Patent Application
20190017999
The present invention relates to a microfluidic chip for co-culture of blood vessels or lymphatic vessels with pericytes. Moreover, the present invention relates to a system that mimics the skin's structural layers by co-culturing skin cells with microvessels, microlymphatic vessels and pericytes in vitro and that analyzes the mechanism of cancer metastasis, the in vivo mechanism of immune cells, or the like based on the co-culture results. In addition, the present invention relates to a method of co-culturing blood vessels and pericytes in vitro and screening a drug candidate based on the co-culture results.
The formation and function of blood vessels is an important process that mediates many physiological and pathological processes. It is known that more than 70 diseases, including cancer development and metastasis, blindness caused by diabetes, senile macular degeneration, psoriasis and rheumatoid arthritis, are caused or exacerbated by abnormalities in blood vessel production and function. Therefore, studies on understanding the mechanisms of these processes and developing drugs and therapies for controlling these mechanisms can lead to overcoming various diseases. Fundamental studies and development of new drugs to overcome vascular diseases require the implementation of an experimental platform that mimics in vitro the formation and function of blood vessels and pathological processes. A system for culturing cells attached to a semi-permeable membrane (Gastroenterology 96(3), 736-749, 1989) can be regarded to be representative of conventional angiogenesis-related research methods or drug test methods, and requires less time and labor costs compared to experiments employing in vivo blood vessels, but has a disadvantage in that it cannot accurately mimic the biological characteristics of actual in vivo blood vessels, making it difficult to accurately predict the biological morphology of actual blood vessels.
The blood vessels in the human body have different characteristics depending on their positions. For example, cerebral and ocular blood vessels form blood-brain barriers (BBB) and blood-retinal barriers (BRB), which have significantly lower vascular permeability than elsewhere. It is believed that these barriers are a major factor that determines drug delivery characteristics, and that they are formed due to the influence of pericytes (which are representative cells surrounding blood vessels) and astrocytes on blood vessels. Thus, in vitro mimicking of this perivascular environment is a major factor in in vitro blood vessel mimicking models for new drug development.
The skin is composed of: an epidermal layer having keratinocytes; a dermal layer located under the epidermal layer and having microvessels and fibrous cells; and a subcutaneous fat layer having blood vessels and fat.
Microvessels extend from the blood vessels of the subcutaneous fat layer to immediately below the epidermal layer, and supply of nutrients and migration of immune cells occurs through the microvessels. Among skin cell lines, keratinocytes form the epidermis, and Langerhans cells are involved in immunity. Cells that immune cells are dendritic cells, and Langerhans cells are one type of dendritic cells. Dendritic cells extravasate from the blood vessel walls in vivo (extravasation) and undergo maturation while migrating to tissues. Exposure to UV rays for a long time in everyday life causes inflammation of the skin, because the skin tissue and the vascular system are connected organically. When keratinocytes in the skin tissue are exposed to UV rays, they induce an immune response to attract immune cells or secrete VEGF to induce new blood vessel formation. There has been a continuing need for a culture platform capable of analyzing the correlation between skin-derived cells and blood vessels and/or lymphatic vessels.
It is known that cancer cells continuously interact with surrounding blood vessels during cancer growth and metastasis (Bergers and Benjamin 2003; Carmeliet and Jain 2000).
As tumor cell clusters proliferate, they secrete factors that stimulate the surrounding blood vessels to go toward the clusters. This phenomenon is known as tumor angiogenesis. Tumor angiogenesis causes nutrients and oxygen to be supplied to tumor cells from blood vessels, causing tumors to grow into malignant tumors. A tumor cell composed of a fully grown ellipsoid passes through surrounding blood vessel cells, circulates in the blood, and passes through the secondary site in a region surrounding blood vessels (Sakai 2007). Extravasating tumor cells grow to form secondary tumor clusters. Most cancer deaths are caused by this recurrent cancer metastasis. Therefore, as a stage involved in the progression of cancer metastasis, studies mainly on tumor angiogenesis and migration through endothelial cells have been extensively conducted. In transwell experiments, cancer cells were grown in a lower chamber, and endothelial cells in extracellular matrices such as collagen or Martrigel were cultured in an upper chamber. Tube formation (Tsujii et al. 1998) and migration of endothelial cells (Abdollahi et al. 2005) can be observed in response to cancer-derived factors. In a model for migration of cancer cells through endothelial cells, the endothelial cells were cultured to confluence in a Petri dish or a porous membrane, and the permeation of the endothelial monolayer could be observed after several hours to several days of culture (Jin et al. 2012; Kramer and Nicolson 1979; Kusama et al. 2006; Lee et al. 2003; Roetger et al. 1998; Zabel et al. 2011).
