JP2013504303A - Lung tissue model - Google Patents

Abstract

The present invention provides an artificial tissue scaffold-free three-dimensional lung model tissue culture that does not include an artificial scaffold. Three-dimensional models of healthy lung tissue and diseased tissue are available. The products according to the invention can be marketed, for example, in the form of tissue cultures, plates or arrays containing such cultures, or kits. The present invention is applicable for testing the effects of compounds on lung tissue, in medical and scientific studies for screening, testing and / or evaluating drugs, and in the diagnosis of lung diseases.
[Selection figure] None

Description

  The present invention relates to an artificial three-dimensional lung model tissue culture that does not contain an artificial scaffold. Includes 3D models of healthy lung tissue and diseased tissue. The products according to the invention can be marketed, for example, in the form of tissue cultures, plates or arrays containing such cultures, or kits. The present invention can be applied to test for the effect of a compound on lung tissue, in medical and scientific studies for screening, testing and / or evaluating drugs, and in some cases in the diagnosis of lung disease.

  Tissue engineering is a rapidly developing field of biomedical research that aims to repair, replace, or regenerate damaged tissue. Because of the recent failure of Phase II clinical drug trials (eg, TGN1412), additional objectives of tissue engineering include the use of human tissue models for safety and efficacy testing of pharmaceutical compounds. Production is included. Tissue engineering in general and in our model specifically utilizes biological morphogenesis, which is an example of self-assembly.

  Cell engineering and tissue engineering are now being pursued from several perspectives. In one aspect, it is used to gain deeper insight into cell biology and physiology.

  In tissue engineering research, two main directions have been developed: tissue scaffold-based systems and tissue scaffold-free systems. While scaffold-based systems in most cases provide artificial 3D structures using biodegradable scaffold materials to facilitate cell interaction, scaffold-free systems are directly Cell-cell interaction and allows the cell to grow on the scaffold material secreted by the cell in the model.

  Patent Document 1 “Three dimensional in vitro model of human prenoplastic breast disease” discloses a three-dimensional in vitro cell culture system useful as a model of precancerous breast disease for screening drugs. The model is by co-culturing pre-cancerous epithelial cells, endothelial cells and breast fibroblasts of breast origin on reconstituted basement membranes in media containing additional additives such as growth factors, estrogens, etc. Prepared. Therefore, in this solution, the basement membrane, preferably Matrigel®, is used as the tissue scaffold. According to the description, in this system, a network of branched vesicular unit vasculature is formed within about 3-7 days.

  This system is about the breast, not the lung, and there is no suggestion to make a lung culture in that way.

  In the disclosure of Patent Document 2 “Engineered animal tissue”, microvascular endothelial cells were obtained from an adult lung and placed between two layers of human dermal fibroblasts present in a three-dimensional collagen gel. As a result, a sandwich structure was formed.

  Vascular endothelial cells were obtained from human lungs, but the artificial tissue prepared by this method was similar to human skin. Therefore, in practice, it cannot be regarded as a lung tissue model.

  In US Pat. No. 6,099,059, “Fetal plumbly cells and uses thereof” teach 3D tissue-like preparations based on epithelial cells, endothelial cells and stromal cells of fetal mice. The authors used mouse embryonic lung cells for preparation to obtain an artificial lung and screened for 3D tissue-like preparations. Appropriate cell-cell interactions were established using Matrigel ™ hydrogel as the matrix. From the description, it seems that when the epithelial cells and fibroblasts were co-cultured, abnormal proliferation of fibroblasts occurred.

  Patent document 4 “Engineered Lung tissue construction for high throughput toxity screening and drug discovery” includes a fetal lung cell, a tissue scaffold comprising a lung tissue, a biocompatible material and a tissue scaffold. Related to things. Fetal lung cells include epithelial cells, endothelial cells and stromal cells. Several applicable biocompatible materials are listed.

  Patent Document 5 “Replication of biological tissue” proposes the preparation of an artificial 3D tissue in a microgravity environment. The tissue is based on human breast cancer cells and is useful as a breast cancer model. In a rotating bioreactor chamber, connective tissue cells are first cultured until a 3D spheroidal structure is formed, and then endothelial and epithelial cells are sequentially added to the culture. It should be noted that this teaching is theoretical and provides a protocol for culturing cells and a protocol for processing cultures, but does not disclose actual experimental results.

  Non-Patent Document 1 was grown as a conventional monolayer (ML) in a culture flask and as a 3D culture in a rotating wall vessel, using a well-documented normal immortalized lung cell line. . A comparison of the differentiation and presence of marker expression between these cultures and control tissues taken from surgical patient specimens was studied. Our goal was to develop both conventional monolayer and 3D cell cultures of known cell lines.

  Recently, Kelly BeruBe and colleagues have developed a 3D tissue engineering model of human lung epithelium for safety testing. They used normal human bronchial epithelial cells [NHBE]. Primary cells obtained from humans were grown on microporous membranes to form three-dimensional (3D) cell cultures. 3D cultures formed tight junctions between cells, ie, cells with active cilia and cells that produce and secrete mucus. These features were very similar to those seen in native human respiratory epithelial tissue and were thought to accurately mimic the human response to tissue damage. This culture actually forms a thick cell layer that forms the mucosal surface (2).

  Despite a wealth of literature on lung models, the prior art only discloses a tissue scaffold-based three-dimensional model, and includes simple tissue scaffold-free lung tissue that includes at least epithelial cells and fibroblasts The model does not appear to be disclosed in the prior art.

  Surprisingly, the inventors have quickly developed a tissue scaffold-free lung tissue model system that, in some aspects, is advantageous over two-dimensional systems or matrix-based systems by simple biochemical methods. I found out that it can be produced. The present invention provides a ready-to-use model organization for various tests. This model is suitable for studying cell-cell interactions in various lung tissues to mimic normal function and disease progression.

US2001 / 0055804A1 US2003 / 0109920 US2008 / 0112890 WO2008 / 100555 WO2004 / 046322

Vertrees, RA, Zwischenberger, JB et al. 2008 Hughes Tracy et al. 2007 Nichols, J. et al. E. and Cortiella J. et al. 2008 Lonza Venders, S.M. p. r. l. Pare Industry de Petit-Rechain B-4800 Venders, Belgium; Biocenter Ltd. , Temsvari krt. 62 H-6726 Szeged, Invitrogen Corporation (Life Technologies Corporation 5791 Van Allen Way, Carlsbad, California 92008 USA) Zhang, Snappenbeck et al. 2005 Hare, K .; J. et al. et al. 1999 Neagu 2006 De Moerloze, Spencer-Dene et al. 2000 Crapo et al, 1982 Nakahata and Toru 2002; Austin and Boyce, 2001 American Type Culture Collection (ATCC; Rockville, MD) Wang, XQ, Li, H et al. 2009 Honig, M .; G. and R.R. I. Hume 1989 Wang, X .; Q. , X. M.M. Duan, et al. 2005 D. J. et al. Giard et al. 1973 http: // www. atcc. org / ATCCAdvancedCatalogSearch / tabid / 112 / Default. aspx American Type Culture Collection (ATCC; Rockville, MD) Wardlaw et al. 2002 Honig and Hume 1989 CFSE Wang, Duan et al. 2005 "Cadherin-switch" Zeisberg M and E.M. G. Neilson 2009 Dobbs, L .; G. 1989 Alcorn, J .; L. , Et al. 1997 Sherbet, G .; V et al. 2009 Guarino, M .; et al. 2009 Zeisberg, M .; and E.E. G. Neilson, 2009 Seike, M .; , Et al. 2009 Belinsky et al. 1992 Wardlaw et al, Molecular Pharmacology, 62, 326-333 2002 Kramer, J .; A. et al. 2007 Innovative Medicine Research Initiative Strategic Research Agenda. 2008, European Technology Platform Alcorn, J .; L. , Et al. , (1997). Primary Cell Culture of Human Type II Pneumonocycles: Maintenance of a Differentiated Phenotype and Transfection with Recombinant Adenovirus. Am. J. et al. Respir. Cell Mol. Biol, 17 (6): p. 672-682 Belinsky et al. , (1992). Role of the alveolar type II cell in the development and progression of pulsed tumors induced by 4- (methylnitrosamino) -1- (3-pyrimidamino) Cancer Res. 52 3164-3173 Bellusci, S.M. , J .; Grindley, et al. (1997). “Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lang.” Development. 124 (23): 4867-4878 Bruno, M.M. D. , R. J. et al. Bohinski, et al. (1995). “Lung cell-specific expression of the murine surfactant protein A (SP-A) gene is moderated by interacts--between the sprout and thirc. Biol. Chem. 270: 6531-6536 Cardoso, W.C. V. (2001). “Molecular regulation of lung development.” Annu Rev Physiol. 63: 471-494 Colvin, J.M. S. A. C. White, et al. (2001). “Lung hypoplasia and neonatal death in Fgf9-null mice identifying this gene as an essential regulator of lumen mesenchyme. Colvin, J.M. S. , B. Feldman, et al. (1999). “Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene.” Dev Dyn. 216 (1): 72-88 De Moerloze, L .; , B. Spencer-Dene, et al. (2000). "An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in messencial-episealous mass3. Dobbs, L .; G. , (1989). Pulmonary Surfactant. Annual Review of Medicine, 40 (1): p. 431-446 Giard D. J. et al. et al. (1973). In vitro culture of human tumors: establishment of cell lines, derived from a series of solid tumors. J. et al. Natl. Cancer Inst. [Bethesda] 51: 1417-1423 Guarino, M .; A. Tosoni, and M.M. Nebuloni, (2009). Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition. Human Pathology, 40 (10): p. 1365-1376 Hare, KJ. , E.C. J. et al. Jenkinson, and G.J. Anderson, (1999). In vitromodels of T cell development. Seminars in Immunology, 11 (1): p. 3-12 Hare, KJ. , E.C. J. et al. Jenkinson, and G.J. Anderson, (1999). In vitromodels of T cell development. Seminars in Immunology, 11 (1): p. 3-12 Honig, M .; G. and R.R. I. Hume (1989). “DiI and diO: versatile fluorescent dyes for neuronal labeling and pathway tracing.” Trends Neurosci. 12 (9): 333-335 Honig, M .; G. and R.R. I. Hume (1989). Trends Neurosci. 12 (9): 333-335 Hughes Tracy et al. , A poster presenting at National Center for Replacement, Refinement and Reduction of animals in Research, Showcasing the 3Rs Portalis House, 28 February 7 Ikeda, A .; , J .; C. Clark, et al. (1995). "Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells." Biol. Chem. 270: 8108-8114 Innovative Medicine Research Initiative Strategic Research Agenda. 2008, European Technology Platform Konigshoff, M.M. , Et al. , (2008). Functional Wnt Signaling Is Increased in Idiopathic Pulmonary Fibrosis. PLoS ONE, 3 (5): p. e2142 Kramer, J .; A. , J .; E. Sagartz, and D.C. L. Morris, (2007). The application of discovery topathology and pathology to the design of safe pharmaceutical candidates. Nat Rev Drug Discov, 6 (8): p. 636-649 Kreda, S .; M.M. , M.M. C. Gynn, et al. (2001). “Expression and localization of epithelial aquaporins in the adult human lang.” Am. J. et al. Respir. Cell. Mol. Biol. 24: 224-234 Lazzaro, D.M. , M.M. Price, et al. (1991). “The transcription factor, TTF-I, is expressed at the onset of the thyroid and lung morphogenesis and the instrated regions of the foet 3 109”. Min, H.C. , D.C. M.M. Danilenko, et al. (1998). “Fgf-10 is required for both limbs and long development and exhibits striking functional similarity to Drosophila branchless.” Genes Dev. 12 (20): 3156-3161 Napolitano, AP, Chai, P et al. (2007). “Dynamics of the self-assembled of complex cellular aggregates on micromolded nonadhesive hydrogens.” Tissue Eng. 13 (8): 2087-94 Neagu, A .; (2006). “COMPUTATIONAL MODELING OF TISSUE SELF-ASSEMBLY.” Modern Physics Letters B 20: 1207-1231 Nichols, J. et al. E. and J. et al. Cortiella, (2008). Engineering of a Complex Organ: Progress Toward Development of a Tissue-engineered Lung. Proc Am Thorac Soc,. 5 (6): p. 723-730 Ornitz, D.M. M.M. and N.N. Itoh (2001). “Fibroblast growth factors.” Genome Biol. 2 (3): 3005 Seike, M .; , Et al. , (2009). Epithelial to Messenical Transition of Lung Cancer Cells. Journal of Nippon Medical School, 76 (4): p. 181-181 Sekine, K .; , H .; Ohuchi, et al. (1999). “Fgf10 is essential for limb and lug formation.” Nat Genet. 21 (1): 138-141 Shannon, J. et al. M.M. and B. A. Hyatt (2004). “Epithelial-Messenical Interactions in the Developing Lung.” Annual Review of Physiology 66: 625-645 Sherbet, G .; V. , (2009). Metastasis promoter S100A4 is a potentially variable molecular target for cancer therapy. Cancer Letters, 280 (1): p. 15-30 Vertrees, RA, Zwischenberger, JB et al. (2008) “Cellular differentiation in three-dimensional long cell cultures.” Cancer Biol Ther. Mar; 7 (3): 404-12 Wang, X .; Q. , H .; Li, et al. (2009) “Oncogenic K-Ras Proliferation Propagation and Cell Junctions in Lung Epithelial Cells through Induction of Cyclogenese-2 and Activation 9”. Wang, X .; Q. , X. M.M. Duan, et al. (2005). “Carboxyfluorescein Diacetate Succinimidyl Esther Fluorescent Dye for Cell Labeling.” Acta Biochimica Biophysica Sinica 37 (6): 379-385 Wang, X .; Q. , X. M.M. Duan, et al. (2005). Acta Biochimica Biophysica Sinica 37 (6): 379-385 Wardlaw et al. , (2002). Transactional regulation of basal cyclooxygenase-2 expression. . . Molecular Pharmacology, 62, 326-333 Yang, M .; C, Y. Guo, et al. (2006). "The TTF-1 / TAP26 complex differentially modulated surfactant protein-B (SP-B) and -C (SP-C) promoters in lang cells." Biochem Biophys Res Commun. 344 (2): 484-490 Zeisberg, M .; and E.E. G. Neilson, (2009). Biomarkers for epithelial-mesenchymal transitions. The Journal of Clinical Investigation, 119 (6): p. 1429-1437 Zhang, X. et al. , T. S. Snappenbeck, et al. (2005). “Reciprocal epithelial-mesenchymal FGF signaling is required for secondary development.” Development. 133: 173-180

