Methods for generation of cytocapsulae and cytocapsular tubes

Abstract

To provide methods for generation of cytocapsulae and cytocapsular tubes.SOLUTION: A method of generating cytocapsulae and cytocapsular tubes includes implanting cells in the top of a 3D matrix, and incubating the cell implanted 3D matrix under conditions where the cells engender the cytocapsulae and cytocapsular tubes. In one aspect, the 3D matrix comprises elastin, laminin, collagen, proteoglycans, or non-proteoglycan polysaccharide, and the proteoglycans include heparan sulfate, chondroitin sulfate, and keratin sulfate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of development of cytocapsulae and cytocapsular tubes, and methods and compositions thereof. [Background technology]

[0002] Cell boundaries separate cells from the external environment, allowing intracellular activities and contents and protecting them from the unwanted effects of environmental factors. Cell boundaries are essential for cell sensing, migration, invasion, rearrangement, proliferation, growth, differentiation, communication with and response to the environment, uptake of nutrients and oxygen, elimination of metabolic waste products, and protection against environmental stresses.

[0003] Cell migration in multicellular organisms is important in embryonic development (Le Douarin NM (1984) Cell migrations in embryos. Cell 38(2):353-360; Reig G, Pulgar E, & Concha ML (2014) Cell migration: from tissue culture to embryos. Development 141(10):1999-2013; Richardson BE & Lehmann R (2010) Mechanisms guiding primordial germ cell migration: strategies from different organisms. Nat Rev Mol Cell Biol 11(1):37-49), organ homeostasis (Acloque H, Adams MS, Fishwick K, Bronner-Fraser M, & Nieto MA (2009) Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease. The Journal of clinical investigation 119(6):1438-1449), tissue regeneration (Bryant DM & Mostov KE (2008) From cells to organs: building polarized tissue. Nat Rev Mol Cell Biol 9(11):887-901), immune response (Woodland DL & Kohlmeier JE (2009) Migration, maintenance and recall of memory T cells in peripheral tissues. Nat Rev Immunol 9(3):153-161; Weninger W, Biro M, & Jain R (2014) Leukocyte migration in the interstitial space of non-lymphoid organs.Nat Rev Immunol 14(4):232-246), wound repair (Gurtner GC, Werner S, Barrandon Y, & Longaker MT (2008) Wound repair and regeneration. Nature 453(7193):314-321), and tumor dissemination (Friedl P & Alexander S (2011) Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147(5):992-1009; Friedl P & Wolf K (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3(5):362-374; Barbolina MV, et al. (2009) Microenvironmental regulation of ovarian cancer metastasis. Cancer Treat Res 149:319-334). Cell migration in three-dimensional (3D) microenvironments experiences heterogeneous cells and extracellular matrices (ECMs) that provide support and cues to guide the direction of locomotion, while essentially forming environmental obstacles that impede cell motility (Thiery JP, Acloque H, Huang RY, & Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139(5):871-890; Franz CM, Jones GE, & Ridley AJ (2002) Cell migration in development and disease.Dev Cell 2(2):153-158). To enhance motility, cells have been known to generate lamellipodia (Murphy DA & Courtneidge SA (2011) The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function. Nat Rev Mol Cell Biol 12(7):413-426), filopodia (Mattila PK & Lappalainen P (2008) Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol 9(6):446-454), podosomes (Tarone G, Cirillo D, Giancotti FG, Comoglio PM, & Marchisio PC (1985) Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp Cell Res 159(1):141-157), and invadopodia (Chen WT (1989) Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. J Exp Zool 251(2):167-185), blebbing (Fackler OT & Grosse R (2008) Cell motility through plasma membrane blebbing. J Cell Biol 181(6):879-884; Ridley AJ (2011) Life at the leading edge. Cell 145(7):1012-1022), focal adhesions (Wehrle-Haller B (2012) Structure and function of focal adhesions.Current opinion in cell biology 24(1):116-124), dendritic projections, and type II epithelial bridges (Zani BG, Indolfi L, & Edelman ER (2010) Tubular bridges for bronchial epithelial cell migration and communication. PLoS One 5(1):e8930). Thus, the dynamics of cell rearrangements in multicellular organisms are unclear.

[0004] Cell morphology, organelles employed, and mechanical and signaling control in 3D cell migration often differ from their 2D counterparts (Baker BM & Chen CS (2012) Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J Cell Sci 125(Pt 13):3015-3024; Wu PH, Giri A, Sun SX, & Wirtz D (2014) Three-dimensional cell migration does not follow a random walk. Proceedings of the National Academy of Sciences of the United States of America 111(11):3949-3954; Friedl P, Sahai E, Weiss S, & Yamada KM (2012) New dimensions in cell migration. Nat Rev Mol Cell Biol 13(11):743-747).Cells migrating in collagen-rich 3D matrices do not display lamellipodia or filopodia, but show highly dendritic protrusions that are absent on the surface of rigid plates (Jayatilaka H, ​​et al. (2017) Synergistic IL-6 and IL-8 paracrine signalling pathway infers a strategy to inhibit tumour cell migration. Nature communications 8:15584; Giri A, et al. (2013) The Arp2 / 3 complex mediates multigeneration dendritic protrusions for efficient 3-dimensional cancer cell migration. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 27(10):4089-4099).

[0005] There is a continuing need in the art for methods and compositions useful for understanding cellular locomotion in 3D extracellular matrices and 3D microenvironments. Summary of the Invention

[0006] The present disclosure addresses this need and is based on the discovery that cells in a controlled 3D microenvironment, such as a 3D extracellular matrix, can generate at least two novel membranous organelles, namely cytocapsulae and cytocapsular tubes. Cytocapsulae provide a tubular pathway for directed cell transport. Multiple cytocapsular tubes interconnect to form a network of tubular meshes for directed cell transport in various directions. Enhanced cap-dependent translation initiation and increased expression of proteins, including ITGB-2, have been found to be associated with the formation of cytocapsulae and cytocapsular tubes as well as the elongation of cytocapsular tubes. Methods and compositions for generating cytocapsulae and cytocapsular tubes in a controlled 3D extracellular matrix provide a powerful tool for understanding the mechanisms underlying cell migration in 3D microenvironments. This is a prerequisite for the development of effective therapies for a wide range of diseases related to cell migration, cell sensing, cell stress protection, cell proliferation, cell differentiation, tumor growth, tumor initiation, tumor metastasis, tumor recurrence, and drug resistance.

[0007] According to one aspect of the present disclosure, there is provided a composition comprising a 3D extracellular matrix, a cell capsule and a cell duct, said cell capsule and cell duct developed by cells embedded in or on said 3D matrix.

