the ATI affinity tag will appear.

Therefore, except that SFPC, differentiation marker thing AQP3, KRT7, TTF1 and SFPA raise in the presence of inoblast.The content of AQP3 and SFTPA but not KRT7 or TTF1 differentiation marker thing further increases under three-dimensional cultivation condition.

S100A4 is that epithelial cell-mesenchymal cell transforms well-known molecular marked compound, and its expression amount is usually higher in metastatic carcinoma [Xiu Erbeite (Sherbet G.V) waits the people, 2009] and pulmonary fibrosis [gal Reno (Guarino M.) waits people, 2009].S100A4 and N-cadherin raise and the parallel downward modulation of E-cadherin [this Burger (Zeisberg M) and Nelson (E.G.Neilson) now; (2009); Sai Ke (Seike; People (2009) such as M.)] also be that epithelial cell-mesenchymal cell transforms the characteristic of (EMT), it characterizes, and epithelial cell dedifferentes process and characteristic is that forfeiture cell adhesion, E-cadherin expression by inhibitation system and cell movement increase.

Dedifferente affinity tag S100A4 and the N-cadherin appears in the primary cell of purifying under the two-dimentional culture condition.

More than dedifferenting affinity tag is reducing in the presence of the inoblast and under three-dimensional condition, is further reducing.

Compared to two-dimentional culture, can in the dimensional culture thing, observe the inflammatory affinity tag that comprises IL1b, IL6 and CXCL8 and reduce.Said affinity tag is significantly downward modulation under three-dimensional cultivation condition; Only exist inoblast to be not enough to reduce its content in the for example two-dimentional culture.

In two kinds of cell cultures, observe SFTPA1 and express, but in three kinds of cell cultures, do not observe (Fig. 5 .c and data not shown).

Instance 6-disease model

Lung tumor model from lung tumor cell system

Described in instance 3, prepare artificial three-dimensional lung tissue culture, change as follows.

Replace epithelial cell (SAEC); Use derives from the II type alveolar epithelial cells (A549) and 5%-20%CL13 or CL30 cell (Lip river, Ward people such as (Wardlaw) of the A/J mouse (adenocarcinoma of lung model [Bie Linsiji people such as (Belinsky) (1992)]) of handling through NNK [4-(methyl nitrosamino-)-1-(3-pyridine)-1-butanone]; Molecular pharmacology (Molecular Pharmacology) 62, combination 326-3332002).CL13 or CL30 cell carry the sudden change of Ki-ras gene.

Carry out a series of experiments, seek the adequate rate of A549 cell and CL13 or CL30 cell.Ratio during the spontaneous formation of tumour is in order to the three-dimensional lung model tissue of preparation as the lung tumor disease model.

In an alternative method of above method, use the patient tumors cell.

Use composition genetically modified and EF in the clone change primary cell aggregate of sublethal exposure

The all cells type all infects, so the same normal in genetic expression and any set three-dimensional lung tissue model.Yet; The cell of aggregate is formed cell (5%-10% of the whole cell numbers of the aggregate)-inoblast (WI-38) or the alveolar epithelial cells (A549) that contain through sublethal exposure, and these cells are genetically modified and produce the EF (Wnt-s, bone morphogenetic protein (BMP)-s, inflammatory and pro-inflammatory cytokine, growth factor etc.) that changes aggregate inner cell microenvironment.

Natural three-dimensional lung cells aggregate

For simulation inflammation symptom, handle natural three-dimensional lung cells aggregate with the bacterial cell wall extracts of various concentration.Use cytokine specific ELISA technology from tissue culture based assays production of cytokines, the changes in gene expression of epithelial cell and inoblast can be quantitative through real-time PCR reactions.

The three-dimensional lung tissue test kit of instance 7-

In this example, the subsequent use 3C lung tissue test kit of preparation test with feature:

1. testing subsequent use lung tissue is transmitted in 96 orifice plates.

2. the lung model that is ready for use on experiment or test organizes little (80000 cells/well) sample to be present in the hole.

3. each is organized all and is made up of mankind's alveolar epithelial cells of former generation and fibroblastic mixed culture (being respectively 25%, 50% and 75% epithelial cell).

4. plate contains 3-3 holes contrasts (only epithelial cell and inoblast).