The present invention provides an artificial three-dimensional lung model tissue culture, the model tissue culture comprising:
a) not including an artificial tissue scaffold,
b) composed of cultured cells or having cultured cell material, where these cells are in direct cell-cell interaction with cells of one or more other cell types comprising this tissue material. And
c) comprising at least lung epithelial cells and stromal cells, preferably lung stromal cells, preferably fibroblasts, wherein preferably the ratio of lung epithelial cells to stromal cells in the model tissue is at least 1: 6, preferably at least 1: 3, and at most 6: 1, preferably at most 3: 1;
d) having a form of one or more cell aggregates, wherein there are many lung epithelial cells on the surface of the aggregates, or lung epithelial cells and stromal cells, preferably fibroblasts , At least partially separated in the agglomerates,
e) Epithelial cells express epithelial differentiation markers.

  a) In a preferred embodiment, the model tissue culture does not include an artificial matrix material to provide a three-dimensional environment for the cells. In one embodiment, the model tissue culture comprises an artificial tissue scaffold material, either a biodegradable or non-biodegradable tissue scaffold material (eg, a porous 3D matrix), a 3D gel matrix. Not included. In one embodiment, the model tissue culture does not or does not include a microporous membrane support.

  b) In a preferred embodiment, the model tissue culture also comprises an extracellular matrix, and the extracellular matrix protein is secreted by at least one of the cell types comprising the tissue, preferably fibroblasts.

c) In a further preferred embodiment, the lung epithelial cells have the following cell types:
-Type I lung cells [alveolar type I cells (ATI)]
-Type II lung cells [alveolar type II cells (ATII)]
At least one of them.

  Preferably, the type II pneumocytes (alveolar epithelial cells with ATII characteristics) are of the following markers: TTF1 transcription factor, surfactant protein A (SFPA), surfactant protein C (SFPC) and aquaporin 3 (AQP3) Express one or more.

  Preferably, the type I lung cells (alveolar epithelial cells with ATI characteristics) are one of the following markers: TTF1 transcription factor, aquaporin 3 (AQP3), aquaporin 4 (AQP4) and aquaporin 5 (AQP5) Or express multiple.

  In various further embodiments, at least one of lung epithelial cells and lung stromal cells is present in the model.

  Preferably, the cell is an amphibian, reptile, avian or more preferably a mammalian cell.

  Preferred avian cells are poultry lung cells.

  Preferred mammalian cells are, for example, herbivore, preferably livestock animal cells, such as sheep, goat, bovine cells, or rodent cells (eg, rabbit or murine cells). Further preferred mammalian cells are omnivorous cells such as porcine cells. Highly preferred cells are human cells.

In a further embodiment, the lung epithelial cells and / or stromal cells are
-Preferably from an established cell line from a commercial source-a healthy donor-a patient donor.

  In a preferred embodiment, the cell is a primary cell. In a preferred embodiment, the cell is a cell that has not been dedifferentiated or has only partially dedifferentiated.

  In a further preferred embodiment, the cells are dedifferentiated cells or are dedifferentiated prior to culturing the cells into a 3D model tissue culture.

  In a preferred embodiment, the lung epithelial cells comprise small airway epithelial cells, preferably small airway epithelial cells having ATII characteristics.

  In a further preferred embodiment, the model tissue culture of the present invention also includes endothelial cells. In a preferred embodiment, the endothelial cells are HMVEC cells or HUVEC cells.

  Optionally, the model tissue culture of the present invention may further comprise additional types of cells selected from macrophages, mast cells, smooth muscle cells.

  d) In one preferred embodiment, the average or typical diameter of the agglomerates is at least 10 μm, 40 μm, 60 μm, 80 μm, 100 μm or 120 μm, and the average or typical diameter of the agglomerates is at most 1000 μm, 800 μm , 600 μm, 500 μm, 400 μm or 300 μm.

  It is very advantageous for the average or typical diameter of the agglomerates to be from 100 to 300 μm, and in a preferred embodiment it is about 200 μm.

  The average size of the agglomerates can be evaluated and calculated or estimated by any experimentally and mathematically correct means. The agglomerates are essentially spherical, but due to deviations from the exact sphere, and depending on the location of the agglomerates at the time of measurement and depending on the measurement method, It is clear that the diameter can be determined. For example, the minimum and maximum diameters can be directly measured and averaged with a microscope that measures the size of several agglomerates. It is convenient to use a microscope for this purpose.

  In a preferred embodiment, the majority of the agglomerates, preferably at least 60%, 70%, 80% or 90% agglomerates, are at least 10 μm, 40 μm, 60 μm, 80 μm, 100 μm or 120 μm in diameter, and at most 1000 μm , 800 μm, 600 μm, 500 μm, 400 μm or 300 μm, and the agglomerate diameter of the above ratio is very advantageously 100-300 μm, in a preferred embodiment it is about 200 μm.

In a preferred embodiment, each agglomerate or culture sample in each container of the kit is at least 10 ³ , preferably at least 10 ⁴ , more preferably at least 2 × 10 ⁴ , 3 × 10 ⁴ , 4 × 10 ⁴ , 5 X 10 ⁴ cells and a maximum of 10 ⁶ , more preferably a maximum of 5 × 10 ⁵ , 4 × 10 ⁵ , 3 × 10 ⁵ , 2 × 10 ⁵ or a maximum of 10 ⁵ cells.

  In a preferred embodiment, lung epithelial cells and fibroblasts are separated based on their surface tension differences. Preferably, most lung epithelial cells are located on the surface of the aggregate.

  Preferably, the majority of lung epithelial cells form a lung epithelial cell inner layer on the surface of the aggregate, and preferably the lung epithelial cell inner layer at least partially covers the surface of the aggregate.

  In a further preferred embodiment, the agglomerates also comprise endothelial cells.

  In a preferred embodiment, the ratio of endothelial cells is higher at the center or central region of the aggregate than at the surface of the aggregate, compared to epithelial cells and fibroblasts, or the ratio of endothelial cells is aggregated It increases from the surface of the mass towards the center of the agglomerate.

  In a preferred embodiment, the agglomerate has a lamellar structure, wherein the core or central region of the agglomerate contains a maximum ratio of endothelial cells, and the intermediate layer or intermediate region of the agglomerate has a maximum ratio of fibroblasts. The outer layer or surface layer of the agglomerate containing cells contains the largest layer of epithelial cells.

e) In a preferred embodiment, the epithelial differentiation marker expressed by tissue cells of an artificial 3D lung model tissue is the following group:
-ATII type differentiation marker, preferably TTF1 transcription factor, cytokeratin 7 (KRT7), surfactant protein A (SFPA), surfactant protein C (SFPC) and aquaporin 3 (AQP3) and / or marker-ATI type differentiation marker, preferably Aquaporin 4 (AQP4) and Aquaporin 5 (AQP5)
At least one marker selected from.

  The marker expressed will also depend on the cell type used in tissue culture.

  The level of any of these markers can be detected at the mRNA or protein level. Thus, the level of the marker can be at the mRNA level and / or the protein level.

  Preferably, in the model tissue culture of the present invention, at least one of the levels of AQP3 and SFTPA is increased, i.e., they are upregulated compared to the control 2D culture.

  Preferably, in the model tissue culture of the present invention, at least one inflammatory marker selected from IL1b, IL6 and CXCL8 is downregulated, ie their levels are 3D compared to the control 2D culture. Decreases under culture conditions.

  Preferably, in a model tissue culture of the invention, the level of at least one of the dedifferentiation markers S100A4 and N-cadherin is compared to purified primary cells under 2D culture conditions or in a control 2D tissue culture. Then decrease.

In various further embodiments, at least one of lung epithelial cells and lung stromal cells is present in the model and, similar to embryonic stage lung development,
The lung epithelial cells secrete one or more fibroblast growth factors selected from FGF4, FGF8, FGF9;
The lung epithelial cells express the cell surface FGFR2b receptor;
-Lung stromal cells, preferably fibroblasts, secrete FGF7 and FGF10 and express FGFR1c and FGFR2c receptors.

  In a preferred embodiment of the tissue culture of the present invention, the ATII type differentiation marker and / or the ATI type differentiation marker is at a level higher than that measured in a two-dimensional tissue culture used as a reference, more preferably It is expressed at a level that is at least 10%, at least 20%, or at least 30% higher.

Preferably, the mRNA level and / or protein level of the epithelial marker in the model tissue is determined from the following reference culture-two-dimensional culture of the same cell type
-A culture of lung epithelial cells only,
-One or more of the cultures of primary fibroblasts, preferably human fibroblasts only, or higher than each such culture.

  Preferably, the level of at least two of the markers is increased.

  In a preferred embodiment, small airway epithelial cells are applied.

  In a further preferred embodiment, small airway epithelial cells exhibiting at least some ATII type characteristics are applied. In this case, the model tissue exhibits increased mRNA and / or protein levels of at least one or more markers selected from the following groups of ATII type differentiation markers, eg, those listed above.

  The artificial three-dimensional lung model tissue of the present invention preferably exhibits reduced expression of one or more pro-inflammatory cytokines and / or one or more EMT markers.

Preferably, the mRNA level and / or protein level of the one or more pro-inflammatory cytokines in the model tissue is determined from the following reference culture—a two-dimensional culture of the same cell composition:
A culture of lung epithelial cells only, where the lung epithelial cells are lower than one or more of the same treated as the model tissue culture or each such culture.

  Preferably, the pro-inflammatory cytokine is selected from the following group: CXCL-8 pro-inflammatory cytokine, IL6, IL1a, IL1b, TNFα.

  In a highly preferred embodiment, the pro-inflammatory chemokine is a CXCL-8 chemotactic factor.

In a preferred embodiment, the model tissue culture comprises lung epithelial cells and lung stromal cells and no endothelial cells, wherein
The level of one or more of the following markers: E-cad, IL-1b and / or IL6 is increased compared to a control comprising uncultured cells;
The level of E-cad is increased compared to the control two-dimensional culture;
-The level of one or more of the following markers: IL-1b, CXCL8, IL6 is reduced compared to the control two-dimensional culture.

In a preferred embodiment, the model tissue culture comprises lung epithelial cells, lung stromal cells and endothelial cells, wherein
The level of one or more of the following markers: E-cad, N-cad is reduced compared to controls containing uncultured cells,
The level of E-cad is increased compared to the control two-dimensional culture;
-The level of one or more of the following markers: N-cad, S100A4 is reduced compared to the control two-dimensional culture.

In a preferred embodiment, the three-dimensional lung model tissue culture is in the following group:
-For example, endothelial cells that mimic the vasculature,
Macrophages,
-Further comprising cells selected from mast cells.

In later stages, this model is cell type:
-Smooth muscle cells,
-Can be expanded using nerve cells.

Disease model:
The present invention also relates to an artificial three-dimensional lung model tissue culture as defined above, wherein the epithelial cells and / or fibroblasts are the model tissue culture of the lung disease model tissue. Pathological cells with pathological features of diseased lung tissue such as

  In a preferred embodiment, the disease involves a condition selected from inflammation, tumor, fibrosis, tissue damage, and the model tissue culture is considered an inflammation model, tumor model, fibrosis model or regeneration model, respectively. become.

  Disease model pathological cells are exposed to cells obtained from patients (patient cells), cell lines with disease characteristics (eg, tumor cell lines); environmental effects (eg, pro-inflammatory substances that produce disease characteristics). Cells that have been subjected to the effects of mutagen and selected for disease characteristics; or genetically modified cells that have been transformed to express a protein or have a gene silenced to have disease characteristics. Can be, but is not limited to.

  In a preferred embodiment, the cells are obtained from a healthy subject and a disease state is induced therein. In this embodiment, for example, tumor induction or potential drug target signaling can be revealed.