Microfluidic technology has a variety of advantages over conventional methods with respect to precise regulation of microenvironmental factors, or high-resolution imaging control of cell migration or cell-cell interactions (Chung et al. 2010; Lee et al. 2014a). An angiogenic process was modeled by attaching endothelial cells to a hydrogel wall and co-culturing the endothelial cells with cancer cells to stimulate the endothelial cells (Kim et al. 2013). The disadvantage of this experiment is that a developmental process begins from endothelial cell clusters deficient in appropriate cell-cell junctions. Since angiogenesis occurs from fully established blood vessels near cancer clusters, this characteristic is not physiologically related to actual in vivo angiogenesis. In another study, vascular walls were modeled by culturing a monolayer of endothelial cells in a microfluidic channel (Jeon et al. 2013; Zervantonakis et al. 2012). Migration through endothelial cells can be modeled by introducing cancer cells into the system. However, this vascular model involves attaching a monolayer of endothelial cells onto a hydrogel without an angiogenic or vasculogenic process. Recently, in order to analyze the extravasation of cancer, a microfluidic model has been developed which generates a vascular network by an angiogenic process (Chen et al. 2013). However, this model did not allow the concentration gradient direction of chemokines to induce chemotactic migration of cancer cells, and the unpredictable geometry of the vascular network has increased the complexity of the experiment.
Accordingly, the present inventors have made efforts to solve the above-described problems occurring in the prior art, and as a result, have found that skin cells, skin blood vessels, skin lymphatic vessels and skin pericytes can successfully co-cultured by using a microfluidic chip of the present invention, thereby completing the present invention.
Using the microfluidic chip of the present invention, it is possible to obtain microvessels having a smooth and continuous boundary, formed by a natural angiogenic process. In addition, the obtained microvessels may be used to model cancer angiogenesis and intravasation. As described above, the microfluidic chip of the present invention can be widely used in the fields of basic cancer biology, drug candidate screening and the like.
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Therefore, the present invention provides a method of cu-culturing blood vessels/lymphatic vessels and pericytes in a microfluidic chip.
The present invention also provides a microfluidic chip for generating tumor angiogenesis, and tumor angiogenesis generated in vitro by use of the microfluidic chip.
The present invention also provides a microfluidic chip for inducing tumor intravasation and extravasation, and tumor intravasation and extravasation induced in vitro by use of the microfluidic chip.
The present invention also provides a skin structural layer cultured in vitro by co-culturing blood vessels/lymphatic vessels and skin cells by use of the microfluidic chip according to the present invention. The skin structural layer may be provided as a skin tissue chip comprising blood vessels and lymphatic vessels. This skin structural layer may be used to mimic the skin immune system and to implement cell migration intravasation and extravasation) through blood vessels and lymphatic vessels. In addition, it may be used to study the change/aging of blood vessels caused by skin damage or to study the passage of immune cells through subcutaneous blood vessels/lymphatic vessels and the mechanism of differentiation of undifferentiated cells after passage through vascular membranes.
The present invention also provides a method of screening a drug candidate by use of the microfluidic chip according to the present invention.
The present invention also provides a biological tissue chip comprising blood vessels/lymphatic vessels and co-culture cells formed in the microfluidic chip of the present invention.
The present invention also provides a method of generating blood vessels/lymphatic vessels and cells, which interact with each other in vitro, by use of the biological tissue chip of the present invention.
To achieve the above objects, in one embodiment, the present invention provides a microfluidic chip comprising: at least one blood vessel channel; at least one cell channel; and at least one medium channel. In the microfluidic chip, the blood vessel channel, the cell channel and the medium channel are disposed adjacent and parallel to one another such that they may be in fluidic communication with one another; both sides or one side of the blood vessel channel is adjacent to the medium channel, both sides or one side of the cell channel is adjacent to the other side of the medium channel; at the interface between the channels adjacent to each other, two or more microstructures, microposts or columns, which have various sectional shapes, for example, a triangular or hexagonal shape, are disposed with a predetermined gap; the medium channel is connected with a medium reservoir such that they may be in fluidic communication with each other, and each of the blood vessel channel and the cell channel is connected with its inlet such that they may be in fluidic communication with each other.
In another embodiment of the present invention, there is provided a biological tissue chip configured such that blood vessels or lymphatic vessels and cells, co-cultured in vitro, interact with each other, the biological tissue chip comprising: at least one blood vessel channel and blood vessels or lymphatic vessels or a combination of blood vessels or lymphatic vessels, formed in the blood vessel channel; at least one cell channel and cells cultured in the cell channel; and at least one medium channel, wherein the blood vessel channel, the cell channel and the medium channel are disposed adjacent and parallel to one another such that they may be in fluidic communication with one another; both sides or one side of the blood vessel channel is adjacent to the medium channel, both sides or one side of the cell channel is adjacent to the other side of the medium channel, and two or more barrier structures or microstructures are disposed at the interface between adjacent two of the channels with a gap; the medium channel is connected with a medium reservoir such that they may be in fluidic communication with each other, and each of the blood vessel channel and the cell channel is connected with its inlet such that they may be in fluidic communication with each other; each of the channels allows the interaction between biochemical substances contained in the channels through the gap; blood vessels or lymphatic vessels are formed from angiogenic or lymphangiogenic cells in the blood vessel channel, and cells are cultured in the cell channel; and the cultured cells interact with the formed blood vessels or lymphatic vessels. In one embodiment, the biological tissue mimics the skin tissue comprising the subcutaneous fat layer, the dermal layer and the horny layer.