5. plate is for example used Sa Lanapu (Saranrap) sealing with plastics film transparent, preferred adhesion.

The tissue quality guaranteed 3 days, comprise be delivered in.

Plate is certainly as the non-adhesion in 96 holes tissue culturing plate at the bottom of the V-arrangement.

Model is organized preparation described in the example 3 strictly according to the facts.Each is organized and all is immersed in the 200 μ l tissue culture medium (TCM)s, and to the lung culture, the best is at 5%CO ₂In the environment, sealing, and at room temperature or on ice transmit.

Quality control: take out a tissue and test vigor from each hole.Through PCR in real time test differentiation marker thing.

When needing, can comprise that two-dimentional control board (be organized in the flat adhesion tissue culturing plate of 96 holes and grow) follows three-dimensional tissue.

Industrial applicability

Hereinbefore, set up the basic parameter and the culture condition of the ATII type lung model of inorganization skeleton, wherein can study the spontaneous self-assembly of cell and the interaction of cell.In theory and application research and in the medicine test, said model is handled and genetic manipulation the system of complex organization easily.Said model also easily through comprise be used for angiopoietic endotheliocyte with in addition other cell type of smooth muscle cell enlarge, wherein can study the interaction of other mutual tissue and cell.

In theory or applied research and in the medicine test, exoskeletal dimensional culture allows system of complex organization simply or is more carried out trouble-free genetic manipulation.This lung tissue's model is particularly suited for studying the spontaneous self-assembly and the cell interaction of cell.Biomedical research and pharmaceuticals all need in vitro, and model improves the toxicity of predicting candidate drug molecule in the preclinical phase or the validity of effect.The policy of [Wayne Kramer people such as (Kramer J.A.), 2007] new settings is also stressed will replace animal model and urge the new clinical Pretesting method of development.[the innovation preliminary strategic research agenda of medical research (Innovative Medicine Research Initiative Strategic Research Agenda) .2008, European technology platform (European Technology Platform).]

Based on above model and use range gene transmission method and variable target gene, the gene that microcomponent of three-dimensional human lung model system is suitable for studying during the pulmonary disorder easily changes, thereby can differentiate the therapy that new medicine target is new with development.These disease models can comprise inflammation model, tumor model, pulmonary fibrosis model or regenerating model.

The three-dimensional model of the lung tissue that can secure good health and diseased tissue.According to product of the present invention can for example be tissue culture, the form that comprises plate or the array or the test kit of said culture sells.

Reference

Claims

1. the three-dimensional lung model tissue culture of a through engineering approaches, wherein said model tissue culture

A) do not contain the artificial organ skeleton, wherein said artificial organ skeleton is a porous three-dimensional matrix; Three dimensional gel matrix or porous-film upholder,

B) constitute by culturing cell,

C) comprise lung's epithelial cell and mesenchymal cell at least, be preferably fibrocyte,

D) have the form of one or more cell aggregations, the said lung of the surface enrichment of wherein said aggregate epithelial cell,

With

2. the three-dimensional lung model tissue culture of through engineering approaches according to claim 1, wherein

The size of said one or more aggregates or mean sizes are at least 80 μ m and 600 μ m at the most, the size of preferred said aggregate or mean sizes between 100 μ m and 300 μ m, and/or

The amount of the cell that said one or more aggregates comprise does

At least 2 * 10 ⁴Individual cell, preferably at least 4 * 10 ⁴Individual cell and

At the most 5 * 10 ⁵Individual cell, preferably at the most 2 * 10 ⁵Individual or at the most 10 ⁵Individual cell.

3. the three-dimensional lung model tissue culture of through engineering approaches according to claim 1 and 2, wherein

Said model tissue culture do not contain any artificial organ skeleton and/or

Said model tissue culture further comprises extracellular matrix, and its extracellular matrix protein is at least a cell type that comprised in the said tissue culture, it is secreted to be preferably said inoblast, and/or

Epithelial cell forms lung's epithelial cell lining at least a portion on the surface of said aggregate, preferred said lung epithelial cell lining at least partly covers the surface of said aggregate.

4. according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the claim 1 to 3, wherein

Said model tissue culture comprises from the primary cell of the cultivation of individuality acquisition, and/or

Said model tissue culture is included in the cell that dedifferentes and when cultivating, break up again before the cultivation, and/or

Said model tissue culture comprises the cell of establishing clone.