  In another embodiment, the tumor model tissue is prepared from immortal cells, eg, from malignant transformed or neoplastic cells or cell lines. In this embodiment, there is no “health” control, but the system is useful in drug testing because the sample in contact with the placebo drug provides a control for the drug treatment sample.

  In one embodiment, neoplastic cells can be obtained from a patient to test the efficiency of the planned treatment. Thus, model tissue culture can be used to establish individual therapy.

Preparation method:
The present invention is also a method for preparing an artificial three-dimensional lung model tissue culture as defined herein comprising:
-Preparing a mixed suspension of at least primary fibroblasts and lung epithelial cells;
-Placing this mixed suspension or an aliquot thereof into a container suitable for pelleting the cells of the suspension;
-Pelleting the cells;
Incubating the pelleted suspension in the presence of CO ₂ for a time sufficient for the cells to form a three-dimensional lung model tissue containing cell aggregates;
-In some cases, model organization
a) Expression of one or more epithelial differentiation markers characteristic of lung tissue, wherein the increased level of expression when compared to a suitable reference culture, eg, as disclosed herein, is a three-dimensional lung model Considered to indicate the formation of tissue culture; and / or
b) Expression of one or more pro-inflammatory cytokines, wherein the decrease in expression level when compared to any suitable reference culture, eg, as disclosed herein, is a three-dimensional lung model tissue culture Assaying for possible formation of
A method comprising:

  Preferably, the container is a container that has not been subjected to tissue culture treatment.

  Preferably, multiple aliquots are placed in multiple containers.

  Preferably, the container is a plate, eg, a well of a 96 well plate or a 384 well plate.

  Preferably, the pelletization is performed at 200 g to 600 g for 1 to 20 minutes, preferably 2 to 10 minutes.

  Preferably, the reporter molecule is supplied to the cell, eg, the cell is stained with a biocompatible dye that signals cell characteristics as disclosed herein.

  The container can be a V-bottom, flat-bottom or U-bottom container, depending on the purpose for which it is used.

  When preparing a mixed suspension, within 18 hours, preferably within 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, more preferably within 4 hours, 3 hours, 2 hours, Most preferably, one or more cells are added to the container within 1 hour or 0.5 hour. Preferably, each type of cell used is added within the period defined above.

In a preferred embodiment, the pelleted suspension is placed in the presence of CO ₂ for 50 hours, 40 hours, 30 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours. Incubate for a period of time or no more than 10 hours. In a preferred embodiment, the pelleted suspension is incubated in the presence of CO _{2 for} a period of 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours or more.

  In one embodiment of the invention, additional cell types are added to the mixed suspension of cells. In one embodiment of the invention, the additional type of cell is at least an endothelial cell. In one embodiment of the invention, the additional type of cell is selected from endothelial cells, smooth muscle cells, neurons, granulocytes, dendritic cells, mast cells, T / B lymphocytes, macrophages. Granulocytes, dendritic cells, mast cells, T / B lymphocytes and macrophages can be added to the culture either in an inactive state or in an immunologically active state.

  Optionally, the method according to the invention comprises dedifferentiating one or more types of cells prior to preparing the mixed suspension.

  Optionally, the method according to the invention comprises propagating one or more types of cells prior to preparing the mixed suspension. This step is particularly necessary when parallel testing of a large number of samples is required, for example, in a solution by HTS (High Throughput Screening).

Screening method:
The present invention is also a method of screening a drug for its effect on lung tissue,
-Preparing an artificial 3D lung model tissue culture as defined herein;
-Taking at least a test sample and a reference sample of the model tissue culture;
-Contacting the test sample with the drug while maintaining the test sample and the reference sample under the same conditions;
-Detecting any change or change in the test sample compared to the reference sample, where if any change or change in the test sample is detected, it is considered to indicate the effect of the drug;
Relates to a method comprising:

  In certain variations of the method, only the direction of change or change is observed, and no physiological values are calculated. In a particular variation, a predetermined threshold is defined based on a calibration curve and compared to this value.

  In a preferred embodiment, the model tissue culture is a model of healthy lung tissue and the adverse effects of the drug are tested, wherein changes or alterations that are harmful to the cells of the test sample are toxic effects or harmful effects of the drug. It is considered an effect.

In a preferred embodiment, the model tissue culture is a lung disease model tissue culture comprising pathological cells with pathological features, where the beneficial effects of the drug are tested, wherein
-Preparing an assay on the model tissue culture to measure or assess its pathological characteristics to obtain the extent of the disease;
-Preparing a reference sample of healthy lung tissue (health reference sample) and / or a reference sample of diseased lung tissue (affected reference sample);
Measuring or assessing pathological characteristics in a health reference sample and / or diseased reference sample and at least one test sample thereof, before and after contacting it with a drug, wherein the degree of disease in the test sample Any change or change in the test sample that changes the to the extent of the disease in the health reference sample and / or from the extent of the disease in the affected reference sample is considered a beneficial effect of the drug. That is, it is more similar to the state of the healthy reference sample than the diseased reference sample.

  In a preferred embodiment, primary cells obtained from a patient are applied. In a preferred embodiment, the primary cells from a given patient are not dedifferentiated or only partially dedifferentiated, and they are removed from that patient in order to prepare a mixed suspension of cells. Used within 5, 4, 3, 2 or 1 day after acquisition or within 12, 10, 8, 6, 4, 3, 2, 1 hour. Model tissue cultures made of primary cells can study the characteristics of a given patient's disease state and test therapeutic drugs and / or regimens.

kit:
The present invention relates to an artificial three-dimensional lung model tissue kit comprising a test plate having a number of side-by-side containers, wherein at least two containers are:
One or more artificial three-dimensional lung model tissue culture samples as defined in any of the preceding claims, wherein each sample is contained in a separate container of the plate;
An appropriate medium for culturing the cells of the model tissue culture,
including.

  Preferably, the subset of containers includes one or more control samples. A control sample can be a pure culture of a particular cell type, eg, an epithelial and fibroblast-only culture, and / or a two-dimensional (2D) culture. In a disease model, the control can be a culture of healthy cells.

Preferably, the artificial 3D lung model tissue kit has one or more of the following features:
The plate is a 96 well plate,
The plate is a V-bottom plate or flat-bottom plate or a plate containing both V-bottom and flat-bottom wells, U-bottom plate can also be applied,
-Culture samples in each container
At least 10 ³ , preferably at least 10 ⁴ , more preferably at least 2 × 10 ⁴ , 3 × 10 ⁴ , 4 × 10 ⁴ , 5 × 10 ⁴ cells, and at most 10 ⁶ , preferably at most 5 × 10 ⁵ , Containing an amount of 4 × 10 ⁵ , 3 × 10 ⁵ , 2 × 10 ⁵ cells, or at most 10 ⁵ cells,
The containers are sealed individually or together and contain a CO ₂ rich environment or atmosphere suitable for lung tissue culture;
-CO _2-rich environment or atmosphere,
At least 2%, 3%, 4% CO ₂ environment,
Up to 10%, 9%, 8% or 7% CO ₂ environment,
Most preferably about 5% CO ₂
including.

  In a preferred embodiment, the sample includes a test sample and a corresponding control sample.

  In a preferred embodiment, the test sample is present on the V-bottom plate or in the V-bottom well of the plate, and the control sample is present on the flat-bottom plate or in the flat-bottom well of the plate.

Definition:
The meaning of “artificial tissue scaffold” in the context of this description is specifically designed to attach cells and / or to assist in the structural three-dimensional arrangement of cells in tissue or cell culture, and A solid support material having a structure useful for adhering and / or to assist in the structural three-dimensional arrangement of cells in tissue or cell culture. Preferably, the artificial tissue scaffold is made prior to culturing the tissue or cells and contacted with the tissue or cells before or while the tissue or cells are being cultured. Thus, artificial tissue scaffolds typically affect the structure (eg, tissue or cell culture 3) by affecting at least a portion of cell interactions (eg, cell-cell interactions) or the cell environment itself. It is a cell growth support structure or cell growth support material that contributes to the (dimensional structure). As a result, when the tissue scaffold is removed from the tissue or cell culture, the tissue or cell culture collapses or becomes disordered.
However, those skilled in the art will recognize that if the artificial tissue scaffold is made of a biodegradable material that is gradually degraded to allow the formation of cell-cell interactions, It is understood that the tissue or cell culture is not in a collapsed or disordered state during this process.

  In some versions, the artificial tissue scaffold is a three-dimensional matrix, preferably a three-dimensional gel matrix or a porous three-dimensional matrix, which preferably has a microspace or pore in which cells are present.

  In some versions, the artificial tissue scaffold itself is a support, preferably a porous membrane support, on the surface to which the cells attach. In this scaffold version, the scaffold is specially designed to attach cells and thereby useful structures for attaching cells, such as cells, so as to promote the formation of a three-dimensional structure. Has a surface that is porous, curved, fringed, or grooved.

Preferably, the artificial tissue scaffold is
-Has a clear three-dimensional structure,
-Porous, preferably highly porous material or matrix;
-A porous membrane;
-A porous three-dimensional matrix;
-Made of biocompatible materials and / or
-Made of polymer.

  In some cases, an “artificial tissue scaffold” is a polysaccharide-based matrix, for example, it is a cellulose-based matrix, such as a methylcellulose matrix.

  In some cases, an “artificial tissue scaffold” has a bead structure, for example, it is a cytodex bead.

  “Three-dimensional tissue culture without an artificial tissue scaffold” is understood herein as a tissue culture having a three-dimensional structure, wherein the three-dimensional structure of the tissue culture is inherently intrinsic. It is formed or contributed by existing cell-cell interactions, not promoted by artificial tissue scaffolds.

  Thus, a three-dimensional tissue culture that does not include an artificial tissue scaffold maintains its three-dimensional structure rather than collapsing or becoming disordered in the absence of an artificial tissue scaffold. Even though a three-dimensional tissue culture that does not contain the artificial tissue scaffold is cultured and formed on a solid support material, the formation of the three-dimensional structure is achieved by the cells attaching to the solid support. It is not encouraged and not caused by it, it can be separated without destroying the three-dimensional structure.

  As used herein, “separation of cells” relates to the spatial separation of at least two types of cells in a tissue or cell culture, and after this spatial separation (ie, separation), the two types The area of the culture, for example, (partial) is different from both the ratio of the same type of cells in the same area of the culture before separation and the ratio of the same type of cells in other areas of the culture ) The volume or surface can be defined or found. Preferably, the difference in surface tension of at least two types of cells greatly contributes to their separation in vitro.

  A “concentration” of a region, eg, the volume or partial volume or surface or partial surface of a culture of a particular cell type is the ratio of a particular cell type to the reference region (eg, other regions of that culture). It should be understood as a phenomenon when it is higher in that area than in. Usually, “enrichment” of an area of culture is the result of cell separation.

  “Inflammation” is an adaptive response induced by harmful stimuli and conditions such as infection and tissue damage.

  Some cytokines accelerate inflammation and are collectively known as “pro-inflammatory cytokines”, but they also induce the synthesis of cell adhesion molecules or other cytokines directly or in certain cell types Modulates the inflammatory response by ability. The main pro-inflammatory cytokines involved in the initial response are IL1-α, IL1-β, IL6, and TNF-α. Other pro-inflammatory regulators include IFN-γ, CNTF, TGF-β, IL12, IL17, IL18, IL8 (CXCL8) and various other chemokines that chemically attract inflammatory cells, and various nerves Regulatory factors are mentioned. The net effect of the inflammatory response is that of pro- and anti-inflammatory cytokines such as IL4, IL10 and IL13, IL16, IFN-α, TGF-β, IL1ra, G-CSF, TNF or IL6 soluble receptors ) Determined by the balance. Activation of IL1-β by various cascades occurs in a large multiprotein complex termed the inflammasome.

  LIF, GM-CSF, IL11 and OSM are additional cytokines that affect the inflammatory process and may be useful in preparing the disease model of the present invention.

  In contrast, “anti-inflammatory cytokines” such as IL10 regulate the inflammatory process so that the inflammatory process is suppressed or alleviated.

  The “average diameter” of the three-dimensional tissue is interpreted as the arithmetic average of several measurements of the three-dimensional tissue diameter generated by the above method.

  “Typical diameter” (median diameter) is a diameter indicating that a given sediment sample is divided into two equal parts by weight, one part containing all agglomerates larger than that diameter, One part contains all the smaller agglomerates.

  A “line” of containers is understood as an arrangement of a plurality of containers of the same size, shape and material. The arrangement can be, for example, an array of containers or a two-dimensional matrix of containers.

  Viruses are often obligate intracellular pathogens that infect cells with a strong specificity for a particular cell type. In the “recombinant viral vector”, genes necessary for the replication phase of the viral life cycle are removed, and the target gene is added to the viral genome. A recombinant viral vector can transduce a cell type to which it normally infects. To create such a recombinant viral vector, a non-essential gene is integrated into the packaging cell line genome or provided in trans on a plasmid. Several viruses have been developed and interest is concentrated in four types; retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses and herpes simplex virus type 1.