[0008] According to another aspect of the present disclosure, there is provided a method for generating cell sacs and cell cystic ducts, comprising embedding cells in or on a 3D matrix and incubating the cell-embedded 3D matrix under conditions such that the cells generate the cell sacs and cell cystic ducts.

[0009] According to yet another aspect of the present disclosure, there is provided a method for producing cell sacs and cell ducts in vitro, comprising embedding cells in a single cell suspension into a top layer of a 3D matrix; and culturing the cell-embedded 3D matrix under conditions such that the cells develop cell sacs and cell ducts in the 3D matrix.

[0010] According to yet another aspect of the present disclosure, there is provided a method for preparing a 3D matrix suitable for the development of cell sacs and cell ducts, said method comprising freezing said 3D matrix and thawing said 3D matrix at a suitable temperature.

[0011] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have the meaning ascribed to them in U.S. patent law, e.g., they may mean "includes," "included," "including," and the like, and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, e.g., they allow for elements not expressly recited, but exclude elements that are recognized in the prior art or that affect a basic or novel characteristic of the invention.

[0012] These and other embodiments are disclosed in or are obvious from, and are encompassed by, the following detailed description of the invention. [Brief description of the drawings]

[0013] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of exemplary embodiments taken in conjunction with the accompanying drawings, which include: [Figure 1A]Figure 1A shows images of cell capsule and cell capsule duct development. Figure 1A shows images of cell capsule and elongated cell capsule duct development. Human mammary epithelial cells (HMECs) embedded on thick 3D Matrigel gel under the indicated conditions develop membranous extracytoplasmic cell capsules and elongated cell capsule ducts. Fluoresence images of cell capsule ducts of single HMECs transiently overexpressing EGFP-PMCA2 and mCherry-β-actin along with cell capsule ducts are shown. HMEC (red arrow), cell capsule (CC, white arrow), and cell capsule membrane (CCM, orange-yellow arrow) are shown. Scale bar = 10 μm. [Figure 1B] Figure 1B shows images of the development of cell sacs and cystic ducts. Quantitative analysis of the length of the cystic ducts. Scale bar = 10 μm. [Figure 1C] Images of cell sac and cystic duct development. FIG. 1C shows an inverted phase contrast bright field microscopy image of ecellularization of cell sacs. A single HMEC, indicated by a blue arrow, develops a giant cell sac (CC, red arrow) that encases the cells. After 1 h, the HMEC detaches from the cell sac, leaving behind an ecellulated cell sac (eCC, white arrow) with no cells inside. The cell sac membrane (CCM, orange-yellow arrow) is shown. Scale bar = 10 μm. [Figure 1D] Figure ID shows images of the development of cell sacs and cell ducts. Schematic diagram of ecellularization of cell sacs. Scale bar = 10 μm. [Figure 1E] Figure 1C shows images of the development of cell sacs and cystic ducts. Figure 1D shows a quantitative assay of cell sac size in diameter. Scale bar = 10 μm. [Figure 1F] Figure 1F shows images of the development of cell sacs and cystic ducts. DIC (left panel) and fluorescence (right panel) images of a large membranous cell sac (CC, red arrow). Scale bar = 10 μm. [Diagram 2]Figure 1 shows the cell cystic duct network of HMECs. Migration and network formation of HMECs in cell cystic ducts. Shown are cell cystic ducts (CT, red arrows), cell cystic duct junctions (CTN), multiple cells migrating in single cell cystic ducts (orange-yellow arrows), and a population of cells without cell cystic ducts (black arrows). [Figure 3A] The diagram shows the structure of the cell cystic duct network. (FIG. 3A) Cell cystic ducts of multiple BCSCs intersect. Cell cystic ducts of multiple BCSCs are interconnected via cell cystic duct nodes (CTNs) to form a crossed morphology. Cells migrate in the cell cystic duct network. Scale bar = 10 μm. [Figure 3B] The diagram shows the structure of the cell cystic duct network. (FIG. 3B) A basic morphology of the cell cystic duct (CT, red arrow) network of BCSCs. Up to five cell cystic ducts are connected via a cell cystic duct node (CTN). In the cell cystic duct networks of various morphologies, multiple cells migrate. Scale bar = 10 μm. [Figure 4] Figure 1 shows real-time imaging of the BCSC cell cystic network system by cytolysis analysis. Bright-field image of the cell cystic network by real-time imaging with cytolysis. Nested cell cystic ducts (CT, red arrows) and cell cystic nodes (CTN) in the cell cystic network are shown. Image was taken 1-2 seconds after treatment with cell lysis buffer. All cell cystic ducts and cell plasma membranes were dissolved and disappeared 3 seconds after treatment with lysis buffer. [Figure 5A] Figure 5. Merger of cell cysts and cell invasion. (Fig. 5A) Bright field image of a merged cell cyst duct connected by two cell cysts generated by two cells. Cell 2 is located inside the merged cell cyst duct. The merged cell cyst duct (MTP, red arrow) and merged cell cyst membrane (MTM, orange-yellow arrow) are indicated. [Figure 5B] Figure 5B shows the merging of cell sacs and cell invasion. (Figure 5B) DIC and fluorescence microscopy images of a giant merging cell sac with multiple cells (cell clusters) located within the merging giant cell sac. The merging cell duct membrane (MTM, orange-yellow arrow) is indicated. [Figure 5C]Figure 5C shows cell cyst merger and cell invasion. (Figure 5C) Quantification of cell invasion. [Figure 5D] Figure 5D shows cell capsule merging and cell invasion. Immunofluoresence microscopy of endogenous syncytin 1 in HMEC. [Figure 5E] Figure 5E shows the merger of cell sacs and the invasion of cells. (FIG. 5E) Western blot image of syncytin 1 expression during the development of cell sac ducts. [Figure 5F] Figure 5F: Cell cyst merger and cell invasion. Knockdown of syncytin 1 reduces cell cyst duct merger in HMECs. [Figure 5G] Figure 5G shows cystic duct merging and cell invasion. Knockdown of syncytin 1 does not affect cystic duct initiation in HMECs. [Figure 6A] ITGB-2 regulates the elongation of the cystic duct. (FIG. 6A) Heat map of transcriptional changes of 26 integrin genes during the development of the cystic duct. [Figure 6B] 6B shows ITGB-2 regulating the elongation of the cell cyst duct (FIG. 6B) The effect of ITGB-2 on the development of cell cysts and the elongation of the cell cyst duct. Description of the Invention

[0014] Aspects of the present disclosure are based on the previously undiscovered observation that cells, when embedded and cultured in a controlled 3D extracellular matrix, can generate two novel extracellular membranous organelles, herein referred to as cytocapsulae and cytocapsular tubes. Cells migrate in the cytocapsulae and generate cytocapsular tubes, which exhibit multifaceted biological functions and provide tubular pathways for directed cell transport within the matrix. Multiple cytocapsular tubes are formed and interconnect to generate a network that supports directed cell transport in various directions within the matrix. The present disclosure proposes a mechanism for directed cell translocation via cytocapsulae in a 3D microenvironment. The disclosed methods and compositions for generating cell sacs and cell cyst ducts will facilitate understanding of the mechanisms underlying the embryonic development of multicellular organisms, organ homeostasis, tissue regeneration, and immune responses, as well as the development of therapeutics for the treatment and management of a wide range of diseases and biological processes, including, but not limited to, cell migration, cell sensing, cell stress protection, cell proliferation, cell differentiation, tumor growth, tumor initiation, tumor metastasis, tumor recurrence, and drug resistance.