5. according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the aforementioned claim, it comprises endotheliocyte in addition.

6. according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the aforementioned claim, wherein said lung epithelial cell comprises at least a following cell type:

I type pneumonocyte (ATI cell),

II type pneumonocyte (ATII cell),

The expressed said epithelium differentiation marker thing of said histocyte of the three-dimensional lung model tissue of wherein said through engineering approaches is preferably selected from following group:

ATII type differentiation marker thing,

ATI type differentiation marker thing.

7. according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the aforementioned claim, wherein

The content of following affinity tag increases with respect to the contrast of the primary cell that comprises non-culturing cell, preferred purifying: TTF1, AQP3, SFTPA, SFTPC, KRT7,

The content of following affinity tag reduces with respect to the contrast that comprises non-culturing cell: CXCL8IL1b, S100A4,

The content of following affinity tag increases with respect to the two-dimentional culture of contrast: AQP3, SFTPA,

The content of following affinity tag reduces with respect to the two-dimentional culture of contrast: CXCL8, IL1b, IL6, S100A4, N-cadherin.

8. according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the aforementioned claim, wherein

Said culturing cell, preferred said lung epithelial cell and/or mesenchymal cell comprise the influenced cell of the pathological characters with diseased lung tissue, make that said model tissue culture is a pulmonary disorder model tissue culture,

Preferably

Wherein said disease comprises inflammation, and said influenced cell expressing surpasses the inflammatory cytokine of normal contents, and said model is the inflammation model,

Wherein said disease is a tumour, and said cell for transform, for example vicious transformation or immortality cell, and said model is tumor model,

Wherein said disease comprises fibrosis, and said model is the fibrosis model,

Wherein said disease comprises the damage of said tissue, and said model is a regenerating model.

9. the method for the three-dimensional lung model tissue culture of a preparation engineeringization, said method comprises following steps:

Prepare at least lung's epithelial cell and mesenchymal cell, be preferably fibrocyte, more preferably former generation fibroblastic mixing suspension,

The cell of the said suspension-s of granulation,

The said cell of granulation,

At CO ₂Cultivating said granulation suspension-s and maintenance under existing is enough to make said cell to form the time of the three-dimensional lung model tissue that comprises one or more cell aggregations,

The said model tissue of optional analysis

A) expression of peculiar one or more epithelium differentiation marker things of model tissue culture, and compared to reference to culture, the expression amount increase is considered to indication and forms three-dimensional lung model tissue culture; And/or

B) one or more as above expression of defined pro-inflammatory cytokine, and compared to being fit to reference to culture (as above define), expression amount reduce and are considered to indicate the three-dimensional lung model tissue culture of formation,

C) size of said one or more aggregates and form.

10. method according to claim 9, wherein

The mean sizes of said cell aggregation is at least 80 μ m and 600 μ m at the most, preferably between 100 μ m and 300 μ m, and/or

The amount of the cell that said one or more aggregates comprise does

11. according to claim 9 or 10 described methods, wherein

Said granulation suspension-s is at CO ₂Exist down and cultivate,

Keep being no more than for 2 weeks, the period and the maintenance that preferably are no more than 12 days, 10 days, 8 days, 7 days, 6 days or 5 days are no less than 10 hours, preferably are no less than the period of 12 hours or 14 hours.

12. according to the described method of arbitrary claim in the claim 9 to 11; Wherein when the preparation mixing suspension; The cell of one or more types is in 18 hours, in preferred 12 hours, more preferably in 4 hours; In extremely preferred 1 hour or add simultaneously in the container, the extremely preferred used cell of each type all adds in said period.

13. according to the described method of arbitrary claim in the claim 9 to 12,

Wherein said container is without container at the bottom of the V-arrangement of tissue culture processing, and/or

Wherein granulation was carried out 1 minute to 20 minutes under 600g at 200g, and preferred 2 minutes to 10 minutes, and/or

Wherein said cell is with the biocompatibility dyeing that is suitable for reporting one or more following cell characteristics: cell state, the for example apoptosis or the moribund condition of phase, cell viability, cell during cell fission; Cell type; Cell position; Vicious transformation; Inflammation.