  “Cancer” is a group of diseases in which a group of cells exhibit uncontrolled growth, invasion (invasion of adjacent tissue and destruction of adjacent tissue), and sometimes metastasis (spread to other parts of the body via lymph or blood). It is. These three malignant features of cancer distinguish cancer from benign tumors that are self-limiting and do not invade or metastasize. 95% of lung tumors are bronchial cancer, bronchial carcinoids, stromal neoplasms, mixed neoplasms.

  “Fibrosis” is the formation or development of excess fibrous connective tissue in an organ or tissue as a repair or reaction process as opposed to the formation of fibrous tissue as a normal component of an organ or tissue . Pulmonary fibrosis is a severe condition characterized by loss of elasticity, pulmonary epithelial cells that are replaced by interstitial myofibroblasts, and the deposition of extracellular matrix proteins in the pulmonary stroma, resulting in reconstruction of the lung structure It is a chronic disease.

A photograph showing the structure of a 3D two-cell microculture. SAEC and NHLF cells were stained with a vital dye called CFSE or DiI, respectively. Cell populations, pure or mixed at various ratios, were pelleted and after 24 hours incubation, aggregates were formed and then transferred to 24-well cell culture plates for imaging. Upper row: phase contrast microscope image; middle row: fluorescence microscope image; lower row: confocal image. SAEC: green channel; NHLF: red channel. Photograph showing a Matrigel-based culture of a mixture of 50% SAEC and 50% NHLF. Photographs showing pelleted microtissue cultures containing different ratios of SAEC and NHLF. Due to the physiological fluorescent markers, SAEC cells are light gray in green or black-and-white copy, whereas NHLF cells are red or dark gray. The graph which shows the mRNA level of TTF-1 in 3D human lung microtissue. TTF1 transcription factor is a characteristic marker of alveolar epithelial cells. 3D fibroblast cultures do not show TTF1 expression, but TTF1 is present in 3D SAEC alone cultures and is increased in 2D SAEC / NHLF co-cultures, showing the beneficial effects of fibroblasts ing. Maximum levels of TTF1 expression were reached in 3D SAEC / NHLF tissue. The graph which shows the mRNA level of the AQP-3 water transporter in 3D human lung microtissue. AQ3 is an ATII epithelial marker in the lung. Although 3D fibroblast cultures do not show AQ3 expression, AQ3 is present in 3D SAEC alone cultures and is increased in 2D SAEC / NHLF co-cultures, showing the beneficial effects of fibroblasts Nevertheless, maximal levels of AQ3 were observed in 3D SAEC / NHLF tissue culture. The graph and photograph which show the gene expression change in a SAEC differentiation marker. A: Relative mRNA levels of AQP3 water transporter in 2D and 3D human lung microtissues. Relative AQP3 expression levels were increased in mixed cell cultures compared to relative AQP3 expression levels in SAEC-only cultures, but no difference was detected between 2D and 3D culture conditions. B: The relative level of KRT7 mRNA expression was increased in mixed cell cultures compared to SAEC-only cultures (independent experiments: n = 3). C: RT-PCR analysis of SFTPA1 and β-actin expression in 2D and 3D cultures. SFTPA1 expression was only detected in SAEC + NHLF co-cultures. The expression of SFTPA1 was always higher in 3D cultures than in 2D cultures. Shown are representative images of three independent experiments. D: After 72 hours of 2D or 3D co-culture with NHLF cells, gene expression changes in SAECs selected by FACS were examined. The levels of differentiation markers AQP3 and TTF-1 in repurified SAEC were markedly upregulated in 3D co-culture compared to 2D co-culture. Data shown are the average of two independent experiments (the purified primary lung cells used in all our experiments were from randomly chosen hosts). The graph which shows the EMT marker in a 3D lung tissue model. A: Relative mRNA levels of S100A4 in 2D and 3D co-cultures. The presence of fibroblasts significantly reduced the level of S100A4 in the SAEC-NHLF co-culture compared to the SAEC-only culture, whereas 2D or 3D culture conditions did not significantly change S100A4 expression. B: E-cadherin (E-cad) relative mRNA levels are increased in 3D cultures in the presence of NHLF. C: Relative mRNA levels of N-cadherin (N-cad) in 2D and 3D human lung microtissues. D: After 72 hours of 2D or 3D co-culture with NHLF cells, gene expression changes in SAECs selected by FACS were examined. The levels of EMT markers S100A4 and E-cad in sorted lung epithelial cells were increased in 3D 2-cell co-cultures compared to 2D co-cultures, whereas N-cad was increased in 3D mixed cultures. It was expressed at a much lower level. The purified primary lung cells used in our experiments were from randomly chosen hosts. Data shown are the average of three independent experiments (panels AC) or two (panel D). The purified primary lung cells used in all our experiments were from randomly chosen hosts. Graph showing inflammatory cytokine and chemokine secretion in human primary lung cell cultures. A: CXCL-8 secretion of 2D and 3D NHLF single cultures was hardly detected in the cell culture supernatant. To a lesser extent than 2D SAEC cultures, 3D SAEC cultures still produced CXCL8. In 2D co-cultures, CXCL-8 expression did not change much, indicating that the presence of fibroblasts could not affect cytokine expression. CXCL-8 expression levels were significantly reduced in the 3D tissue system in both pure SAEC and SAEC-NHLF co-cultures. A and B: IL-1b and IL-6 mRNA expression levels in human primary lung cell cultures, respectively. Compared to 2D cultures, inflammatory mRNA levels of the inflammatory cytokines IL-1b and IL-6 are always lower in 3D cultures. In pure fibroblast cultures, IL-1b mRNA expression was not detected, whereas IL-6 levels were much lower and expression changes were less noticeable (see also Table 2). D: Similar to the mixed cell culture samples, the levels of inflammatory cytokines IL-1b and IL-6 were also significantly reduced in SAEC purified from 3D cultures than in SADs of 2D cultures. Data shown are the average of three independent experiments (panels AC) or two (panel D). The purified primary lung cells used in all our experiments were from randomly chosen hosts. The photograph which shows the structure of 3D 3 cell type | mold microculture which consists of SAEC, NHLF, and HMVEC. SAEC, NHLF and HMVEC were stained with the vital dyes CFSE, DiI, or DiD, respectively, and then aggregated. After 24 hours of incubation, spontaneously rearranged 2-cell or 3-cell microcultures were carefully transferred to 24-well cell culture plates for imaging. A: 2-cell culture, B: 3-cell culture. Upper row: phase contrast microscope image; middle row: fluorescence microscope image; lower row: confocal image. SAEC: green channel; NHLF: red channel; HMVEC: blue channel. The graph which shows the gene expression change in a 3 cell type culture. A: Compared to 2D cultures, expression levels of AQP3 and KRT7 were increased and S100A4 and N-cad were decreased in 3D cultures. B: Comparison of changes in expression of molecular markers in 3D SAEC-NHLF two-cell culture and SAEC-NHLF-HMVEC three-cell culture. AQP3 and E-cad mRNA levels increased, S100A4 and N-cad decreased, indicating that tissue differentiation was maintained in a three-cell type model. The purified primary lung cells used in all our experiments were from randomly chosen hosts. Flow chart of preparation of a lung tissue kit supplied in a 96 well plate and ready for testing. Photo showing adenoviral gene delivery to SAEC in a two cell type model. Due to GFP expression, SAEC appears green on the surface of the 3D tissue model. Prior to aggregation, fibroblasts were prestained red with a physiological dye. Similarly, RT-PCR reactions have shown effective GFP gene delivery to this model. GFP can be detected in model cultures transduced by adenovirus.

  The inventors have created a simple artificial three-dimensional lung model tissue culture that is useful as a lung tissue model and can be readily used in various test methods.

  In creating the model, we considered several aspects of tissue features, including the main features of lung tissue types, cell-type interactions in embryonic lung development and technical advances in tissue engineering.

  Both scaffold-based systems and scaffold-free systems were tested as described herein. Surprisingly, analysis of lung tissue-specific markers showed that a three-dimensional scaffold-free system showed significant similarity to native lung tissue.

  In the prior art, abnormal proliferation of fibroblasts has often occurred when attempting to use these types of cells in model tissues (Patent Document 3). In light of the results presented herein, it can be inferred that the cause of such abnormal growth may have been the lack of aggregate formation. Cells that are distant from the agglomerate may have adhered to the surface of the container and may begin to propagate until contact inhibition is achieved thereby.

  The lung tissue model developed so far has been maintained in a culture state for a long time using a special scaffold material (Non-patent Document 3). In contrast, the model presented here allows easy manipulation and uses simple experimental setups and relatively short incubation times. Furthermore, no special laboratory equipment is required. The system is suitable for use with human cells, including human primary cells and non-transformed human cells.

  It should be understood that in certain systems, the scaffold (eg, matrix) is biodegradable. However, because the scaffold affects or defines the structure or shape of the tissue culture, in such cases, these systems are considered scaffold-free systems even after the scaffold is degraded. Should not be considered. In addition, in vitro systems such as the present invention typically do not seem to dissolve the degradable membrane.

  Furthermore, dissolution of the biodegradable scaffold takes a long time and is much longer than the time it takes to prepare and use the tissue culture of the present invention.

  Cells that are not dedifferentiated cells can also be applied in the present invention, but the number of cells will be reduced. Therefore, this embodiment is mainly useful when a small number of cells is sufficient in a planned test, for example, when the sensitivity of the test is sufficiently high. In rapid testing, the preparation of the model tissue culture of the present invention can be initiated from purified differentiated cells. Such cells can be freshly prepared from the subject. This version of the method is particularly useful, for example, in patient-specific testing of drugs or compounds or when trying to test the effect of an active agent in certain disease situations (eg, for potential manufacturers) .

  Primary cells can also be obtained from commercial sources, such as Non-Patent Document 4.

  If a large number of samples are required, the cells should be propagated before preparing the model tissue culture sample. Dedifferentiation can occur during this process. This is true for screening (eg, HTS) applications. For patient-specific tests, a smaller number of samples is sufficient.

  If the cells differentiate within the clump, propagation slows or stops, after which the clump does not increase significantly.

  If a small number of samples is sufficient, no preliminary breeding is necessary. Typically, this can occur in patient sample studies for a few drugs or when trying to observe certain phenomena (eg, signal transduction processes). However, the screening process requires a large number of samples, and cell propagation should take place before preparing the model tissue culture.

  In a preferred embodiment of the invention, adult dedifferentiated epithelial cells are used. In adult dedifferentiated epithelial cells, differentiation may be brought about simply by co-culture in the presence of fibroblasts, which is further suitable for 3D conditions, particularly suitable for the formation of 3D aggregates. It was not known in the art to increase. Accordingly, in a preferred embodiment, model tissue culture preparation begins with dedifferentiated cells.

  Primary cells maintained in culture, for example in 2D tissue culture, exhibit certain dedifferentiation markers. Therefore, such cells can also be applied in the present invention. Examples of dedifferentiation markers include S100A4, N-cadherin, and inflammation markers. Therefore, more cells can be applied.

  Pluripotent cells or undifferentiated cells can be allowed to differentiate after addition of tissue components and / or factors.

Cell interactions in lung tissue:
Cell dedifferentiation and redifferentiation:
Both stem cells and tissue-specific progenitor cells can go through a directional tissue-specific differentiation stage and are therefore an ideal source for making organ-specific tissue culture materials. Unfortunately, both cell types are only available in limited numbers in differentiated tissues.

  When tissue models need to be built regularly according to experimental and / or test requirements, tissue-specific differentiated cells are a better source of primary material simply because there are many. This model system utilizes, at least in part, the phenomenon that differentiated cells under two-dimensional culture conditions can be dedifferentiated and can be redifferentiated using appropriate culture conditions.

Cell interaction:
Lung development and epithelial damage repair are tightly regulated by a delicate balance of stimulatory and inhibitory genes that appear to co-regulate the functions of lung stem cells and adult progenitor cells. For example, FGF receptor tyrosine kinase signaling is essential for respiratory organogenesis and is negatively regulated by a family of inducible FGF pathway inhibitors (Non-Patent Document 5). In addition, FGF signaling is required for the formation of new alveoli, protection of alveolar epithelial cells from injury, and putative alveolar stem / progenitor cell migration and proliferation during lung repair. Conversely, TGFβ receptor serine-threonine kinase signaling via Smad2, 3 and 4 can inhibit lung morphogenesis and inhibit postnatal alveolar development, but excess TGFβ via Smad3. Signaling causes interstitial fibrosis.

  Therefore, there is a need for a slightly more complex but interrelated system of interaction between the stroma and the epithelium. The inventors hypothesized that a purified lung mixture can be used to create a basic lung model.

  Therefore, initially only two cell types were used, both commercially available (Lonza), primary human fibroblasts (NHLF) and small airway epithelial cells (SAEC with ATII characteristics). Surprisingly, these two cell types have been found to provide enough of the necessary factors to form a three-dimensional lung tissue model. One skilled in the art can further develop this model by adding additional cell types, such as those listed herein.

Separation of cell types in mixed culture (sorting):
Our microscopic examination has demonstrated that spontaneous tissue reorganization— “sorting” —occurs in 3D lung primary cell cultures. The inventors used herein a simple centrifugation method to agglutinate cells, similar to the method of preparation of fetal thymus organ culture (Non-Patent Document 6). Here, evidence shows that 3D co-cultures of primary lung epithelial cells and fibroblasts are advantageous in maintaining SAEC more differentiated than either 2D or 3D in vitro single cultures. Has been. Inclusion of NHLF not only facilitated epithelial differentiation, but 3D microtissue aggregation and structure compared to SAEC-only cultures (FIG. 3) or SAEC-HMVEC (FIG. 8a) cultures, It became much harder and more compact.