[0015] As observed, the life cycle of cell sacs and cell sac ducts developed in 3D extracellular matrices according to the methods described herein can be outlined as follows: In the first stage, single / individual cells develop small, round, extracellular, membranous cell sacs that encapsulate the cells. Subsequently, the cell sacs undergo multiple developmental phases. First, the cell sacs enter a stage of ecellularization. As a result of ecellularization, the cell sacs become completely separated from the expelled cells and become acellular cell sacs. Incomplete separation of the cell sacs and the expelled cells has also been observed. In the case of incomplete separation, the expelled cells remain connected to the acellular cell sacs and may re-invade the connected acellular cell sacs by autoinvasion, reforming the closed cell sac with the intraluminal cells. Second, the cell sacs grow and form giant (approximately 100-250 μm in diameter / long axis), round or ovoid cell sacs. The giant cell sacs may shrink slightly to form shrunken cell sacs that encapsulate the intraluminal cells. On the other hand, other cells from the surrounding environment can invade the single giant cell sac containing the cells, leading to a single cell sac with multiple cells inside. Ecellularization of these giant cell sacs generates large acellular cell sacs that shrink and contract to form large contracted concave (or irregularly shaped) disks. Third, cells migrate in the cell sacs, deforming the cell sac membrane and generating elongated cell sac ducts. Non-autoinvasion of surrounding cells allows multiple cells to invade and migrate into the cell sac ducts. Cell migration (single or multiple cells) in homogeneous and membrane-enclosed cell sac ducts is faster than that observed in heterogeneous environments composed of heterogeneous extracellular matrix (ECM) and other cells and structures. Presumably, in cell sac ducts, cell migration is free of the obstruction of ECM, cells and structures. The cell sacs form an interconnected, branched, seamless, membranous tubular network, providing a tubular meshwork system for directional 3D cell transport / locomotion in various directions. The process of ecellularization generates acellular cell sacs and cell sacs. All acellular cell sacs and cell sacs undergo a rapid autolysis phase.

[0016] According to one aspect, a composition is provided comprising a 3D extracellular matrix, a cell sac, and a cell cystic duct. In one embodiment, the cell sac and cell cystic duct are generated by a single cell embedded in or on the 3D matrix. In one embodiment, the cell is embedded on the top surface of the 3D matrix. Prior to embedding, the cells are present in a concentration of about 1×10 to about 1×10 5 The cells are processed into a single cell suspension at a density of 100 / ml.

[0017] According to one embodiment, the cell sacs and ducts encapsulate the cells that give rise to the cell sacs and ducts. According to another embodiment, the cell sacs and ducts contain a plurality of cells that invade the cell sacs and ducts from the surrounding environment. According to yet another embodiment, the cell sacs and ducts undergo ecellularization to support the encapsulated cells and form acellular cell sacs and ducts that are devoid of cells.

[0018] According to certain embodiments, a 3D matrix is ​​provided in which a plurality of cells in approximately a single cell suspension are added into or on top of the 3D matrix. Incubation of the cell-embedded 3D matrix under controlled conditions results in single cells developing cell sacs and cell sac ducts.

[0019] According to one embodiment, the 3D matrix is ​​porous. Porosity can be obtained as a result of polymerization and / or crosslinking of the molecules used to make the matrix material. Porosity is controlled by varying the crosslink density, chain length and percentage of copolymerized branched monomers according to methods known to those skilled in the art. According to one embodiment, the 3D matrix is ​​porous to the extent that additional reagents can diffuse or otherwise move through the matrix. Porous matrices may be made according to methods known to those skilled in the art. Further control of molecular sieve size and density is achieved by adding additional crosslinkers such as functionalized polyethylene glycol.

[0020] According to another embodiment, the 3D matrix is ​​viscous. The viscosity of the 3D matrix can be adjusted according to any means known in the art.

[0021] According to one embodiment, the 3D matrix material is chemically inert and thermally stable to allow for various temperatures. According to one embodiment, the 3D matrix material is optically transparent. According to one embodiment, the 3D matrix material is optically transparent to allow for 3D imaging techniques known to those skilled in the art. According to one embodiment, the matrix is ​​sufficiently optically transparent or has otherwise suitable optical properties for deep 3D imaging for high throughput information reading.

[0022] According to one embodiment, the materials used to form the matrix are biodegradable. According to another embodiment, the materials used to form the matrix are compatible with a wide range of in situ biological and non-biological specimens.

[0023] According to one embodiment, the matrix material can be a semi-solid medium that can be composed of polyacrylamide, cellulose, alginate, polyamide, cross-linked agarose, cross-linked dextran, or cross-linked polyethylene glycol. In a particular embodiment, the semi-solid medium can be attached to a solid support, such as a microscope slide, a culture plate, or a flow cell. The solid support can be attached to the bottom surface of the semi-solid medium.

[0024] Matrix-forming materials include, but are not limited to, polyacrylamide, cellulose, alginate, polyamide, cross-linked agarose, cross-linked dextran, or cross-linked polyethylene glycol. The matrix-forming materials can be formed by polymerization and / or cross-linking of the matrix-forming materials using methods specific for the matrix-forming material and methods, reagents, and conditions known to those skilled in the art.

[0025] Matrix-forming materials may further include, but are not limited to, elastin, laminin, proteoglycans (such as heparan sulfate, chondroitin sulfate, and keratin sulfate), and non-proteoglycan polysaccharides (such as hyaluronic acid). In certain embodiments, the matrix-forming materials may also include proteins, including, but not limited to, fibrous, FACIT, short chain, and basement membrane proteins. In other embodiments, the matrix-forming materials may further include signaling proteins, including, but not limited to, focal adhesion kinase (FAK), talin, vinculin, paxillin, α-actinin, and GTPases. Biodegradable, biocompatible polymers may be used as matrix materials, including, for example, but not limited to, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-polyglycolic acid copolymers (PLG).