In another embodiment of the present invention, the microfluidic chip 10 comprises a first channel 110, a second channel 120, a third channel 130, a fourth channel 140, and a fifth channel 150, which are sequentially disposed over a substrate, wherein the first channel is adjacent to one side of the second channel, the other side of the second channel is disposed adjacent and parallel to one side of the third channel, the other side of the third channel is disposed adjacent and parallel to one side of the fourth channel, and a portion or all of the other side of the fourth channel is disposed adjacent and parallel to one side of one or more fifth channels; and when two or more fifth channels are provided, the fifth channel is divided into two cells 170 and 180 by a barrier 160 provided in direction perpendicular to its interface with the third channel, and the cells may comprise fifth channels 151 and 152, respectively. Two or more microstructures 610 at an interface formed between adjacent channels in the microfluidic chip 10 are disposed with a predetermined gap 620, and each of the channels allows the interaction between biochemical substances contained in the channels through the gap.
In still another embodiment of the present invention, the microfluidic chip 20 comprises: a first channel 210 configured to be in fluidic communication with a first medium reservoir 201; a second channel 220 configured to be in fluidic communication with a second medium reservoir 202 and disposed parallel to the first channel 210; a blood vessel channel 230 configured to be in fluidic communication with a blood vessel channel inlet 203 and disposed between the first channel 210 and the second channel 220 and adjacent to one side of the first channel 210 and the second channel 220; and a cell channel 240 configured to be in in fluidic communication with a cell channel inlet 204 and adjacent to the other side of the second channel 220 and disposed parallel to the second channel 220. The microfluidic chip 20 comprises two or more barrier structures 660 protruding from the blood vessel channel 230, wherein each of the barrier structures 660 comprises: a first barrier 601 disposed parallel to the first channel 210; a second barrier 602 formed to extend from the first barrier 601 to the second channel 220; a third barrier 603 formed to extend from the second barrier 602 and disposed parallel to the first barrier 601; and at least one protrusion 604 formed in a space surrounded by the first barrier 601, the second barrier 602 and the third barrier 603, wherein the interface between the channels allows the interaction between biochemical substances in the channels.
In still another embodiment of the present invention, a microfluidic channel 30 comprises: a first channel 310 configured to be in fluidic communication with a first medium reservoir 301; a second channel 320 configured to be in fluidic communication with a second medium reservoir 302 and disposed parallel to the first channel 310; a first blood vessel channel 330 configured to be in fluidic communication with a first blood vessel channel inlet 303 and adjacent to one side of the first channel 310; a second blood vessel channel 340 configured to be in fluidic communication with a second blood vessel channel inlet 304 and adjacent to the other side of the second channel 330; a first cell channel 350 configured to be in fluidic communication with a first channel inlet 305 and adjacent to the other side of the first channel 310 and disposed parallel to the first channel 310; and a second cell channel 360 configured to be in fluidic communication with a second cell channel inlet 306 and adjacent to the other side of the second channel 310 and disposed parallel to the second channel 320.
In the microfluidic chip of the present invention, the height and width of the channel are not particularly limited, and may be determined flexibly according to the type of material to be injected into the channel, and the purpose and conditions of the experiment. In one embodiment, the height of the channel 10 μm to 1000 μm, preferably 500 μm to 800 μm.
In still another embodiment of the present invention, there are provided microvessels produced using the microfluidic chip 10. Specifically, production of the microvessels is performed by supplying a mixture of fibroblasts (e.g., lung fibroblasts (LF)) and fibrin to the fifth channel 150 and supplying a mixture of endothelial cells and fibrin to the third channel 150. During formation of the microvessels, the first channel 110 and the second channel 120 are maintained in an empty state. The second channel 120 should contain only air during formation of the microvessels in order to inhibit the formation of concentration gradient of growth factors from the second channel 120 and to prevent endothelial cells from moving toward an empty channel or from forming sprouts. To form microvascular cells having a smoother and more continuous boundary, endothelial cells are injected at a concentration of 2 to 10×106 HUVECs/m, preferably 4 to 8×106 HUVECs/ml, most preferably 6×106 HUVECs/ml. At a low HUVEC concentration of 3×106 cells/ml (
In still another embodiment, the present invention provides tumor angiogenesis using the microfluidic chip 10. Specifically, microvessels are formed according to the present invention, and then a mixture of a cell line having angiogenic ability (e.g., the U87MG cell line) and fibrin is injected into the first channel, followed by injection of fibrin into the second channel.
In still another embodiment, the present invention provides a platform for mimicking the immune function of a skin structural layer using the microfluidic chip 10.
In still another embodiment, the present invention provides a method of screening an anticancer drug candidate. Specifically, the screening method of the present invention comprises: treating a cell line, which has the angiogenic ability to induce tumor angiogenesis, with a sample; and determining that when tumor angiogenesis is not generated, the sample is an anticancer drug candidate.
In still another embodiment, the present invention provides tumor intravasation using the microfluidic chip 10. Specifically, after tumor angiogenesis is generated according to the present invention, cancer cells (e.g., MDA-MB-231 cells) are attached to a fibrin gel in the second channel 120, and a medium for cancer cell growth is supplied to the first channel 110. Next, when growth factor-free medium (EBM) is supplied to the first channel 110, cancer intravasation is induced by the chemotactic migration of cancer cells to the microvascular wall.