This two-cell co-culture system consisting of human small airway epithelial cells (SAEC) and normal human lung fibroblasts (NHLF) requires the presence of exogenously added ECM to form and maintain 3D structures. There was no (Figure 3). By pelleting cell suspensions of one cell type of SAEC and NHLF and various ratios of cell mixtures, the generation of pulmonary microtissues can be achieved with fibroblasts to maintain a compact and stable 3D structure. It became clear that existence was necessary (FIG. 3). By morphological examination of 3D microtissue, the separation of the two cell types in the mixed culture is characteristic of 3D microtissue, fibroblasts that form the inner core part, while epithelial cells cover the outer layer (Fig. 3).
The phenomenon of separation or “sorting” is based on the adhesion energy characteristics of different cell types and has not been described so far for primary human lung tissue. Spontaneous cell “sorting” is based on the difference in cohesion between different cell types. The most cohesive core or central region of the lung microtissue is formed by the NHLF population surrounded by less cohesive SAEC. The process of segregation in primary differentiated human lung tissue is particularly interesting because even the differentiated human adult cells are the basis of the idea that they maintain the ability to actively explore their own microenvironment. Cells in the 3D co-culture can exchange positions with neighboring cells and thus structurally reorganize the tissue. This process also requires reorganization of the extracellular matrix. Study of various SAEC / NHLF ratio models in the 3D microtissue model reveals that a 1: 1 ratio is sufficient for epithelial cells to coat the inner core of the fibroblasts, thus 1: A further analysis of this model was performed using a setting of 1. However, this model may be useful with other epithelial-stromal cell ratios. The ratio sufficient for complete coverage can vary to varying degrees depending on the cell type.

  Without being bound by theory, the present inventors believe that the separation of cells in this model is due to the different aggregation of cell types where the difference in surface tension plays a major role.

  Any cell can actively explore its own microenvironment and swap positions with neighboring cells or reorganize the nearby extracellular matrix. The latter process is known to require both mechanical traction and enzyme activity by matrix metalloproteases (MMPs). It is a known experimental fact that mixed cell types of specific cell composition can be separated in aggregates based on different adhesion energy properties.

  In the isolated cell aggregate of the hanging drop culture, the most aggregated population occupies the central region, which is surrounded by the less aggregated population. A measure of tissue aggregation is cell surface tension. Therefore, the selection hierarchy can be predicted by the surface tension, which is an experimentally detectable amount. Therefore, in that regard, early attempts have been made in the art, and this sorting hierarchy can predict to some extent whether new cell types can be included (Non-Patent Document 7).

  However, the surface tension coefficient is not known for certain cell types of human lung. In the prior art, nothing is said about whether the phenomenon of tissue sorting occurs in other cultures or only in hanging drop cultures. The inventors have surprisingly demonstrated for the first time herein that epithelial cell and fibroblast separation occurs in a mixed culture of pelleted alveolar epithelium and fibroblasts. I made it.

Microagglomerate size and structure:
Even the very small but structured agglomerates showed tissue characteristics and were therefore unexpectedly found by the inventors to be suitable for interaction studies and for testing compound or environmental effects.

  Small agglomerates have several advantages, for example, no special reaction vessels are required, their size and ratio of various cell types are reproducible, so the interaction is more easily controlled. With small agglomerates, the necrosis inside the tissue agglomerates can be considered substantially absent. Furthermore, a surprisingly uniform size distribution can be achieved, and this size distribution makes small agglomerates very suitable for parallel testing.

  Therefore, if tissue characteristics appear and thereby the interaction can be investigated, preferably the size of the agglomerates should be kept small according to the present invention.

  If the agglomerates are too small, the exact form as disclosed herein may not form and the agglomerates may not have tissue-like features. If the agglomerates are too large, their size can deviate from the average.

  In addition, necrosis may occur inside the agglomerates because of longer culture times and less perfusion of the agglomerates.

  In the absence of special additives, for example, when receiving only cell culture media, aggregate growth is controlled by cell contact inhibition.

  However, the size depends on the number of cells in the aggregate. Those skilled in the art will appreciate that if the above requirements are met, the size and cell number of the agglomerates can vary within the limits set forth herein.

  It has been found that the addition of endothelial cells does not significantly change the size of the aggregate.

Cell types useful in the present invention:
Fibroblasts:
Fibroblasts are the most versatile connective tissue cell family and, in fact, the most common cell type. Fibroblasts are an important structural element of tissue integrity. Fibroblasts are involved in repair and regeneration processes in almost all human tissues and organs (including lungs). Its primary function is to secrete extracellular matrix (ECM) proteins that provide a tissue scaffold for normal repair events such as epithelial cell migration.

  Fibroblasts, or distinct subpopulations thereof, exhibit tissue-specific functions when immune regulatory cells secrete chemokines and cytokines that can elicit an immune response by attracting inflammatory and immune cells. Fulfill. Fibroblasts from different anatomical sites display a number of common phenotypic characteristics. However, fibroblasts show a distinct phenotype at different anatomical sites. Characteristic expression of fibroblast growth factor and receptor is also characteristic of lung fibroblasts (Non-patent Document 8).

  Based on fibroblast physiology, we can create an artificial tissue scaffold-free tissue system that mimics several aspects of distal lung tissue, We have found that the model is not necessarily the only way to create a three-dimensional lung culture. Without being bound by theory, the inventors believe that the fact that lung fibroblasts secrete ECM contributes significantly to this result.

Lung epithelial cells (lung cells):
Lung cells (lung epithelial cells or alveolar epithelial cells or AEC) are epithelial cells lining the normal alveolar basement membrane (ie, the peripheral gas exchange region in the distal airway of the lung). Lung cells or AECs can be subdivided into type I and type II lung cells.

  The characteristic markers of the two alveolar epithelial cell types are easily traceable and can be monitored during experiments using, for example, RT-PCR reactions or immunohistochemistry.

Type 1 lung cells:
Type 1 lung cells [alveolar type 1 lung cells, type 1 alveolar cells, alveolar type 1 cells (abbreviated as ATI cells), or small alveolar cells, squamous alveolar cells, membranous lung cells, or 1 Type alveolar epithelial cells] are complex branched cells having a large number of cytoplasmic plates which are gas exchange surfaces in the alveoli. These cells are metabolically active and have cell surface receptors for various substances, including extracellular matrix (ECM) proteins, growth factors, and cytokines. About 95% of the alveolar surface is covered with type I lung cells.

Type 2 lung cells:
Type 2 lung cells (alveolar type 2 lung cells, alveolar type 2 cells; ATII cells, abbreviated as T2P) are type 2 alveolar epithelial cells (also abbreviated as AEC and EPII cells), type 2 granular lung cells, 2 Cubic epithelial cells, also called type cells, type 2 alveolar cells, septal cells, or large alveolar cells, large alveolar cells, or granular lung cells. These cells arise from immature epithelial cell precursors. Alveolar type 2 lung cells are thought to be alveolar epithelial progenitor cells. Alveolar type 2 lung cells can self-renew and differentiate into squamous type 1 lung cells. Type II cells constitute only 4% of the alveolar surface area, but are cubic cells that occupy 60% of alveolar epithelial cells and 10-15% of total lung cells (Non-patent Document 9).

Type 3 alveolar epithelial cells:
Type 3 alveolar epithelial cells differ from flat type 1 cells and cubic type 2 cells due to the presence of apical microcilia and the absence of lamellar secretory granules. These cells are also called alveolar brush cells.

Endothelial cells:
Endothelial cells are elliptical cells that line the lumen of all blood vessels as a single squamous cell layer. Endothelial cells are derived from hemangioblasts and hemangioblasts.

Macrophages:
Macrophages are cells derived from bone marrow-derived monocytes (bone marrow-derived macrophages) that have homed to the tissue. Macrophage differentiation from unipotent and bipotent progenitor cells in the bone marrow is controlled by various cytokines. Further differentiation occurs in the tissue and the resulting macrophage population is called resident macrophages.

Mast cells:
Mast cells arise from pluripotent CD34 (+) precursors in the bone marrow (Non-patent Document 10). Immature mast cells take their typical granular form when they migrate into the tissue.

  These cells also express Fc-εR1 and stop the expression of CD34 and Fc-γR2. Most mast cells in the lung and intestinal mucosa produce only tryptase (denoted MCT) or chymase. Mast cells play a central role in immediate allergic reactions by releasing potential mediators.

Smooth muscle cells:
Smooth muscle cells are highly specialized multifunctional contractile cells that regulate the lumen of hollow organs, either transiently (reversible contraction) or chronically (due to fibrosis and muscle hypertrophy). is there. Smooth muscle cells play an important role in angiogenesis, shape the vessel wall and maintain vascular tone.

Further cell observation:
Addition of endothelial cells resulted in stable aggregates containing differentiated cells. As can be seen based on the expressed markers, inclusion of endothelial cells in the model tissue culture does not reduce the degree of differentiation. These aggregates appear to maintain a lamellar structure in which the endothelial cells are located.

Embodiment:
Preparation of 3D model tissue culture:
The method of the present invention uses at least lung epithelial cells and stromal cells, preferably fibroblasts. To obtain a live culture, the cells are cultured separately, then mixed in the appropriate ratio and the presence of CO ₂ under appropriate conditions as understood based on the present disclosure and methods of the art. Co-culture below. By determining the ratio of cells and selecting the conditions, abnormal growth of one cell type by another cell type can be avoided.

  In a preferred embodiment, the cells are obtained from a human subject as primary cells and are dedifferentiated or used immediately. For example, dedifferentiation can be performed by known methods (passaging, removal of other cell types, addition of growth factors). A cell is considered dedifferentiated if it can become confluent.

  Pelleting the co-cultured cell mixture is an important step for establishing cell-cell contact and for creating the proper distance between cells. The most convenient way to pellet the cells is to apply centrifugation. Selecting a suitable means for pelletization is well possible within the ability of one skilled in the art based on the teachings provided herein.

  In principle, in this model, a lung model tissue close to the native lung tissue can be obtained using any of the cells listed above. Each applied cell type must be able to grow under conditions useful to obtain a three-dimensional model tissue as disclosed herein and be associated with other cell types of the model. I must. These elements should be tested in preliminary experiments. For convenience, initially a relatively small ratio of additional cells should be applied, and then the ratio of additional cell types can be increased until a ratio similar to that in vivo is usually achieved.

  In preferred embodiments, additional cell types that can be included in the model are, for example, endothelial cells and smooth muscle cells.

Disease model:
Based on the above model and using a variety of gene delivery methods and a variety of target genes, the above system can identify genetic changes during lung disease that can lead to the identification of new drug targets and the development of new therapies. Easily adapted to study, where
The disease includes inflammation, the affected cell, preferably the epithelial cell, expresses an inflammatory cytokine (above normal levels), the model is an inflammation model,
The disease is a tumor, the cell is a transformed cell, such as a malignant transformed cell or an immortal cell, the model is a tumor model,
The disease includes fibrosis, the model is a fibrosis model,
The disease includes tissue damage and the model is a regeneration model.

  Disease models can be used in drug trials.

Cells obtained from the patient:
In one embodiment, lung cells are obtained from a patient and cultured according to the present invention. In this embodiment, it is preferred that no or only partial dedifferentiation is allowed. Thereafter, in a rapid preparation method, a 3D model tissue culture can be formed to test drugs proposed for treatment of the patient or to test planned treatment regimens. Among other things, the advantage of this embodiment is that a pure and parallel sample culture with a uniform composition and size can be prepared. The sample also does not contain any pathogens and can be purified as needed.

Model prepared from healthy cells:
In a preferred embodiment, a disease model is prepared starting with healthy cells and factors that affect the disease characteristics (symptoms) of the cells are added later.

  For example, a tumor model is prepared from healthy cells, factors that affect malignant transformation are added, and / or genes that cause malignant transformation are expressed therein. It has been observed that the level of Wnt protein (eg, Wnt5) is increased in lung tumor tissue. Accordingly, carcinogenic factors such as EGF (epidermal growth factor), IGF (insulin-like growth factor), insulin, Wnt factor (eg, Wnt5) or a cocktail thereof are added to the cell mixture or culture of the present invention. Therefore, it is assumed that a tumor model can be prepared.

  In an alternative to this method, neoplastic cells are added to a medium in which a model culture according to the present invention is present but separated by a semipermeable membrane. Thereby, factors produced by neoplastic cells induce neoplastic (malignant) transformation of the cultured cells of the present invention.

Lung tumor model made of lung tumor cell lines:
A lung tumor model can be prepared from a lung tumor cell line. Such cell lines are readily available in NPL 11 by searching for tumor cell lines.

  It is wise to experiment to find the appropriate conditions for culturing cells and optimizing the ratio of cell types used in the cell mixture.

Inflammation model:
For inflammation models, monocytes and / or macrophages can be added to the model culture of the present invention, preferably during the preparation process.

  In this model, pretreatment with LPS or WNT5A is sensible.