[0026] According to certain embodiments, matrix is ​​used with solid support.For example, said matrix can be polymerized so that one surface of said matrix is ​​attached to solid support (e.g., glass surface), while the other surface of said matrix is ​​exposed or sandwiched between two solid supports.According to one embodiment, said matrix can be contained in a container.

[0027] The solid support of the present disclosure can be made into various shapes. In certain embodiments, the solid support is substantially planar. Examples of solid supports include slides, microtiter plates, flow cells, cover slips, plates such as microchips, containers such as microcentrifuge tubes and test tubes, tubes, sheets, pads, films, etc. Furthermore, the solid support can be, for example, biological, non-biological, organic, inorganic, or a combination thereof.

[0028] Certain characteristics of the matrix have been found to be crucial for the development of the cell capsules and cystic ducts in the matrix. For example, the degree of polymerization, protein concentration, and viscoelasticity of the matrix affect the development of cell capsules and cystic ducts. The present disclosure contemplates a wide range of degrees of polymerization of the matrix, from as low as about 10% polymerization to full 100% polymerization. The degree of polymerization of the matrix is ​​also related to the density and viscoelasticity of the matrix. Additionally, proteins present in the matrix or subsequently added to the matrix also affect the development of cell capsules and cystic ducts. The present disclosure contemplates a final protein concentration in the matrix ranging between about 2-12 mg / ml. The present disclosure further contemplates a pH range of 4-8 for the matrix. The degree of polymerization, gradient, density, and viscoelasiticity of the matrix affect the hard-region-directed movement of cell migration within the matrix. In one embodiment, the 3D matrix is ​​Matrigel.

[0029] Cells according to the present disclosure include eukaryotic cells, animal cells, plant cells, insect cells including but not limited to Drosophila cells, C. elegans (C. elegant) cells, and the like. Exemplary cells include any cell, human or otherwise, including abnormal or normal cells. Particular cells include human cells, non-human cells, human stem cells, differentiated cells, induced pluripotent stem cells (iPSCs), genetically modified cells, human pluripotent cells, epithelial cells, endothelial cells, immune cells, muscle cells, mouse stem cells, primary cell lines, immortalized cell lines, primary and immortalized fibroblasts, HeLa cells and neurons, and tumor cells. In one embodiment, the cell is a mammalian cell. In another embodiment, the cell is a human mammary epithelial cell. In a particular embodiment, the cell is a stem cell, whether adult or embryonic. In one embodiment, the cell is a pluripotent stem cell. In another embodiment, the cell is an induced pluripotent stem cell. In yet another embodiment, the cell is a human induced pluripotent stem cell. According to a particular embodiment, said cell is in vitro, in vivo or ex vivo.

[0030] According to another aspect, the present disclosure provides a method for the generation of cell sacs and cystic ducts. In one embodiment, the method includes embedding cells in or on a 3D matrix and incubating the cell-embedded 3D matrix at a temperature suitable for the generation of the cell sacs and cystic ducts. The incubation temperature suitable for the generation of the cell sacs and cystic ducts is approximately 37°C and varies for certain cell types. In one embodiment, the 3D matrix is ​​frozen prior to embedding. In certain embodiments, embedding is performed at a temperature ranging between about 1-45°C, 1-37°C, 1-6°C, and 2-4°C. In other embodiments, the thickness of the 3D matrix ranges between about 1-1000 μm, 2-100 μm, 5-50 μm, and 5-10 μm.

[0031] According to certain aspects, the cell sacs and cystic ducts encapsulate the cells that give rise to the cell sacs and cystic ducts. According to other aspects, the cell sacs and cystic ducts allow the egress of the encapsulated cells that give rise to the cell sacs and cystic ducts. According to yet other aspects, the cell sacs and cystic ducts allow the entry of multiple cells from the surrounding environment.

[0032] According to one embodiment, the cells are about 1×10 to 1×10 5 It is presented as a single cell suspension with a density between 100 and 150 / ml.

[0033] The membranes of the cell sacs and cell cysts have certain characteristics. According to one embodiment, the membranes of the cell sacs and cell cysts contain the plasma membrane protein Ca 2+ According to another embodiment, syncytin 1 regulates cell cyst merging and cell invasion. According to yet another embodiment, growth factors regulate cell cyst development and cell cyst duct development. According to yet another embodiment, ITGB-2 mediates cell migration in cell cyst ducts and regulates cell cyst duct elongation. According to yet another embodiment, matrix metalloproteinase mediates cell cyst duct elongation.

[0034] In certain embodiments, the 3D matrix comprises bioactive and / or bioinert agents including, but not limited to, collagen, matrigel matrix material, elastin, laminin, proteoglycans (such as heparun sulfate, chondroitin sulfate, and keratin sulfate), non-proteoglycan polysaccharides (such as hyaluronic acid), proteins (such as fibrous, FACIT, short chain, and basement membrane proteins), and signaling proteins (such as FAK, talin, vinculin, paxllin, α-actinin, and GTPases).

[0035] According to another aspect, a method for producing cell sacs and cell ducts in vitro is provided. In one embodiment, the method includes embedding cells in a single cell suspension into a top layer of a 3D matrix; and culturing the embedded cells in a suitable medium and at a suitable temperature, where each single cell develops cell sacs and cell ducts in the 3D matrix.

[0036] According to yet another aspect, a method for preparing a 3D matrix suitable for the development of cell sacs and cell ducts is provided. In one embodiment, the method comprises freezing the 3D matrix and thawing the 3D matrix at a suitable temperature. Suitable temperatures for thawing the matrix are between about 1-45°C, 1-37°C, 1-6°C, and 2-4°C. EXAMPLES