In another embodiment of the present invention, the endothelial cells have proper cell-cell junctions and shows an elongated shape as in vivo, and the medium can be perfused through the vascular lumen, and it has two three-dimensional vascular structures opened toward the fourth channel 140. In addition, the present invention makes it possible to successfully model cancer angiogenesis and the inhibition of the angiogenic pathway by treatment with an anti-vascular endothelial growth factor (anti-VEGF; bevacizumab). In addition, one embodiment of the present invention shows the regulation of tumor intravasation rate by treatment with tumor necrosis factor-alpha (TNF-a).
In the present invention, extracellular matrix or extracellular matrix/angiogenic cells are filled in the blood vessel channel is filled with, thereby forming a three-dimensional angiogenic region. The angiogenic region provides a space in which angiogenic cells can three-dimensionally proliferate and differentiate to form blood vessels. At the interface between the blood vessel channel and the medium channel, specific barrier structures 660 or other microstructures 610 are arranged with a gap. When extracellular matrix or extracellular matrix/angiogenic cells are injected into the blood vessel channel, the extracellular matrix or extracellular matrix/angiogenic cells form an angiogenic region due to surface tension without escaping through the microstructures. On the other hand, since various biochemical substances contained in the channels can move through the gap between the microstructures, various angiogenic factors, nutrients, etc. contained in other channels can be supplied to the blood vessel channel and/or the cell channel.
In addition, blood vessels generated in the angiogenic region may extend toward the gap formed between the plurality of microstructures and communicate with a channel adjacent to the blood vessel channel, thereby forming an inlet and outlet for blood vessels. Thus, it is possible to observe and image the reaction of blood vessels in real time by transferring various biochemical and biophysical substances and signals directly into the vascular lumens through the channel communicated with the gap between the plurality of microstructures. Accordingly, it is possible to control the number of inlets and outlets of the vascular network to be generated, by changing the arrangement, number, size, etc. of the microstructures of the vascular channel, in particular, the barrier structures 660. It is obvious that parameters such as the shape of the microstructures are not limited to the embodiment shown in the drawings but can be appropriately adjusted according to the purpose and configuration of the experiment.
The liquid extracellular matrix or extracellular matrix/angiogenic cells prior to curing injected into the vessel channel form a meniscus by capillary action and surface tension between the plurality of microstructures. Below a threshold pressure level, the meniscus does not advance to an adjacent channel due to surface tension. The range of pressure, in which the meniscus can stop between the plurality of microstructures without advancing to the side channel adjacent thereto, is influenced by the spacing between and height of the microstructures. Thus, the threshold pressure level can be adjusted by optimizing these two parameters (the spacing between the microstructures and the height of the microstructure). For a specific method of adjusting the threshold pressure level, reference may be made to the content disclosed in Carlos P. Huang et al. (Engineering microscale cellular niches for three-dimensional multicellular co-cultures, Lab Chip, 2009, 9, 1740-1748).
By adjusting the spacing between the microstructures and the height of the microstructures, the meniscus can be effectively stopped between the structures when extracellular matrix or extracellular matrix/angiogenic cells are injected into the vessel channel, thereby precisely controlling the filling of the extracellular matrix between the channels that are not completely physically separated from each other. This is also true when extracellular matrix or extracellular matrix/co-culture cells are filled in the cell channel.
In one embodiment of the present invention, the number of the barrier structures 660 in the blood vessel channel is 1 to 15, preferably 5 to 8. In another embodiment, one protrusion 604 is preferably included per barrier structure 660. The shape of the protruding portion 604 is preferably configured such that the length of one side in the direction of the blood vessel channel inlet is shorter than that of the other side in the opposite direction, that is, the length of the other side in the direction of an empty channel, if any. In another embodiment of the present invention, the second barrier 602 is preferably formed to extend perpendicularly from the first barrier 601, and the third barrier 603 may include a bent portion 605 in the direction of a space surrounded by the first barrier 601, the second barrier 602 and the third barrier 603. For example, the bent portion 605 may be bent toward the connection between the first barrier 601 and the second barrier 602. In another embodiment, when an empty channel is present, two microstructures are preferably disposed between the blood vessel channel and the empty channel. The microstructures may have the same shape as that of the protrusion 604 in the blood vessel channel.
Extracellular matrix or extracellular/co-culture cells are filled in the cell channel, thereby forming a three-dimensional cell culture region. At the interface between the cell channel and the medium channel, a plurality of microstructures may also be arranged with a gap. In this case, when extracellular matrix or extracellular/co-culture cells are injected into the cell channel, the extracellular matrix or extracellular/co-culture cells form a cell culture region in the cell channel due to surface tension without escaping through the microstructures. The cell culture region may provide a space in which the co-culture cells may be cultured three-dimensionally. This three-dimensional multicellular co-culture can physiologically mimic in vivo environments and can promote the production and secretion of various signal substances from the co-culture cells. Meanwhile, since various kinds of biochemical substances such as angiogenic factors contained in the medium channel and various signal substances generated in the cell channel can move through a gap between the plurality of microstructures, the active interaction of biochemical substances between the cell channel and other channel adjacent thereto may occur.