  The cytokine production of activated macrophages and the production of other factors such as Wnt5 affect tissue culture and allow an inflammation model.

  If one wants to examine an inflammation model system, neutrophil cells can be provided separated from lung aggregates with a membrane in a suitable chamber. In this case, neutrophil migration and MMP production can also be measured.

  In an alternative embodiment, the disease model lung cell line is cultured according to the present invention. In this embodiment, the drug can be tested for efficacy against the disease.

  In this disease model, the use of overexpressed genes should be avoided, rather an inducible promoter should be applied.

Inflammation model with native 3D lung cell aggregates:
To mimic inflammatory conditions, native three-dimensional lung cell aggregates can be treated with various materials that elicit inflammatory responses.

Such materials are, for example,
Chemical substances that cause acute inflammation, such as vasoactive amines, eicosanoids, etc.
Pro-inflammatory polypeptides such as growth factors, hydrolases, etc.
Reactive oxygen species,
Pro-inflammatory cytokines, such as IFN-γ and other cytokines,
Bacterial cell wall extract.

  Inflammatory status, for example, by detecting cytokine expression by biochemical assays, immunological assays (eg, ELISA), PCR-based methods (eg, real-time PCR), or expression analysis, eg, by application of a gene chip Inspect.

Genetic modification of primary cells:
Recombinant viral delivery vectors (r-adenovirus and r-lentiviral vectors) can be used to genetically modify both epithelial and stromal cells, and these gene delivery methods do not impair the ability of the cells to aggregate. Characteristic genes for inflammation, tumors, fibrosis and regeneration can be overexpressed or silenced constitutively or inducibly, and changes in tissue morphology, cellular responses, gene and protein expression can be expressed in three dimensions Can study in the environment.

  For example, one or more genes known to promote tumorigenesis are transferred to a lung cell line, such as an alveolar type I or type II cell line, preferably a type II cell line, or a fibroblast cell line. Can be introduced. Such a gene can be, for example, an oncogene (eg, a ras gene) or a gene or gene set (eg, a COX-2 gene) that is characteristic of a tumor expression pattern. Although expression of the ras gene alone may not be sufficient to transform cells, preferably immortal cells, proliferation is likely to increase (Non-Patent Document 12), which is why the disease of this model Features can be provided.

Modification of secretory factor composition in primary cell aggregates using genetically modified and sublethal irradiated cell lines:
In this example, all cell types remain uninfected, so gene expression is normal as in any given 3D lung tissue model. However, the cellular composition of the aggregate is genetically modified to produce secretory factors (Wnt, bone morphogenetic protein (BMP), inflammatory and pro-inflammatory cytokines, growth factors, etc.) that modify the cellular microenvironment within the aggregate. Containing cells (5-10% of the total number of aggregates) irradiated with a sublethal dose of either the fibroblast cell line (WI-38) or the alveolar epithelial cell line (A549) or both . Irradiation at sublethal doses can reduce cell proliferation and suppress abnormal growth by other cell types of one cell type.

Product:
The invention also provides a kit comprising a plurality of samples of 3D model tissue culture.

  Preferably, the container is a well of a plate (eg, a 96 well plate or a 384 well plate).

  The 3D model tissue can be a model of healthy tissue or a disease model (disease model kit).

  The plate includes, for convenience, a number of side-by-side containers or wells, where the number of containers contains one or more artificial three-dimensional lung model tissue culture samples in a suitable medium.

  The container can be, for example, a flat bottom, U bottom or preferably a V bottom container on a plate that allows parallel testing of multiple samples.

  In order to prevent cells from adhering to the wall of the container, the container is preferably a container that has not been subjected to a tissue culture treatment.

  In a preferred embodiment, each container contains a single agglomerate. In a preferred embodiment, the culture sample in each vessel contains an amount of cells as defined in the brief description of the invention.

The containers are preferably sealed, individually or together, and contain a CO ₂ rich environment or atmosphere suitable for lung tissue culture as defined in the brief description of the invention.

  Usually, this disease model requires the same environment.

  The following cellular characteristics: cellular state, eg, cell phase, cell viability, apoptotic or moribund state; cell type; cell location; malignant transformation; biocompatible suitable to inform about one or more of inflammation It is preferable to stain cells with a sex dye.

Contrast:
As a control sample, the kit contains a culture of epithelial cells and fibroblasts only. Preferably there are at least 3-3 well controls (epithelial cells and fibroblasts, respectively) on the plate.

  Preferably, an additional control that is 2D lung tissue is used to identify or evaluate features specific to 3D tissue.

  Thus, a 2D control plate (preferably a flat bottom adherent tissue culture plate) can be included with 3D tissue upon request. Alternatively, the plate may also include as a control a well of 2D lung tissue, preferably in a flat bottom well.

  Thus, one embodiment of the present invention uses a plate that includes both a V-bottom well for 3D tissue and a flat or U-bottom well for 2D tissue.

Example 1-Materials and Methods:
Primary SAEC, NHLF and lung HMVEC cells were purchased from Lonza. All cell types were isolated from the lungs of several randomly selected hosts of different genders and ages. We used SAGM, FGM or EGM-2 media for initial expansion of SAEC, NHLF or lung HMVEC, respectively, as recommended by the manufacturer. All cell cultures were incubated at 37 ° C. in an atmosphere containing 5% CO ₂ . For 2D and 3D cultures, pure or mixed cell populations were cultured in a 50-50% mixture of SAGM (Small Airway Growth Medium, Lonza) and complete DMEM. For 2 and 3 cell cultures containing HMVEC cells, the appropriate growth factor supplement for HMVEC cells was added to a 50-50% mixture of SAGM and DMEM. Cell culture media compositions were prepared according to the manufacturer's instructions. For 2D and 3D cultures, cells were mixed at the indicated ratios and distributed to flat bottom 6 well plates or 96 well V bottom plates (Sarstedt), respectively. V-bottom plates were centrifuged at 600 × g for 10 minutes at room temperature immediately after seeding the cells.

SAEC and NHLF were stained with the following physiological fluorescent dye: DiI (Non-patent Documents 13 and 14) so that cell migration in the culture could be followed. Cells with or without Matrigel were pipetted into V-bottom 96 well plates that had not been treated for tissue culture and incubated in a 37 ° C. CO ₂ incubator for 1 hour. Following incubation, the cells were pelleted at 2000 rpm for 5 minutes at room temperature, after which the resulting cell pellet was incubated overnight (5% CO ₂ , 37 ° C.).

The A549 strain was established in 1972 by Non-Patent Document 15 by explant culture of lung cancerous tissue from a 58 year old male. A549 cells are adenocarcinoma human alveolar basal epithelial cells. A549 cells are classified into the squamous epithelial segment of epithelial cells. Cells seeded in the above culture medium at a concentration of 2 × 10 ⁴ cells / cm ² become 100% confluent in 5 days.

  A549 cells are available as CCL-185 in the American Type Culture Collection (ATCC; Rockville, MD), and Ham's F-12 medium (GIBCO BRL, Grand Island) containing 10% fetal calf serum (FCS; GIBCO BRL). , NY) or according to the supplier's recommendations.

  The WI-38 cell line was developed in 1962 from lung tissue taken from a therapeutically aborted fetus of about 3 months gestational age. Cells released by trypsin digestion of lung tissue were used for primary culture. The cell morphology is fibroblast-like. Karyotype is 46, XX; normal diploid female. The longest life span of 50 population doublings of this culture was obtained in the repository. A thymidine labeling index of 86% was obtained after reversion. G6PD is isozyme B type. This culture of WI-38 is expanded from frozen cells of passage 9 obtained from the submitter.

  WI-38 cells available as CCL-75 in NPL 16 are grown according to the supplier's recommendations. CL13 or CL30 cells (17) were cultured in Dulbecco's modified Eagle / F12 medium containing 5% fetal calf serum and 25% μg / ml gentamicin.

Human cells are preferably maintained at 37 ° C. in a humid atmosphere containing CO ₂ as necessary.

Fluorescence and confocal microscopy:
Prior to 2D and 3D culture, SAEC, NHLF and HMVEC were stained with the physiological fluorescent dyes CFSE, DiI and DiD (all from Molecular Probes), respectively. Cells were washed twice in PBS and incubated for 10 minutes at 37 ° C. with CFSE, DiI or DiD at a concentration of 0.5 μg / ml. Cells were washed with DMEM + 10% FCS to remove excess dye. 2D and 3D cultures were prepared using fluorescently labeled cells as indicated above. After overnight incubation, the 3D cell culture was carefully removed from the V-bottom plate and transferred to a coverslip bottom dish (MatTek). Lung tissue microcultures were examined by fluorescence microscopy (Olympus IX-81 microscope) or confocal microscopy (Olympus FV1000 confocal imaging system).

Cell sorting:
SAEC and NHLF were stained with CFSE and DiI according to the manufacturer's instructions (Molecular probes). Cells were mixed and cultured for 72 hours in 2D and 3D systems. Stained cells were dissociated with gentle trypsin treatment followed by PBS + EDT treatment. Dissociated cells were selected using a BD FACSVantage cell sorter and placed in a tube containing a lysis buffer (Miltenyi Biotech) for mRNA preparation.

cDNA synthesis and quantitative RT-PCR:
Total RNA was prepared from 2D and 3D cell cultures using the NucleoSpin RNAII kit (Machery-Nagel) with DNase digestion on the column. Messenger RNA was prepared from sorted SAEC samples using the μMACS mRNA isolation system (Miltenyi Biotech). CDNA was prepared from the RNA samples using the MMuLV reverse transcriptase kit (Thermo Scientific). Real-time quantitative PCR experiments were performed using an ABsolute QPCR SYBR Green Low ROX master mix (ABGene) and an Applied Biosystems 7500 thermal cycler system. Primers are listed in Table 1.

Recombinant adenovirus vector:
A complete gene or GFP-only sequence of interest is amplified by a PCR reaction using a forward (5 ′): 5 ′-3 ′, reverse (3 ′): 5 ′-3 ′ primer sequence, and a shuttle vector And then cloned into an adenovirus vector by homologous recombination. Adenovirus was produced by transfecting linearized plasmid DNA into the 293 packaging cell line (American Type Culture Collection, Rockville, MD) using Lipofectamine 2000 (Invitrogen). The resulting plaques were amplified and adenovirus was purified and concentrated using an adenovirus purification kit (BD Biosciences).

Adenovirus infection of epithelial cells:
Adenovirus containing GFP or gene of interest-GFP was added to 2D or 3D SAEC. 1 × 10 ⁶ cells were resuspended in 250 μl of cell culture medium and 50 μl of virus at 37 ° C. for 90 minutes.

CXCL-8 assay:
The CXCL-8 (IL-8) content of 2D and 3D cell culture supernatants was measured using a Quantikine CXCL-8 / IL-8 ELISA kit (R & D Systems). A sandwich ELISA assay was performed according to the manufacturer's instructions. Briefly, similarly diluted cell culture supernatant samples and CXCL-8 standards were distributed into wells pre-coated with anti-CXCL-8 monoclonal antibody. After a 2 hour incubation at room temperature, the plates were washed 4 times with the provided wash buffer. Thereafter, polyclonal anti-CXCL-8 antibody conjugated with HRPO was added for 1 hour. After the final wash step, TMB substrate solution was added to the wells. Optical density was determined using an iEMS reader MF (Thermo Labsystems) at 450 and 570 nm and the data was analyzed using Ascent software.

Data quantification:
Quantitative real-time RT-PCR data was analyzed using the 7500 system SDS software by the ΔCt (dCt) and relative quantity (RQ) method as proposed by Applied Biosystems. All samples were prepared in duplicate. Briefly, Ct values were determined for each sample using an automatic threshold level determined by 7500 system SDS software. The ΔCt (dCt) value was determined according to the following formula: dCt (target gene) = Ct (target gene) −Ct (housekeeping gene). The change in gene expression is expressed by the following formula:
RQ = 2− ^ddCt (where ddCt value was calculated as ddCt = dCt (sample) −dCt (reference sample))
It is shown as an RQ value calculated using.

  The OD was compared to a standard curve calculated from seven different concentrations of CXCL-8 ranging from 31.2 to 2000 pg / ml to determine the CXCL-8 content of the cell culture supernatant. Two replicates were made to distribute the samples and the average was used for further data analysis.

Example 2-3 Experiment to develop a three-dimensional lung tissue model:
Hanging drop model:
In order to simulate human lung structure, we performed 3D of 100,000 cells in a random mixed, approximately equal volume of distinct fibroblast population (NHLF) and small airway epithelial cell population (SAEC). Start with cell clumps. Within 1 day of incubation in the hanging drop assay, the cells produced loose tissue structures. FIG. 1 shows a hanging drop culture of a mixture of 50% SAEC and 50% NHLF.

  However, the formation was not stable and it was impossible to transfer the produced microtissue from the initial culture conditions to another test plate without irreparable damage to the tissue structure.