[0037] The following examples are presented for the purpose of illustrating various embodiments of the present invention and are not meant to limit the present invention in any manner. The examples, together with the methods described herein, are currently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the present invention. These and other uses that are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0038] Example I Development of cell sacs and cell ducts To investigate the dynamics of cell boundaries and 3D cell migration, normal primary human mammary epithelial cells (HMECs) were embedded in 3D Matrigel matrix, a reconstituted extracellular matrix (ECM) surrogate, according to certain embodiments of the present disclosure. Single spherical HMECs in 3D Matrigel developed extracellular bubble-like capsules with round / irregular shapes of various sizes that encapsulated the cells, whereas cells in a 2D environment did not (Figure 1A). Approximately 96% of single HMECs (1x10 in 6-well plates) were embedded in 3D Matrigel matrix, a reconstituted extracellular matrix (ECM) surrogate, according to certain embodiments of the present disclosure. 3 HMECs (n = 3 / well) developed extracellular capsules at 12 h. The diameter / long axis of the extracellular globule / utricle increased significantly, reaching a maximum of 250 μm. To confirm whether the capsule surface was membranous, we transfected HMECs with enhanced green fluorescent protein (EGFP) and the plasma membrane protein Ca. 2+ We transiently overexpressed a fusion with ATPase 2 (EGFP-PMCA2). Indeed, EGFP-PMCA2 was distributed in both the plasma membrane and the extracellular capsule membrane of the cells, proving that the extracellular capsules are enclosed by a membrane (Figure 1A). These extracellular capsules could elongate to form long tubes of various lengths (Figure 1B). Over time, single capsules automatically ecellularized, leaving acellular closed capsules of multiple morphologies (spherical, ovoid, or irregular) with taut membranes (Figures 1C-1D). These capsules were of various sizes and could grow up to 100 μm in diameter (Figure 1E). These capsules were membranous (Figure 1F). These observations demonstrated that the capsule membrane is independently located outside the plasma membrane and that acellular capsules can exist without cells. We named this previously unappreciated extracellular membranous capsule generated by a single mammalian cell a capsule.

[0039] Example II Ecellularization of cell capsules In most cases, after ecellularization of the cell pouch, the cells are completely separated from the acellular cell pouch, which collapses to form a shrunken concave disk (FIG. 1C). Occasionally, ecellularization of the cell pouch results in incomplete separation of the expelled cells from the acellular cell pouch.

[0040] After ecellularization, the growth of the acellular cysts was terminated, strongly suggesting that the development and growth of cysts depended on intraluminal cells. Meanwhile, the huge gap / distance between the plasma membrane and the extracellular cyst membrane indicated that cyst components derived from intraluminal cells were released / delivered to the outside of the plasma membrane for the construction of cysts (Figure 1C). Then, the acellular cysts continued to shrink and contract, and the cyst membrane folded, forming a huge contracted concave disk, or a short flat tube. After about 0.5-1 h, the membrane of the acellular cysts degraded, and the cysts autolyzed. These observations suggested that the life cycle of cysts went through several successive and distinct phases: from initiation, to growth, ecellularization, shrinkage and shrinkage, membrane degradation, and autolysis.

[0041] Cell contact at high cell density reduces the development of cell sacs and significantly increases their degradation. Inhibition of cell contact at high cell density does not completely suppress the development of cell sacs or induce their degradation. Cell contact at high cell density reduces the growth of cell sacs in diameter.

[0042] Over time, single cells migrate bidirectionally in their cysts, deforming the cyst membrane and generating elongated cysts to form membranous tubes of various lengths (Figure 2). The intraluminal cells were then expelled from the cyst ducts. Acellular cyst ducts were generated by ecellularization of the cyst ducts. The long acellular cyst ducts then autoshrink, and the membrane folds significantly to form contracted, coiled, twisted, membrane-condensed filaments. The membrane of the long, shrunken acellular cyst duct then degrades, followed by autolysis of the duct. These sequential developmental stages of the cyst ducts demonstrated that the life cycle of the cyst ducts proceeds sequentially from single cell migration in their cysts to deformation of the cyst membrane, elongation of the cyst duct, formation of the cyst duct, ecellularization, and autolysis of the acellular cyst duct.

[0043] Cell ducts generated by a single HMEC can reach lengths up to 820 μm. Increased cell density reduces the average length of cell ducts but not their average width (approximately 11 μm in diameter / width). Furthermore, high cell density leads to a decrease in the average life span of cell ducts. Furthermore, high cell density reduces the average density (in length and number) of cell ducts. Some cell ducts can tolerate a compressed microenvironment.

[0044] Next, we investigated the role of HMLER (CD44 高 / CD24 低 ) FA We evaluated breast cancer stem cells in a subpopulation (Yi T, et al. (2014) Quantitative phosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1 / CXCR4 in breast cancer stem cells. Proceedings of the National Academy of Sciences of the United States of America 111(21):E2182-2190). 2 and 1 × 103 At a low cell density of 10 cells / well, approximately 99% of BCSCs developed cell sacs in 3D Matrigel in 15 hours. BCSC cell sacs have a higher ability to withstand the inhibition of cell contact. BCSC cell sac ducts are statistically longer than HMEC cell sac ducts, and a single BCSC cell sac duct can reach up to 1000 μm in length.

[0045] Example III Cysts and cyst ducts are extracellular organelles The inventors have discovered that cell sacs and cystic ducts are not extensions of plasma membrane or connected cell surfaces. They are generated by a single cell, surrounded by their own generated membrane that is different from the plasma membrane, and encapsulate the cell that generated them. Cell sacs and cystic ducts have unique features, including extracellular membranous sacs (or ducts) that encase cells, and allow ecellularization and cell entry, in addition to multifaceted biological functions. On the other hand, cell sacs provide long tubular ways for the directional transport of multiple cells, which cell sacs do not. Cell sacs and cystic ducts are extracellular membranous organelles that encase cells, rather than extensions of plasma membrane. Cell sacs and cystic ducts do not exist in 2D cell cultures. Cell sacs and cystic ducts have multifaceted biological functions, including providing a support scaffold and a covering for the encapsulated cells, and allowing ecellularization and cell entry. Cells can migrate in cell sacs, cell cystic ducts, and cell cystic duct networks. Autolysis of cell sacs and cystic ducts does not affect cell survival, proliferation, and growth. Ecellularized cell sacs and cystic ducts can exist for up to 98 hours. Cell sacs and cystic ducts can accommodate multiple cells, such as tens of cells. Cell sacs and cystic ducts interconnect to form an open tubular network for directional cell translocation in multiple directions. These characteristics of the cell sacs and cystic ducts demonstrated that they are two novel facultative organelles that are distinct from all previously described organelles.

[0046] Example IV Formation of a cystic network At 6 hours, a single primary normal HMEC generated large membranous short cystic ducts in the indicated Matrigel matrix (Figure 2). At 10 hours, HMECs migrated in short cystic ducts and generated elongated cystic ducts (Figure 2). At 36 hours, the long cystic ducts of multiple HMECs interconnected and formed a network via connective nodes (Figure 2). At 68 hours, HMECs aggregated to form a cell cluster, and all HMEC cystic ducts had disintegrated, with no cystic ducts remaining (Figure 2).