The medium channel that provides medium provides a passage allowing the flow of a fluid, wherein the fluid may include cell culture medium, various angiogenic factors, and the like. One of the medium channels is placed in contact with one side of the blood vessel channel and the cell channel, and the other medium channel is placed adjacent to the other side of the blood vessel channel, and thus these medium channels supply cell culture medium, angiogenic factors and the like to the blood vessel channel and the cell channel. This allows long-term cell culture of angiogenic cells and co-culture cells, and provides a passage through which the two cell groups (angiogenic cells and co-culture cells) in the blood vessel channel and the cell channel
allows passage of both vascular channels and two cell groups (angiogenic and co-culture cells) in the cell channel can interact through a paracrine mechanism. Paracrine interaction between angiogenic cells and co-culture cells promotes the morphogenesis of the angiogenic cells through the vascular network, and also has an important effect on the expression of specific properties and function of formed blood vessels.
In addition, one of the medium channels physically separates between angiogenic cells and co-culture cells, thereby making it easy to independently observe and analyze the angiogenic cells and the co-culture cells. In particular, it provides a space allowing various biochemical and biophysical materials and signals to be injected into blood vessels communicating with the medium channel through the gap between the plurality of microstructures.
The microfluidic chip according to the present invention can be fabricated by, for example, a lithography method, a molding method or the like, but is not limited thereto. In one embodiment, photoresist is deposited on a silicon wafer to a thickness of 50 to 100 μm, and ultraviolet light is irradiated thereto through a pattern formed on a light transmissive film to cure the photoresist, thereby forming a structure. Polydimethylsiloxane (PDMS) may be poured and cured on the formed structure which is then bonded to thin glass, thereby fabricating an angiogenic device.
According to the present invention, co-cultivation of angiogenic cells and pericytes is possible, and the two cell groups (angiogenic and co-culture cells) in the blood vessel channel and the cell channel can interact through a paracrine mechanism. The paracrine interaction between angiogenic cells and co-culture cells promotes the morphogenesis of the angiogenic cells through the vascular network, and also has an important effect on the expression of specific properties and function of formed blood vessels. In addition, one of the medium channels physically separates between angiogenic cells and co-culture cells, thereby making it easy to independently observe and analyze the angiogenic cells and the co-culture cells. In particular, it provides a space allowing various biochemical and biophysical materials and signals to be injected into blood vessels communicating with the medium channel through the gap between the plurality of microstructures. This allows to mimic the interaction between various biochemical/biophysical substances and blood vessels, and to observe the response of blood vessels to various stimuli and to image and quantify this response in real time.
In another aspect, the present invention is directed to an in vitro angiogenesis method comprising a step of sequentially or simultaneously injecting one or more, selected from the group consisting of angiogenic cells, extracellular matrices, cell culture media, angiogenic factors and co-culture cells, into one or more independent channels of the microfluidic chip according to the present invention, culturing angiogenic cells, and inducing blood vessel formation. The blood vessel formation in the present invention may be an angiogenesis process and/or a vasculogenesis process.
Injection of extracellular matrix, cells, cell culture medium, various angiogenic factors, etc. into each channel, does not require a separate external laboratory device, and may be simply performed by pipetting.
In addition, angiogenic cells and co-cultured cells may be injected as a mixture with extracellular matrix, but only the extracellular matrix may be injected depending on the purpose of the experiment. The extracellular matrix injected into each channel is cured by a rise in temperature, chemical action, light irradiation, etc. depending on the type of extracellular matrix injected.
In another embodiment of the present invention, there is provided a method for generation of blood vessels/lymphatic vessels and cells, which interact with each other in vitro, the method comprising: sequentially or simultaneously injecting one or more, selected from the group consisting of angiogenic cells, lymphangiogenic cells, extracellular matrices, cell culture media, angiogenic factors, lymphangiogenic factors and co-culture cells, into one or more independent channels of the microfluidic chip according to the present invention; culturing angiogenic cells; inducing blood vessel formation; and culturing co-culture cells.
In another embodiment of the present invention, there is provided a method for generation of blood vessels or lymphatic vessels and cells, which interact with each other in vitro, the method comprising the steps of: (a) injecting extracellular matrix and angiogenic or lymphangiogenic cells into the blood vessel channel of the microfluidic chip according to the present invention; (b) injecting extracellular matrix or extracellular matrix and co-culture cells into the cell channel; and (c) injecting cell culture medium, angiogenic or lymphangiogenic factor, or a combination of cell culture medium and angiogenic or lymphangiogenic factor into the medium channel, inducing blood vessel or lymphatic vessel formation in the blood vessel channel, and culturing the co-culture cells in the cell channel.
In still another embodiment of the present invention, there is provided a method for generation of blood vessels or lymphatic vessels and cells, which interact with each other in vitro, the method comprising the steps of: (a) injecting extracellular matrix or extracellular and co-culture cells into the blood vessel channel of the microfluidic chip according to the present invention, and forming a cell adhesion surface for cell adhesion at the interface between the blood vessel channel and the medium channel; (b) injecting angiogenic cells into the medium channel, and allowing the angiogenic cells to adhere to the cell adhesion surface; (c) injecting extracellular matrix or extracellular matrix and co-culture cells into the cell channel; and (d) injecting cell culture medium, angiogenic factors, or cell culture medium and angiogenic factors into the medium channel, culturing in the angiogenic cells in the blood vessel channel, and inducing blood vessel formation.