14. according to the described method of arbitrary claim in the claim 9 to 13, wherein

The mixing suspension of the said epithelial cell of lung at least and lung's mesenchymal cell comprises from the primary cell of the cultivation of individuality acquisition, and/or

The mixing suspension of the said epithelial cell of lung at least and lung's mesenchymal cell is included in the cell and the said cell that dedifferente before the cultivation to be broken up when cultivating again, and/or

The mixing suspension of the said epithelial cell of lung at least and lung's mesenchymal cell comprises the cell of establishing clone.

15. according to the described method of arbitrary claim in the claim 9 to 14, wherein

Said culturing cell, preferred said lung epithelial cell and/or mesenchymal cell comprise the influenced cell of the pathological characters with diseased lung tissue, make that said model tissue culture is a pulmonary disorder model tissue culture.

16. preferably according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the claim 1 to 7, it can be through obtaining according to the arbitrary method in the described method of arbitrary claim in the claim 9 to 15.

17. a method that is directed against medicine to the effect screening of medicaments of lung tissue, said method comprises following steps:

Provide according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the aforementioned claim,

Get at least one specimen and the reference sample of said model tissue culture,

Said specimen is contacted with medicine, keeps said specimen and said reference sample simultaneously under the same conditions,

Compare with said reference sample, detect any variation or the change of said specimen,

If wherein detect any variation or the change of said specimen, think to indicate the effect of said medicine so.

18. method according to claim 17; Wherein said model tissue culture is the undesirable action of healthy lung tissue model and testing drug, wherein the deleterious variation of the cell of said specimen or change is considered to the poisonous or undesirable action of said medicine.

19. method according to claim 17, wherein said model tissue culture are to comprise the pulmonary disorder model tissue culture of the influenced cell with pathological characters and the beneficial effect of testing drug, wherein

The analysis of said pathological characters is measured or assessed to said model tissue culture, measure to obtain disease,

The reference sample (healthy reference sample) of healthy lung tissue and/or the reference sample (ill reference sample) of diseased lung tissue are provided,

In said healthy reference sample and/or said ill reference sample, and in said at least a specimen, contact front and back with said medicine, measure or assess said pathological characters, wherein at it

Disease described in the said specimen is measured shift to disease described in the said healthy reference sample measure with or the said specimen measured away from disease described in the said ill reference sample in any variation or the change beneficial effect that is considered to said medicine.

20. the three-dimensional lung model of through engineering approaches is organized test kit, it comprises the test panel with vessel array, and wherein at least two containers contain

The sample according to the three-dimensional lung model tissue culture of the described through engineering approaches of arbitrary claim in the aforementioned claim of one or more types, each sample is put into the container out of the ordinary of said plate,

Supply the appropriate culture medium of the cell of the said model tissue culture of cultivation.

21. the three-dimensional lung model of through engineering approaches according to claim 20 is organized test kit, it has one or more feature:

Wherein said plate is 96 orifice plates,

Wherein said plate is the V-arrangement base plate,

The amount of the cell that culture samples comprises described in each container does

At least 10 ³Individual, preferably at least 10 ⁴Individual, more preferably at least 2 * 10 ⁴Individual, 3 * 10 ⁴Individual, 4 * 10 ⁴Individual, 5 * 10 ⁴Individual cell and

At the most 10 ⁷Individual, preferably at the most 10 ⁶Individual, more preferably at the most 5 * 10 ⁵Individual, 4 * 10 ⁵Individual, 3 * 10 ⁵Individual, 2 * 10 ⁵Individual or at the most 10 ⁵Individual cell,

Wherein said container distinctly or together seals, and contains the enrichment CO that is suitable for the lung tissue culture ₂Environment or atmosphere,

Wherein said enrichment CO ₂Environment or atmosphere comprise

At least 2%, 3%, 4%CO ₂Environment,

At the most 10%, 9%, 8% or 7%CO ₂Environment,

Extremely preferably about 5%CO ₂

22. the purposes according to the described three-dimensional lung model tissue culture of arbitrary claim in the claim 1 to 7, it is used for

To the effect of compound to lung tissue, the parllel screening compound, for example toxotest, seek candidate compound etc.,

To the treatment potentiality of compound, compound is carried out the patient-specific test, as long as said model tissue culture is to obtain from former generation patient lung cells,

Study the interaction of cell in the said lung tissue,

Gene variation during the research pulmonary disorder etc.