[0047] At 4 hours, breast cancer stem cells (BCSCs) had already generated large short cell cystic ducts in the 3D Matrigel matrix shown (Figure 3A). BCSCs migrated bidirectionally in the cell cystic ducts, deformed the cell cystic membrane, shaped the ductal morphology, and generated long curved cell cystic ducts (Figure 3B). Multiple BCSC cell cystic ducts connected to form various morphologies, including closed rings. Many cancer cells migrated in cell cystic ducts with various morphologies, including flow-shaped and linear (Figure 3). Then, BCSCs aggregated to form tumor spheres with spherical or irregular shapes. Next, we quantified the length of BCSC cell cystic ducts over time. BCSC cell cystic ducts could reach a length of up to 1000 μm. Multiple cell cystic ducts could connect to form longer ducts. The number of cell cystic ducts decreased over time, indicating that the length of cell cystic ducts is dynamically and tightly controlled (Figure 4).

[0048] The above data demonstrated that cytocapsular ducts generally undergo successive but distinct phases: initiation of cytocapsular tunes, cell migration in the cytocapsular tunes, elongation of the cytocapsular tunes and formation of cytocapsular ducts, connection and network formation of multiple cytocapsular tunes, cell migration in the cytocapsular tune network, cell aggregation and formation of cell clusters / clumps, and degradation of cytocapsular ducts.

[0049] Two or more HMEC cell cystic ducts of various lengths interconnect and form a tubular network through connective nodes. At 38 hours, BCSCs generated more cell cystic duct connective nodes than HMECs. Furthermore, the average number of cell cystic ducts per connective node of BCSCs is greater than that of HMECs. The density of cell cystic ducts of BCSCs is approximately three times higher than that of HMECs. These data demonstrated that BCSCs have a more aggressive ability to generate interconnected cell cystic duct networks. The life span of BCSC cell cystic ducts with isolated or connected cells is significantly longer than that of acellular BCSC cell cystic ducts, indicating that intraluminal cells promote the maintenance of ducts against degradation. Compared with isolated BCSC cell cystic ducts with cells, connected BCSC cell cystic ducts with cells remain statistically intact for a longer period of time. These results suggested that the maintenance of cell cystic ducts is tightly controlled and mediated by intraluminal cells. Importantly, the cystic ducts of BCSCs interconnected to form tubular networks in tumors in vivo, and multiple cells migrated in these tubular networks. These data demonstrated that the cystic duct networks provided a membranous tubular meshwork for directed cell migration in various directions.

[0050] Example V The cystic duct is a highway for cell transport Cells migrate in a membranous cell cystic duct network, where tubular cell cystic ducts provide a highway for cell translocation (Figures 2-3). We used rapid cell lysis and real-time imaging techniques to investigate the cell cystic duct network structure. The cell cystic ducts of BCSCs extensively and actively interconnect, significantly forming crossings, open rings, closed rings, and many other irregular morphologies, providing an extra-large tubular network for directed cell relocation of cancer cells to distant destinations in multiple directions (Figure 4). Both single cells and multiple cells migrate faster in cell cystic ducts than in 3D environments. The cell cystic ducts form a highway for directed 3D cell transport.

[0051] Example VI Syncytin 1 regulates cell cyst duct merging and cell invasion Cell sacs of different cells can merge to form giant cell sacs, and cells can invade cell sacs of other cells (Figure 5A). Cells can invade cell sacs of other cells, resulting in a single giant cell sac with multiple cells or cell populations inside (Figure 5B-5C). Next, we explored the molecular mechanisms underlying cell sac fusion, merger, and cell invasion. An endogenous membrane fusion protein, syncytin 1, was expressed in HMECs, cell sacs, and cell sac ducts. Transient knockdown of syncytin 1 in HMECs significantly reduced cell sac duct elongation, connection, but not cell sac initiation (Figure 5D-5F). Furthermore, the protein level of syncytin 1 was increased during cell sac duct elongation (Figure 5G). These data demonstrated that syncytin 1 regulates cell sac duct merger and cell invasion.

[0052] Example VII Integrin subunit beta-2 (ITGB-2) promotes cell cyst development Integrins are cell surface support molecules essential for cell adhesion, attachment, migration and invasion. We examined 26 integrin gene transcripts in BM-MSCs, HMECs and BCSCs during the development of cystic ducts. ITGB-2 significantly increased during the process of cystic duct elongation (Fig. 6A). Furthermore, transient knockdown of ITGB-2 using gene-specific shRNA resulted in a significant decrease in cystic duct elongation, but knockdown of ITGA-8 did not (Fig. 6B). These data demonstrated that ITGB-2 mediates cell migration in cystic ducts and regulates cystic duct elongation.

[0053] In summary, this disclosure provides that single cells develop two novel organelles, the extracellular membranous cyst and the cyst duct, and that the cyst duct provides a tubular highway for 3D cell transport. Cell locomotion in multicellular organisms is crucial for embryonic development, tissue formation, organ homeostasis, immune response, wound healing, tissue regeneration, and tumor metastasis.

[0054] The emergence and development of cell sacs and cystic ducts strongly depends on both the 3D environment and cellular activity, and in this study we focus on them only when the biochemical, biophysical and biomechanical characteristics of the 3D matrix (polymerization, density and viscoelasiticity, etc.) are in a precisely controlled manner. The spatiotemporal appearance, self-degradation and auto-decomposition of cell sacs and cystic ducts all contribute to the difficulty of recognizing and identifying these previously unknown organelles.

[0055] The cell sac membrane provides a substantial biomembranous scaffold that supports cell adhesion, attachment, detachment, morphological transition, and motility plasticity. On the other hand, the cell sac encases cells and physically shields them from the microenvironment outside the cell sac. This may facilitate the interconversion of the tight spherical cells into a loose irregular morphology and serve as a protective covering against environmental stress. The cell sac ducts provide membranous tubes for the directed migration and bidirectional locomotion of multiple cells. More importantly, the cell sac ducts interconnect and form a tubular network, which significantly increases the direction of cell migration, amplifies the cell spreading area, and increases the efficiency of directed 3D cell migration. Although inhibition of cell contact at high cell density reduces the development of cell sacs, increases the degradation of cell sacs, and reduces the duration of cell sac ducts, there are still approximately 1.2‰ of normal cells (HMECs) and up to 1.1% of breast cancer stem cells that develop cell sacs and long sac ducts for the directed migration of multiple cells. Thus, compared to the morphology that all cells inevitably experience with the compacted and heterogeneous 3D ECM and heterogeneous obstacles posed by cells in vivo, a morphology in which a small proportion of cells develop uniform membranous cysts that serve as highways for the directed transport of multiple cells is an efficient morphology, or at least an alternative pattern. Cell sacs and cysts exhibit potential multifaceted biological functions and should be further studied to elucidate other functions.