In still another embodiment of the present invention, there is provided a system capable of mimicking the skin immune system by culturing immune cells in the cell channel of the microfluidic chip according to the present invention.
In the present invention, when angiogenic cells are adhered to the extracellular matrix or extracellular matrix and co-culture cells on the cell adhesion surface, the angiogenic cells contained in the cell culture medium are injected into the channel, and the angiogenic device is tilted by an angle of about 90° for about 10 to 40 minutes, so that the angiogenic cells can be adhered to the intended location.
In the present invention, in order to promote or control the culture and morphogenesis of angiogenic cells depending on the type of co-culture cells and the purpose of the experiment, angiogenic factors or other factors that influence cell responses may be added to extracellular matrix or cell culture medium. Angiogenic sprouts form blood vessels that can grow and spread from the angiogenic region having angiogenic cells attached thereto. In order to provide a directional concentration gradient of angiogenic factors that induce angiogenesis from angiogenic cells, the location of adhesion of angiogenic cells, the injection of co-culture cells, and the introduction of angiogenic factors into the medium channel, may be suitably selected depending on the purpose of the experiment.
In the present invention, the term “angiogenic cells” refers to cells that form blood vessels by interaction with an angiogenic factor, an angiogenic inducer contained in cell culture medium, co-culture cells, and the like. These angiogenic cells can form blood vessels through vasculogenesis and/or angiogenesis.
In the present invention, the angiogenic cells are not particularly limited and may be appropriately selected depending on the purpose of the experiment, but may preferably be one or more selected from the group consisting of endothelial cells, epithelial cells, cancer cells, stem cells, stem cell-derived cells, and endothelial progenitor cells. For example, the angiogenic cells may be endothelial cells derived from various tissues of the body, for example, human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells, human brain microvascular endothelial cells, human lymphatic endothelial cells, and the like. In addition, cancer cells may also be used to study cancer growth and metastasis mechanisms, etc. The culture cells that are used in the present invention may be vascular endothelial cells derived from various species other than humans, for example, porcine, murine or bovine species.
In addition, the angiogenic cells may be mutated cells, transfected cells, or mutated and transfected cells.
In the present invention, the extracellular matrix is not particularly limited, but may be one or more selected from then group consisting of collagen gel, fibrin gel, Matrigel, self-assembled peptide gel, polyethylene glycol gel, and alginate gel.
In the present invention, the co-culture cell may be cells that secretes biochemical substances necessary for angiogenesis, such as an angiogenic inducer and the like, through interaction with angiogenic cells. In the present invention, the co-culture cells are not particularly limited, but may be one or more selected from the group consisting of cells of the immune system, astrocytes, glial cells, mesothelial cells, fibroblasts, smooth muscle cells, cancer cells, pericytes, neuroglial cells, stem cells, stem cell-derived cells, and cells that interact with vascular endothelium. For example, when the blood vessels to be generated are brain blood vessels, the co-culture cells are preferably astrocytes, glial cells, mesothelial cells, or fibroblasts, and when the blood vessels to be generated are blood vessels other than brain blood vessels, the co-culture cells are preferably fibroblasts or smooth muscle cells. In addition, cancer cells may also be used as co-culture cells to study the relationship between cancer and angiogenesis. In addition, cells of the immune system may include T cells, B cells, macrophages, NK (natural killer) cells, or dendritic cells. In the present invention, co-culturing of cells of the immune system can complete an in vitro system that mimics the skin immune system. The type and combination of cells to be cultured and the culture method may be selected according to the purpose of the experiment.
In the in vitro angiogenesis method of the present invention, the co-culture cells may be mutated cells, transfected cells, or mutated and transfected cells.
In the in vitro angiogenesis method of the present invention, in order to quantitatively measure angiogenesis and functional effect and efficacy or to screen a new drug having the property of promoting or inhibiting angiogenesis, the extracellular matrix or the cell culture medium may include one or more selected from the group consisting of drugs, soluble factors, insoluble factors, biomolecules, proteins, nanomaterials, and siRNA (small interfering RNA).
In the in vitro angiogenesis method of the present invention, the angiogenic factors are not particularly limited, but may be vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and the like.
As used herein, the term “microstructure” is a micro-sized structure and refers to a collection of the microstructures 610, the gap 620, and channels and structures defined by them. The sectional shape of the microstructures 610 formed at the interface between the channels may be circular, triangular, or hexagonal, but is not limited thereto, and may also be a micropost or column shape. In one embodiment, the size (width) of the gap formed between the microstructures is 10 μm to 100 μm, preferably 50 μm to 90 μm, and more preferably 60 μm to 80 μm.
In the microfluidic chip 10 of the present invention, the first channel 110 is an upper channel; the second channel 120 is a bridge channel; the third channel 130 is a blood vessel channel; the fourth channel 140 is a medium channel; and the fifth channel 150 is an LF channel; and these terms have the same meaning as above.
As used herein, the term “empty channel” means a channel that allows the presence of gas therein but does not allow the presence of a liquid and a solid therein.