Pelleted, matrigel-containing model:
In order to improve the stability of the mixed lung microtissue, a 1: 1 ratio of SAEC and NHLF was pelleted and grown in the presence of Matrigel. Many 3D lung tissue models and other tissue models use Matrigel to create a three-dimensional structure in which various cell types can grow and interact with each other. SAEC and NHLF were stained with the physiological fluorescent dye DiI (Non-Patent Documents 18 and 19) so that cell migration in the culture could be followed. Cells and Matrigel were pipetted into a V-bottom 96-well plate that had not been treated for tissue culture and left in a 37 ° C. CO ₂ incubator for 1 hour. Following incubation, cells were pelleted at 2000 rpm for 5 minutes at room temperature, after which the resulting cell pellet was incubated overnight (5% CO ₂ , 37 ° C.).

  FIG. 2 shows a Matrigel-based culture of a mixture of 50% SAEC and 50% NHLF. It is clear that despite the presence of Matrigel, SAEC and NHLF were unable to form a stable 3D structure. Furthermore, the small spherical tissue structure containing mainly epithelial cells appeared to be distant from the tissue / matrigel mass.

Example 3-Pelleted, artificial tissue scaffold-free model:
As a next step in simulating 3D culture conditions of human lung, a random mixture of the same number of epithelial cells (SAEC) and fibroblasts (NHLF) was pelleted in two steps without matrigel. The cells were pipetted and placed in a V-bottom 96-well plate that had not been treated for tissue culture and left in a 37 ° C. CO ₂ incubator for 1 hour. Following incubation, the cells were pelleted at 2000 rpm for 5 minutes at room temperature, after which the resulting cell pellet was incubated overnight (5% CO ₂ , 37 ° C.). Cells before mixed culture were prepared by diluting 1: 1000 dilution of physiological fluorescent dye DiI (1 mg / ml stock in DMSO) and CFSE (1 mg / ml stock in DMSO) in PBS (phosphate buffered saline pH 7.2). Was stained. Under these culture conditions, the cells form stable aggregates in which the most adherent fibroblasts (red or dark gray) are the less adherent epithelial cell types (green or light gray). ) (Figure 3). The agglomerate diameter is about 200 μm.

  In further experiments, the ratio of SAEC cells to NHLF cells was systematically changed and the following SAEC: NHLF ratios were obtained: 0/100%, 25/75%, 50/50%, 75/25%, 100, respectively. Cultures were prepared using a ratio of 0/0%, otherwise as described above. FIG. 3 shows pelleted microtissue cultures containing different ratios of SAEC and NHLF. As is apparent from the figure, the most prominent 3D structural aggregate with a surface epithelial cell inner layer is formed when the same amount of epithelial cells and fibroblasts is applied, the aggregate being a 25/75% ratio. And slightly smaller and less well-defined, but with regular epithelial cell lining, but with excess SAEC cells, ie when the ratio of epithelial cells to fibroblasts is 75/25% A more uniform epithelial lining is formed. Pure cultures do not form 3D structured aggregates (epithelial cells) or have a much smaller aggregate size (fibroblasts).

Example 4-2 Characterization of a cell-type tissue scaffold-free 3D lung tissue model:
Differentiation markers:
The molecular characterization of this model was based on epithelial differentiation markers using real-time PCR analysis. mRNA was purified from cell aggregates to produce cDNA. Results were analyzed using TTF1 (FIG. 4a), AQ3 (FIG. 4b) and AQ5 specific primers compared to β-actin as an internal control.

  FIG. 4a shows TTF-1 mRNA levels in 3D human lung microtissue. TTF1 transcription factor is a characteristic marker of alveolar epithelial cells. Although 3D fibroblast cultures do not show TTF1 expression, TTF1 is present in 3D SAEC alone cultures and increased in 2D SAEC / NHLF co-cultures, indicating the beneficial effects of fibroblasts. Maximum levels of TT1 expression were reached in 3D SAEC / NHLF tissue.

  FIG. 4b shows AQP-3 water transporter mRNA levels in 3D human lung microtissue. AQ3 is an ATII epithelial marker in the lung. Although 3D fibroblast cultures do not show AQ3 expression, AQ3 is present in 3D SAEC alone cultures and increased in 2D SAEC / NHLF co-cultures, indicating the beneficial effects of fibroblasts Nevertheless, maximal levels of AQ3 were observed in 3D SAEC / NHLF tissue culture.

  Thus, the above markers showed an inducible increase in ATI type differentiation, further supported by the lack of increased ATI type marker expression.

  The purified differentiated cell type used by the inventors in the experiment was obtained from a commercial source. Although these cell types were based on differentiated tissue, once purified and maintained under 2D culture conditions, these cells almost immediately showed signs of dedifferentiation as indicated by increased levels of S100A4. (FIG. 6 and Table 2). As soon as SAEC was co-cultured with NHLF, S100A4 and N-cadherin levels were significantly reduced, but E-cadherin levels were increased. Non-Patent Document 20 shows that it is more prominent in 3D culture conditions than in 2D culture conditions (FIG. 6), and apart from the presence of NHLF, it was shown that 3D structures are also required to reduce SAEC dedifferentiation. .

  These changes are also characteristic of epithelial-stromal transition (EMT) characteristic of the epithelial dedifferentiation process.

Pro-inflammatory cytokine expression:
When induced by lung infection or alveolar epithelial damage (continuous destruction of the epithelial cell layer), pro-inflammatory cytokines are produced by the alveolar epithelium and attract inflammatory cells, including neutrophils. CXCL-8 protein from cell supernatants of 2D alone and co-cultures and 3D tissue co-cultures prepared as seen in FIG. 3 to study the expression of the pro-inflammatory cytokine CXCL-8 The level was examined. Using commercially available ELISA test kits (R & D Laboratories), CXCL-8 expression levels are significantly reduced in 3D tissue systems compared to conventional 2D tissue cultures (FIG. 7a) and when epithelial cell ratio is maximal It was clarified that minimal levels were detected, suggesting that CXCL-8 expression is induced by disruption of the epithelial cell layer.

  In the diagram of FIG. 7, CXCL-8 secretion is shown in a human in vitro 3D lung model. It was found that 3D single cultures of fibroblasts do not secrete CXCL-8, but 3D SAEC (to a lesser extent than 2D SAEC cultures—data not shown) still produce cytokines. 2D co-cultures did not significantly change CXCL-8 expression, indicating that the presence of fibroblasts cannot affect cytokine expression. CXCL-8 expression levels are markedly reduced in 3D tissue systems when the epithelial-fibroblast ratio is 75/25% and the epithelial cells are essentially completely covered by fibroblasts, and CXCL- It was suggested that 8 expression can be induced by disruption of the alveolar epithelial cell layer. This reduction in CXCL-8 expression was somewhat inconspicuous at the 50/50% and 25/75% ratios.

  Quantitative real-time RT-PCR analysis was also used to examine mRNA levels of the inflammatory cytokines IL-1β and IL-6. When PCR was performed from a mixture of SAEC-NHLF mRNA, both IL-1β and IL-6 mRNA levels were significantly down-regulated in 3D cultures compared to the respective 2D cultures (FIG. 7B, respectively). And 7C). Once SAEC cells were selected from 2D and 3D NHLF co-cultures, the decrease in IL-1β and IL-6 expression in SAECs cultured in 3D conditions was even more pronounced (FIG. 7D).

Example 5-Three-cell type model containing epithelial cells, endothelial cells and fibroblast components:
To further improve the complexity of our lung tissue culture model, we added primary human lung-derived microvascular endothelial cells (HMVEC) to SAEC and NHLF cells and co-cultured SAEC and NHLF. 2D and 3D tissue microcultures similar to those were prepared.

  Microstructural morphological examination by fluorescence and confocal microscopy revealed that HMVEC can successfully co-culture with both SAEC and NHLF cells in 3D conditions (FIG. 8a). Interestingly, in co-cultures with either SAEC or NHLF, HMVEC formed a compact core inside the microculture. When three cell types (1: 1: 1) were cultured together in 3D conditions, they adhered together and formed a remarkably compact and stable 3D structure (FIG. 8b).

  Molecular characterization of 3 cell cultures revealed that the levels of AQP3 and KRT7 expression were significantly increased in 3D cultures compared to levels measured in 2D culture conditions (FIG. 9a and Table 2). ). The levels of EMT markers S100A4 and N-cad increased when cells were maintained in 2D culture, but were stabilized in 3D culture. A slight decrease in E-cad in 3D cultures was not detected under 2D culture conditions (Figure 9a and Table 2). Quantitative RT-PCR was performed from mixed mRNA of three cell types, but in order to find the optimal proportion of the three cell types that helped to build tissue from purified tissue elements, 3D cultures of the three cell types Further analysis of the object is necessary.

Table 2 shows gene expression changes in human primary lung cell cultures. The numbers are RQ values calculated according to the formula RQ = 2− ^ddCt , where the ddCt value is calculated as ddCt = dCt (sample) −dCt (reference sample). When the data was presented as dCt values, except for the selected SAEC ^** , it was calculated as follows: dCt = Ct (target gene) -Ct (housekeeping gene). A portion of the cells collected before preparing the various cultures was always used as a reference sample. The ΔCt (dCt) value was calculated as follows: dCt (target gene) = Ct (target gene) −Ct (housekeeping gene). Except in the case of sorted SAEC ^** samples (in this case actin was used as the housekeeping gene), 18S ribosomal RNA was used as the housekeeping gene.

Abbreviations:
N. A. : Data not available and expression level could not be determined. N. D. : No specific PCR product was detected by real-time QPCR. N. D. ^* : No specific PCR product was detected by conventional PCR. N. C: No specific expression level was obtained in parallel wells or samples by real-time QPCR. Low ^* : A relatively low expression level was detected by conventional PCR. High ^* : A relatively high expression level was detected by conventional PCR.

Summary of results for cell markers and factors secreted by cells:
We have shown that 3D co-culture of primary lung epithelial cells and fibroblasts is advantageous for maintaining SAEC more differentiated state than either 2D or 3D in vitro single culture. Offered here. Including NHLF not only facilitated epithelial differentiation, but 3D microtissue aggregation and structure compared to SAEC-only cultures (FIG. 3) or SAEC-HMVEC cultures (FIG. 8a), It became much harder and more compact.

  The TTF1 transcription factor is a characteristic general marker of embryonic and postnatal alveolar epithelial cells in ATII cells. Cytokeratin is a component of the intermediate filament of the cytoskeleton, and its expression pattern is important for cell lineage identification. In our experiments, lung epithelial markers TTF-1 and cytokeratin 7 (KRT7) showed increased expression levels in 3D co-cultures.

Type II lung cells facilitate transepithelial migration of water (via members of the aquaporin protein (AQP) family). The ATII marker aquaporin 3 shows increased levels in the presence of fibroblasts (FIG. 5). Surfactant protein secretion is a unique feature of ATII lung epithelial cells (Non-Patent Documents 21 and 22).
In our experiments, SAEC cells in a single culture were unable to express surfactant protein, but surfactant protein A1 mRNA was eventually in 3D and in lesser amounts in 2D co-culture. Was expressed (FIG. 5c). However, surfactant proteins B and C were eventually not detected by our 3D co-culture system (data not shown). The inventors also examined the expression of the ATI markers aquaporins 4 and 5 in both 2-cell and 3-cell cultures, but no expression of these molecules was eventually detected. This is probably because the cells used herein (SAEC) are rather type ATII. In addition, ATI differentiation requires a significant amount of time (data not shown). ATI markers appear when cells with ATI-type characteristics are used with slightly longer incubation times.

  Thus, with the exception of SFPC, the differentiation markers AQP3, KRT7, TTF1 and SFPA were upregulated in the presence of fibroblasts. AQP3 and SFTPA levels were further increased under 3D culture conditions, but KRT7 or TTF1 differentiation markers were not increased.

  S100A4 is a well-known molecular marker of epithelial-stromal transition, and its expression level is often high in metastatic carcinoma (Non-patent document 23) and pulmonary fibrosis (Non-patent document 24). Up-regulation of S100A4 and N-cadherin and concomitant down-regulation of E-cadherin (Non-patent Documents 25 and 26) are also characteristic of epithelial dedifferentiation and loss of cell adhesion, suppression of E-cadherin expression, And epithelial-stromal transition (EMT) characteristics characterized by increased cell migration.

  The dedifferentiation markers S100A4 and N-cadherin appeared in purified primary cells under 2D culture conditions.

  The above dedifferentiation markers decreased in the presence of fibroblasts and further decreased in 3D conditions.

  A decrease in inflammatory markers including IL1b, IL6 and CXCL8 could be observed in 3D cultures compared to 2D cultures. The marker was markedly down-regulated under 3D culture conditions, for example, the presence of fibroblasts simply in 2D culture was not sufficient to reduce its level.

  SFTPA1 expression was observed in 2-cell cultures but not in 3-cell cultures (FIG. 5c and data not shown).

Example 6-Disease model:
Lung tumor cell line derived lung tumor model:
An artificial three-dimensional lung tissue culture is prepared as described in Example 3 with the following modifications.

  Type II alveolar epithelial cells (A549) instead of epithelial cells (SAEC), and NNK [4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone], a model of lung adenocarcinoma A combination of 5-20% CL13 or CL30 cells (Non-patent Document 28) derived from treated A / J mice (Non-patent Document 27) is used. CL13 or CL30 cells carry a mutation in the Ki-ras gene.