[0056] The enlargement of the cysts depends on the activity of intraluminal cells, while acellular cysts terminate their growth. On the other hand, acellular cysts and cyst ducts undergo rapid auto-degradation and self-decomposition. In addition to intracellular organelles, eukaryotic cells contain extracellular organelles, such as exosomes, that are released or shed into the microenvironment (Keller S, Sanderson MP, Stoeck A, & Altevogt P (2006) Exosomes: from biogenesis and secretion to biological function. Immunology letters 107(2):102-108). Interestingly, the lumen of the cyst ducts contains a large number of different single-layered membrane-enclosed vesicles. These membranous vesicles may thus be functionally linked to carriers or cargoes that deliver / shuttle cyst components of cellular origin and to receptacles containing lysates whose release leads to tightly controlled autolysis of cysts and cyst ducts.

[0057] The phenomenon that acellular cystic ducts shrink and contract into contracted, shortened, twisted and folded filaments after ecellularization is consistent with the absence of an intraluminal skeleton within the cystic duct. The absence of a microfilament network in the lumen of the cystic duct is consistent with single and multiple cells actively migrating in the cystic duct. On the other hand, the presence of a microfilament scaffold beneath the cystic duct membrane is consistent with the fact that long cystic ducts can be pulled by cells and cross the ECM surface without cutting, interruption or obstruction.

[0058] Cell migration in the cyst induces deformation and elongation of the cyst membrane and the formation and elongation of the cyst duct. The assembly of the membrane and other components in the cyst and duct requires many different proteins, suggesting that mRNA translation and / or protein biosynthesis are essential for the development of the cyst and duct.

[0059] In summary, the two novel organelles revealed in this study, the cell sac and cell duct, exhibit multifaceted biological functions including providing tubular pathways and networks for cell transport, relocation and migration, which may provide insights into understanding processes involving cell protection and translocation in the development and pathogenesis of diseases, including tumor metastasis.

[0060] Example VIII Materials and Methods Cells and reagents Primary normal human mammary epithelial cells (HMEC) were ordered from ATCC (PCS-600-010™). HMLER (CD44 高 / CD24 低 ) FA BCSC subpopulation cells were prepared as previously described (27). FITC-conjugated anti-CD44 (BD Biosciences; G44-26) and phycoerythrin-conjugated anti-CD24 (BD Biosciences, ML15) antibodies were used for cell sorting using flow cytometry. MEGM™ Mammary Epithelial Cell Growth Medium BulletKit™ (Clonetics™ MEGM™ Mammary Epithelial Cell Growth Medium and SingleQuots™ Kit Package) was ordered from Lonza (CC-3150). Matrigel™ Basement Membrane Matrix (CB-40234) was purchased from Corning. BD Matrigel Matrix Growth Factor Reduced (GFR, Cat. No. 356230) was ordered from BD Bioscience.

[0061] Time-lapse DIC microscopy, transient transfection, quantitative real-time PCR, and transient gene knockdownTime-lapse DIC (differentiation interference contrast) microscopic analysis of cell capsule elongation and cell migration was performed using a Nikon Ti motorized inverted microscope and a digital Hamamatsu ORCA-ER cooled CCD camera with a 20x lens. HMEC cultures with cell capsules in Matrigel matrix (>40 μm deep) were analyzed by TEM. EGFP-hPMCA2z / b (Addgene, #47584) and / or mCherry-β-actin (#54967) plasmids were co-transfected into HMECs using lipofectatine® 2000. Quantitative real-time PCR assays were performed using gene-specific primers (IDT Company), iQ SYBR® Green Supermix (Bio-Rad), and 7900HT Fast Real-Time PCR.

[0062] Reagents and antibodies EGFP-hPMCA2z / b (#47584) and mCherry-β-actin (#54967) plasmids were ordered from Addgene. Anti-GAPDH (catalog no. 2118S, diluted 1:1000 in Western blot assay) antibody was ordered from Cell Signaling Technology. DAPI (4,6-diamidine-2-phenylindole dihydrochloride, diluted 1:1000 in immunofluorescence assay) was ordered from KPL. Anti-γ-actin (γ-actin, monoclonal, ab123034, diluted 1:1000 in immunofluorescence assay), anti-pan-cadherin (polyclonal, ab140338, diluted 1:1000 in immunofluorescence assay) antibodies were ordered from Abcam.

[0063] Transient transfectionHMECs were cultured in MEGM at 37°C in a humidified atmosphere of 5% CO2. EGFP-hPMCA2z / (Addgene, #47584) and / or mCherry-β-actin (#54967) plasmids were co-transfected into HMECs using lipofectatine® 2000 (Life Technologies, #11668027) according to the manual. Two days after transfection, cells were used for assays.

[0064] Development of cell sacs and cell ducts HMECs (with or without transfection), BM-MSCs, and HMLER (CD44 高 / CD24 低 ) FA Subpopulations of BCSCs were cultured in MEGM medium at the indicated cell densities (i.e., 5 × 10 in 6-well plates). 2 cells / well, or 1.2 x 10 cells / well in a 10 cm dish if not indicated. 4 Cells were then plated on the Matrigel matrix layer at 100 μm depth (30 μm). 3D Matrigel layers (>40 μm deep) were prepared by quickly adding cold thawed Matrigel matrix (thawed overnight on ice in a cold room at 4 °C; protein concentration 4–12 mg / ml) to pre-cooled 6-well plates (with or without cold microcover glass), followed by adding cold MEGM (4 °C) and incubating for 5–25 min in a hood at room temperature (25 °C). Cells were then embedded on the 3D Matrigel gel surface and cultured in a humidified incubator (37 °C, 5% CO2). Cells in (or that have invaded) the Matrigel gel in the various layers develop cell sacs and cell cyst ducts. Various stages of developed cell sacs and cell cyst ducts were used in this study.

[0065] Time-lapse DIC microscopy and imagingTime-lapse DIC (differential interference contrast) microscopic analysis of cell capsule elongation and cell migration was performed using a Nikon Ti motorized inverted microscope and a digital Hamamatsu ORCA-ER cooled CCD camera with a 20x lens. The time-lapse microscope was equipped with DIC, phase contrast, and epifluorescence optics, a Prior Proscan III motorized stage and shutter, a full focus system, and an OkoLab 37°C, 5% CO2 cage microscope incubator (OKO Lab). Images were acquired every 30 seconds for 10-36 hours. All images were acquired using MetaMorph software. Tracks of cell capsule elongation up to 2 hours were acquired using MetaMorph and ImageJ software. Cell capsule elongation velocity was also calculated using length and time measurements. Movies were generated (15 frames per second, fps) using images acquired via time-lapse and MetaMorph software.