In the present invention, the term “blood vessels/lymphatic vessels” means “blood vessels or lymphatic vessels, and blood vessels or lymphatic vessels”, but it should be understood that it includes lymphatic vessels or blood vessels, even blood vessels or lymphatic vessels are used alone.
As used herein, the term “biochemical substances” is understood to include various substances, including cells, proteins, peptides, amino acids, cofactors, neurotransmitters, antioxidants, cofactors, lipids, carbohydrates, hormones, antibodies, antigens, or the like, which are required for biological functions and activities in the biochemistry field. The term is also understood to include common chemical compounds.
As used herein, the term “biological tissue chip” means a chip capable of mimicking in vivo tissue, which includes blood vessels/lymphatic vessels formed in the microfluidic chip of the present invention, as well as cells co-cultured therewith.
Using the microfluidic chip of the present invention, it is possible to obtain microvessels having a smooth and continuous boundary, formed by a natural angiogenic process. In addition, the present invention makes it possible to mimic a perivascular environment, which provides a basic environment for research on brain-blood vessel barriers (BBB), brain-retinal barriers (BRB), cancer angiogenesis and cancer intravasation, immune cells, skin cells, and the like. Thus, the present invention provides a more substantial means for forming vascular structures, which may be applied to various experimental models, including extravasation models of extracellular efflux of cancer cells or leukocytes, and mechanotransduction experiments on fluid movement through the vascular lumen. In addition, the present invention makes it possible to mimic experiments on the passage of immune cells through subcutaneous blood vessels, as well as differentiation processes after the passage of undifferentiated cells through vascular membranes. The microfluidic chip of the present invention may be widely used to screen drug candidates.
Hereinafter, the constituent elements and technical features of the present invention will be described with reference to examples below. However, these examples are only to illustrate the present invention, and the scope of the present invention is not limited by these examples.
Cell Culture, Immunostaining and Reagents
Human umbilical vein endothelial cells (HUVEC, Lonza) were cultured in endothelial cell growth medium (EGM-2, Lonza). Normal human lung fibroblasts (LF, Lonza) were cultured in fibroblast growth medium (FGM-2, Lonza). Human glioblastoma cells (U87MG, ATCC, Virginia) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 U/ml). MDA-MA-231 cells were purchased from the ATCC (Manassas, Va.). MDA-MB-231 cells were transfected with a pEGFP plasmid, and cells were selected using 1 mg/ml G418 (A.G. scientific, Inc.). MDA-MA-231 GFP cells obtained from monoclones were incubated in RPMI1640 (WELGENE, Korea) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Gibco, BRL) and 250 μg/ml G418 (A.G. scientific, Inc.). All cells were incubated in a humidified incubator at 37° C. under 5% CO2. For immunostaining, endothelial cells were imaged using mouse monoclonal antibodies specific for human ZO-1 (Alexa Fluor594, clone ZO1-1A12, molecular probe), CD31 (AlexaFluor1647, clone WM59, Biolegends), VE-cadherin (eBioscience) and claudin-5 (Invitrogen), and the nucleus was stained with Hoechst 33342 (molecular probe). Bevacizumab (Avastin, Genentech) was diluted to 500 μg/ml, and microvessels introduced with cancer were treated with the dilution in cancer angiogenesis experiments. Recombinant human TNF-alpha (PetroTech) was diluted to 5 ng/ml, and microvessels were treated with the dilution at 24 hours before introduction of cancer.
Fabrication of Microfluidic Chip
A master mold was made by casting photoresist onto a silicon wafer. A 80-μm thickness mold was made according to the standard photolithography protocol (Xia, Y. et al. 1998) for SU-8100 (Microchem, US) photoresist. PDMS (Dow Corning, US) was poured onto the prepared master mold and cured in a dry oven at 80° C. PDMS and a cleaned coverslip were bonded together by plasma treatment (Femto Science, KR). To make the surface hydrophobic, the bonded device was kept in an oven at 80° C. for 48 hours or more.
Hydrogel and Cell Loading
HUVECs (used at 6×106 cells/ml in most experiments, and used at 3 or 9×106 cells/ml in some experiments) and LFs (7×106/ml) were mixed with a fibrinogen solution (2.5 mg/ml fibrinogen, 0.15 U/ml aprotinin and 0.5 U/ml thrombin) and injected into the third channel 130 (blood vessel channel) and the fifth channel 150, 151 or 152 (LF channel), respectively (
Microscopy
For microvascular DIC (Differential Interference Contrast Microscope) imaging, the Nikon AE31 microscope was used. For 3D z-stack and cross-sectional imaging, stained samples were imaged using a confocal microscope (Olympus FV1000). Confocal images were analyzed using IMARIS software (Bitplane, Switzerland). For fluorescence imaging, FITC-dextran-injected samples were imaged using the IX81 inverted microscope (Olympus).