  A series of experiments is performed to find the appropriate ratio of A549 cells to CL13 or CL30 cells. A 3D lung model tissue is prepared using the ratio at which the tumor spontaneously forms and is used as a lung tumor disease model.

  An alternative to the above method uses patient tumor cells.

Modification of secretory factor composition in primary cell aggregates using genetically modified and sublethal irradiated cell lines:
All cell types remain uninfected, so gene expression is normal as in any given 3D lung tissue model. However, the cellular composition of the aggregate is genetically modified to produce secretory factors (Wnt, bone morphogenetic protein (BMP), inflammatory and pro-inflammatory cytokines, growth factors, etc.) that modify the cellular microenvironment within the aggregate. Cells irradiated with radiation below the lethal dose of either the fibroblast cell line (WI-38) or the alveolar epithelial cell line (A549) (5-10% of the total number of cells in the aggregate).

Native 3D lung cell aggregate:
To mimic inflammatory conditions, native three-dimensional lung cell aggregates are treated with various concentrations of bacterial cell wall extracts. Cytokine production is determined from tissue culture media using cytokine-specific ELISA techniques, and gene expression changes in both epithelial cells and fibroblasts can be quantified by real-time PCR reactions.

Example 7-3D lung tissue kit:
In this example, a ready-to-test 3C lung tissue kit of the following characteristics is prepared.
1. Lung tissue ready for examination is supplied in a 96-well plate.
2. There is a small sample (80000 cells / well) of lung model tissue in the well that can be readily experimented or tested.
3. Each tissue consists of a mixed culture of human primary alveolar epithelium and fibroblasts (25, 50 and 75% epithelium, respectively).
4). Plates contain 3-3 well controls (epithelial and fibroblasts only).
5. The plate is sealed with a transparent, preferably adhesive, plastic foil (eg with Saran Wrap®).

  Tissue quality is guaranteed for 3 days, including delivery.

  The plate itself is a V-bottom 96 well non-adherent tissue culture plate.

Model tissue is prepared as described in Example 3. Each tissue is immersed in 200 l tissue culture medium optimal for lung culture in a 5% CO ₂ environment, sealed, and placed on room temperature or ice.

  Quality control: One tissue is taken from each well and examined for viability. Differentiation markers are examined by real-time PCR.

  If desired, 2D control plates (tissues grown on flat bottom 96-well adherent tissue culture plates) can be included with 3D tissue.

References:
Non-Patent Documents 31-69

  The basic parameters and culture conditions described above have been established for an ATII type tissue scaffold-free lung model, in which spontaneous self-assembly of cells and cell interactions can be studied. it can. This model allows for easy handling and genetic manipulation of complex tissue systems in both theoretical and applied research and pharmaceutical testing. This model is also easily expanded by additional cell types to include endothelial cells for vascularization, as well as smooth muscle cells, in which case further study of interrelated tissue and cell interactions be able to.

  Scaffold-free 3D culture allows for smooth genetic manipulation of simple or more complex tissue systems in both theoretical and applied research and pharmaceutical testing. This lung tissue model is particularly suitable for studying spontaneous self-assembly and cell interactions of cells. Both biomedical research and pharmaceutical companies need an in vitro model that enhances the effectiveness of predicting the toxicity or efficacy of drug candidate molecules in the preclinical phase (29). The newly established guidelines also emphasize the replacement of animal models and encourage the development of new preclinical test methods (Non-patent Document 30).

  Based on the above model and using various gene delivery methods and various target genes, our 3D human lung microtissue model system will lead to the identification of new drug targets and the development of new therapies Can be easily adapted to study genetic changes during lung disease. These disease models can include inflammation models, tumor models, pulmonary fibrosis models, or regeneration models.

  Three-dimensional models of healthy lung tissue and diseased tissue are available. The products according to the invention can be marketed, for example, in the form of tissue cultures, plates or arrays containing such cultures, or kits.

Claims (22)

An artificial 3D lung model tissue culture comprising:
a) does not include an artificial tissue scaffold that is a porous 3D matrix, 3D gel matrix or porous membrane support;
b) composed of cultured cells,
c) comprises at least lung epithelial cells and stromal cells, preferably fibroblasts;
d) including one or more cell aggregates having a surface on which a large number of lung epithelial cells are present,
e) An artificial three-dimensional lung model tissue culture characterized in that the epithelial cells express an epithelial differentiation marker. The size or average size of the cell clumps is at least 80 μm and at most 600 μm, preferably 100-300 μm,
And / or the amount of cells is
At least 2 × 10 ⁴ cells, preferably at least 4 × 10 ⁴ cells,
The artificial three-dimensional lung model tissue culture according to claim 1, which is at most 5 x 10 ⁵ cells, preferably at most 2 x 10 ⁵ or at most 10 ⁵ cells. The model tissue culture does not include an artificial tissue scaffold and / or
The model tissue culture includes an extracellular matrix, and the extracellular matrix protein is secreted by at least one of the cell types contained in the tissue culture, preferably by fibroblasts, and / or
The epithelial cells form a lung epithelial cell inner layer on at least a part of the surface of the cell aggregate, and preferably the lung epithelial cell inner layer at least partially covers the surface of the cell aggregate. The described artificial 3D lung model tissue culture. The model tissue culture comprises cultured primary cells obtained from the subject and / or
The model tissue culture includes cells that have been dedifferentiated before culture and cells that have been redifferentiated after culture, and / or
The artificial three-dimensional lung model tissue culture according to any one of claims 1 to 3, wherein the model tissue culture contains cells of an established cell line. The artificial three-dimensional lung model tissue culture according to any one of claims 1 to 4, comprising an endothelial cell. The lung epithelial cells comprise at least one of cell types: type I lung cells (ATI cells), type II lung cells (ATII cells),
The artificial three-dimensional lung according to any one of claims 1 to 5, wherein the epithelial differentiation marker expressed by tissue cells of the artificial three-dimensional lung model tissue is preferably either an ATII type differentiation marker or an ATI type differentiation marker. Model tissue culture. Markers: levels of TTF1, AQP3, SFTPA, SFTPC, KRT7 are increased compared to controls containing uncultured cells, preferably purified primary cells,
Markers: CXCL8, IL1b, S100A4 levels are reduced compared to controls containing uncultured cells,
Markers: AQP3, SFTPA levels are increased compared to control 2D cultures,
The artificial three-dimensional lung model tissue culture according to any one of claims 1 to 6, wherein the levels of the markers: CXCL8, IL1b, IL6, S100A4, and N-cadherin are decreased as compared to the control two-dimensional culture. The cultured cells, preferably lung epithelial cells and / or stromal cells comprise pathological cells having pathological characteristics of affected lung tissue, wherein the model tissue culture is a lung disease model tissue culture, preferably
The disease is accompanied by inflammation, the pathological cells express inflammatory cytokines above normal levels, and the model is an inflammation model,
The disease is a tumor, and the cell is a transformed cell selected from the group comprising malignant transformed cells or immortal cells, and the model is a tumor model,
The disease is associated with fibrosis and the model is a fibrosis model,
The artificial three-dimensional lung model tissue culture according to any one of claims 1 to 7, wherein the disease is accompanied by tissue damage and the model is a regeneration model. A method for preparing an artificial three-dimensional lung model tissue culture comprising:
Preparing a mixed suspension of at least lung epithelial cells and stromal cells, preferably fibroblasts, more preferably primary fibroblasts;
Pelleting the cells of the suspension;
Pelleting the cells,
Incubating the pelleted suspension in the presence of CO ₂ for a time sufficient for the cells to form a three-dimensional lung model tissue comprising one or more cell clumps;
If necessary, model organization
a) an increase in the expression level when compared to the reference culture is considered to indicate the formation of a three-dimensional lung model tissue culture, the expression of one or more epithelial differentiation markers characteristic of the model tissue culture, and / or ,
b) a decrease in expression level when compared to a suitable reference culture is considered to indicate the formation of a three-dimensional lung model tissue culture and the expression of one or more pro-inflammatory cytokines;
c) assaying for the size and morphology of one or more agglomerates, comprising preparing an artificial three-dimensional lung model tissue culture. The average size of the cell clumps is at least 80 μm and at most 600 μm, preferably 100-300 μm and / or
The amount of cells in one or more cell aggregates is
At least 2 × 10 ⁴ cells, preferably at least 4 × 10 ⁴ cells, and
10. The method according to claim 9, wherein there are at most 5 x 10 < ⁵ > cells, preferably at most 2 x 10 < ⁵ > or at most 10 < ⁵ > cells. Pelleted suspension
In the presence of CO ₂ ,
A period of 2 weeks or less, preferably a period of 12, 10, 8, 7, 6 or 5 days or less, and
The method according to claim 9 or 10, wherein the incubation is performed for a period of 10 hours or longer, preferably for a period of 12 or 14 hours or longer. In preparing the mixed suspension, the one or more types of cells are placed in a container within 18 hours, preferably within 12 hours, more preferably within 4 hours, particularly preferably within 1 hour or simultaneously. The method according to any one of claims 9 to 11, particularly preferably, each type of cell used is added within that period. The container is a V-bottomed container that has not been processed for tissue culture and / or
Pelletization is performed at 200-600 g for 1-20 minutes, preferably 2-10 minutes and / or
Suitable to inform the cell about one or more of the following cellular characteristics: cellular state of the cell, eg, cell phase, cell viability, apoptotic or moribund state; cell type; cell location; malignant transformation; inflammation The method according to claim 9, wherein the method is dyed with a new biocompatible dye. At least the mixed suspension of lung epithelial cells and lung stromal cells comprises cultured primary cells obtained from the subject and / or
At least the mixed suspension of lung epithelial cells and lung stromal cells contains cells that have been dedifferentiated prior to culture, and the cells are redifferentiated by culture and / or
The method according to any one of claims 9 to 13, wherein at least the mixed suspension of lung epithelial cells and lung stromal cells contains cells of an established cell line. The cultured cells, preferably lung epithelial cells and / or stromal cells include pathological cells having pathological characteristics of diseased lung tissue in which the model tissue culture becomes a lung disease model tissue culture. The method in any one of. An artificial three-dimensional lung model tissue culture obtained by the method according to any one of claims 9 to 15, and preferably the artificial three-dimensional lung model tissue culture according to any one of claims 1 to 7. A method of screening for the effect of a drug on lung tissue,
Preparing an artificial three-dimensional lung model tissue culture according to any one of claims 1 to 8, 16;
Collecting at least a test sample and a reference sample of the model tissue culture;
Contacting the test sample with the drug while maintaining the test sample and the reference sample under the same conditions;
Detecting any change or change in the test sample as compared to the reference sample, and evaluating any change or change in the test sample as indicating the effect of the drug when detected. . 18. The model tissue culture is a model of healthy lung tissue, and the adverse effect of the drug is tested, and a change or change that harms the cells of the test sample is evaluated as a toxic effect or adverse effect of the drug. Method. The model tissue culture is a lung disease model tissue culture containing pathological cells with pathological features, and the beneficial effects of the drug are tested,
To obtain the extent of the disease, prepare an assay on the model tissue culture that measures or evaluates pathological features,
Preparing a reference sample of healthy lung tissue (health reference sample) and / or a reference sample of diseased lung tissue (affected reference sample);
Measuring or assessing pathological characteristics in a health reference sample and / or diseased reference sample and at least one test sample before and after contacting it with a drug;
Any change or change in the test sample that changes the degree of disease in the test sample to the degree of disease in the health reference sample and / or changes from the degree of disease in the affected reference sample is assessed as a beneficial effect of the drug. The method of claim 17. At least two containers in which each sample of the artificial three-dimensional lung model tissue culture according to any of claims 1 to 8, 16 is contained in a separate container of a plate;
An appropriate medium for culturing cells of the model tissue culture;
An artificial three-dimensional lung model tissue kit comprising a test plate having a plurality of containers. Whether the plate is a 96-well plate,
Whether the plate is a V-bottom plate,
The culture sample in the container
At least 10 ³ , preferably at least 10 ⁴ , more preferably at least 2 × 10 ⁴ , 3 × 10 ⁴ , 4 × 10 ⁴ , 5 × 10 ⁴ cells, and
An amount of at most 10 ⁷ , preferably at most 10 ⁶ , more preferably at most 5 × 10 ⁵ , 4 × 10 ⁵ , 3 × 10 ⁵ , 2 × 10 ⁵ cells, or at most 10 ⁵ . Containing cells of
The containers are sealed individually or together and contain a CO ₂ rich environment or atmosphere suitable for lung tissue culture,
An environment or atmosphere rich in CO ₂
Contains at least 2%, 3%, 4% CO ₂ environment, at most 10%, 9%, 8% or 7% CO ₂ environment, particularly preferably about 5% CO ₂ ,
The artificial three-dimensional lung model tissue kit according to claim 20, which has one or more of any of the following characteristics. Parallel screening for the effects of compounds on lung tissue selected from the group including toxicity testing, search for candidate compounds,
A patient-specific test for its therapeutic ability of the compound, provided that the model tissue culture is obtained from primary patient lung cells,
Study cell interactions in lung tissue,
Study of genetic changes during the period of the disease selected from the group comprising lung disease,
Use of the three-dimensional lung model tissue culture according to any one of claims 1 to 7 for any of the above.

Images