[0066] Image acquisition Differential interference contrast (DIC) and fluorescent images of fixed cells (with and without cell sacculus) were acquired using an 80i upright microscope and a digital Hamamatsu ORCA-ER cooled CCD camera equipped with a 20x or 40x lens. Bright-field phase-contrast images were acquired using a Nikon digital camera. Cell sacculus initiation ratio / high performance field (HPF, 200x) and number of elongated cell sacculus / HPF were quantified. All images were acquired using MetaMorph image acquisition software and analyzed using ImageJ software.

[0067] Total RNA extraction and quantitative real-time PCR HMECs and BCSCs (1.2 × 10 ) with and without detectable cell capsules were cultured at the indicated times using TRIzol® (Thermo Fisher Scientific). 4Total RNA was extracted from 1000 cells / 10 cm dish. The samples used were plated on a layer of Matrigel matrix (approximately 10 μm thick). Total RNA was extracted as described in the manual. Quantitative real-time PCR assays were performed using gene-specific primers (IDT Company), iQ SYBR® Green Supermix (Bio-Rad), and 7900HT Fast Real-Time PCR according to the manufacturer's instructions. GAPDH was used as a control and three independent experiments were performed. Data analysis and heat map figures were calculated and generated as previously described.

[0068] Western blot HMECs and BCSCs (1.2 × 10 ) with and without cysts were cultured at the indicated times using radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and protease inhibitors (Roche). 4 Total protein was extracted from 1000 cells / 10 cm dish. Samples used were embedded in a layer of Matrigel matrix. Total protein was electrophoresed in 10% or 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride Immobilon™-P membranes. Polyvinylidene fluoride membranes were probed with primary antibodies (anti-GAPDH (Cell signaling Technology, 2118, 1:1000) for 4 hours at 4°C, then washed with 0.1% Tween / TBS. Membranes were incubated with appropriate peroxidase-conjugated secondary antibodies for 1 hour at 25°C or 1 hour, and washed three times before signal detection. Development was performed with ECL™ Western Blot Detection Reagents.

[0069] Quantification and statistical analysis In all figures: no significant difference, ns, P>0.05; * P<0.05; ** P<0.01; *** P<0.001.

[0070] Other embodiments will be apparent to those skilled in the art. It should be understood that the foregoing description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention is not limited to the above examples, but is encompassed by the following claims. All publications and patent applications cited above are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application was specifically indicated to be incorporated by reference herein.

Claims

1. 1. A method for generating cell sacs and cell cystic ducts, comprising: Embedding cells on top of the 3D matrix, and incubating the cell-embedded 3D matrix under conditions in which the cells develop the cell sacs and cell cyst ducts; The method comprising:

2. 10. The method of claim 1, wherein the cell-embedded 3D matrix is ​​incubated at a temperature between about 4°C and about 37°C.

3. The method of claim 1 , wherein the 3D matrix is ​​frozen prior to embedding.

4. The method of claim 1, wherein the embedding is performed at a temperature between about 2 and 6°C.

5. The method of claim 1 , wherein the 3D matrix is ​​biodegradable.

6. The method of claim 1 , wherein the 3D matrix comprises elastin, laminin, collagen, proteoglycan, or non-proteoglycan polysaccharides.

7. The method of claim 6, wherein the proteoglycans comprise heparan sulfate, chondroitin sulfate, and keratin sulfate.

8. The method of claim 6 , wherein the non-proteoglycan polysaccharide comprises hyaluronic acid.

9. The method of claim 1, wherein the 3D matrix further comprises proteins including fibrous proteins, FACIT proteins, short chain proteins and basement membrane proteins.

10. 2. The method of claim 1, wherein the 3D matrix further comprises signaling proteins including focal adhesion kinase (FAK), talin, vinculin, paxillin, α-actinin, and GTPase.

11. The method of claim 1 , wherein the 3D matrix is ​​Matrigel.

12. The method of claim 1, wherein the polymerization, density, protein concentration, and viscoelasticity of the 3D matrix affect the development of the cell capsules and cell ducts.

13. The method of claim 1, wherein the thickness of the 3D matrix is ​​between about 2 and about 100 μm thick.

14. The method of claim 1 , wherein the cell sacs and cell cystic ducts enclose the cells from which the cell sacs and cell cystic ducts give rise.

15. The method of claim 1 , wherein the cell sacs and cell cystic ducts allow escape of the encapsulated cells giving rise to the cell sacs and cell cystic ducts.

16. The method of claim 1 , wherein the cell sacs and cell cystic ducts allow the entry of multiple cells from the surrounding environment.

17. The cells are, prior to implantation, from about 1×10 to about 1×10 ５ The method of claim 1, wherein the cell is presented as a single cell suspension at a density of 100 / ml.

18. The method of claim 1, wherein the membranes of the cell sacs and cell cyst ducts comprise the plasma membrane protein Ca2+ATPase 2.

19. The method of claim 1, wherein syncytin 1 regulates cell cyst duct merging and cell invasion.

20. The method of claim 1, wherein ITGB-2 mediates cell migration in the cell cystic duct and regulates the elongation of the cell cystic duct.

21. The method of claim 1 , wherein the 3D matrix further comprises a bioactive and / or bioinert agent.

22. 10. The method of claim 1, wherein the cell comprises a mammalian cell, an insect cell, or a C. elegans cell.

23. 23. The method of claim 22, wherein the mammalian cell is a human cell.

24. 24. The method of claim 23, wherein the human cell is a human mammary epithelial cell.

25. 2. The method of claim 1, wherein the cells include differentiated cells, iPS cells, genetically modified cells, human pluripotent cells, epithelial cells, endothelial cells, immune cells, muscle cells, mouse stem cells, primary cell lines, immortalized cell lines, primary and immortalized fibroblasts, HeLa cells and neurons, and tumor cells.

26. The method of claim 1 , wherein the cells are embedded in a top surface of the 3D matrix.

27. A method for producing cell sacs and cell cysts in vitro, comprising the steps of: Embedding cells in a single cell suspension into the upper layer of a 3D matrix; and Culturing the cell-embedded 3D matrix under conditions in which the cells develop cell sacs and cell ducts in the 3D matrix; The method comprising:

28. 1. A method for preparing a 3D matrix suitable for the development of cell sacs and cell ducts, comprising: freezing the 3D matrix; and thawing the 3D matrix at 2-6°C; The method comprising:

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