Data Analysis
To quantify the success rate of the smooth and continuous boundary of microvessels, the length of the vessel boundary was measured, and compared with the straight-line length of the blood vessel channel along the microvessel by use of image J. When the boundary length value was within ±10% of the linear length of the vascular channel, the chip was regarded as success. All microvessels having disconnected blood vessel walls were regarded as failure. Calculation of the permeability coefficient was performed using the method disclosed in the prior art (Non-Patent Document 19: Lee et al. 2014b). Specifically, FITC-dextran solution was introduced into microvessels and fluorescently imaged in every 15 seconds using multi-stage time-lapse mode in Metamorph. The acquired time-lapse image was analyzed using Image J, and the permeability coefficient was calculated using Equation 1 below:
P=1/lw×(dl/dt)/li
where lw is the length of the vessel wall that separates between perivascular region and microvessel region, li is the mean intensity in the microvessel region, I is the total intensity in the perivascular region.
For the quantification of cancer angiogenesis, sprout area and the number of sprouts were manually quantified by using Image J. For quantification of cancer intravasation, the microvessels were stained with CD31 and fluorescence imaged, and cancer cells at the apical side of the microvessel were manually counted using the IMARIS software.
In previous works, the present inventors have described in detail the formation of a perfusable microvessel network in a microfluidic device using a co-culture system of HUVEC and LF. The HUVEC sprouts were stimulated by LF, which opened their lumens to both sides of the channel, allowing fluid passage through the vessels. However, the structure of microvessel in the previous model was unpredictable, and other cell types could not be introduced into the perivascular region, as the perivascular regions were filled with gel or PDMS (polydimethylsiloxane) wall. Therefore, the present inventors generated a microvessel with more predictable geometric characteristics and with perivascular regions that could be filled with other cell types after the generation of the microvessels with vasculogenic process. However, in this Example, the present inventors positioned two openings of the third channel 130 (blood vessel channel) on the same side (lower side), and interfaced the upper portion of the third channel 130 (blood vessel channel) with an empty channel (
The first channel 110 (upper channel) and the second channel 120 (bridge channel) were empty during microvessel growth. The interface with the empty channel prevented HUVECs in the upper region from migrating or generating sprouts toward the upper direction of the third channel 130 (blood vessel channel), and resulted in the generation of a smooth vessel wall, parallel to the interface.
Formation of smooth and continuous vessel boundaries is important in reproducible data analysis and the use of microvessels in further studies. Thus, the present inventors have optimized the HUVEC cell concentration to form microvessels having smooth and continuous boundaries. The present inventors tested three conditions (3, 6, and 9×106 HUVECs/ml and 7×106 LF/ml) using 4-5 chips per condition, and results are expressed as the mean of three independent experiments.
After formation of the microvessel (days 7-8), nuclei (blue) and CD31 (red) were immunostained and imaged with a confocal microscope (
The present inventors introduced 20 kDa FITC-dextran solution into the microvessel to verify the patency of the lumen, and visualized the behavior of the lumen using fluorescence microscopy. The FITC-dextran filled the microvessel lumen without severe focal leakage through the wall (
The present inventors analyzed the angiogenic potential of cancer cells by using the microvessels having a smooth vessel wall. After formation of the microvessels using the microfluidic chip 10 according to Example 2 (day 7-8), the present inventors introduced U87MG, a glioblastoma cell line having high angiogenic potential, and a fibrin mixture into the first channel 110 (upper channel), while the second channel 120 (bridge channel) was filled with fibrin gel only. The microvessel was regarded as pre-existing at the cancer site, whereas the U87MG cells in the perivascular region secrete angiogenic factors to induce production of angiogenic sprouts toward the microvessels. Three different concentrations (control 0/ml, 2.5×106 and 5×106) of U87MG cells with or without bevacizumab (bev) were used, and an anti-VEGF antibody used widely for targeting angiogenesis was used. The angiogenic sprouting from the microvessels was analyzed, and the mean values of six chips per condition were calculated.
Three days after introduction of U87MG cells into the chip, the microvessels were imaged, and the number and coverage area of angiogenic sprouts under each condition were quantified. As shown in
Next, the number and coverage area of sprouts. The microvessels with cancer cells exhibited significantly increased sprout numbers and coverage areas compared to the control (
As a vessel to assess the transendothelial migration of MDA-MB-231 cancer cells, the microvessel obtained in the present invention was used. The cells are conventionally used for the assessment of transendothelial migration and have aggressive metastatic potential. As shown in
Using the microfluidic chip 20 (
For a microfluidic chip 30 (
In addition, two fibrin hydrogels were patterned adjacent to each other, and blood vessel cells or lymphatic vessel cells were cultured alone in the fourth channel 340, and in vivo blood vessel/lymphatic vessel formation processes were observed. Over 2-3 days, a blood vessel or lymphatic vessel network was first formed in the fourth channel 340, and then over 2-3 days, angiogenesis could be induced from the generated blood vessel network. This confirms that it is possible to sequentially and accurately mimic in vivo blood vessel formation processes in which a blood vessel network is formed through angiogenesis following vasculogenesis. In addition, it is also possible to mimic the process of lymphatic vessel formation and reproduce lymphatic vessels in this context. The results are shown in
In this Example, the skin's structural layer was cultured horizontally. In this Example, the microfluidic chip 20 having the structure shown in
In this Example, the immune function of the skin's structural layers was mimicked. In this Example, the microfluidic chip 10 having the structure shown in
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2015/009379 | 9/4/2015 | WO | 00 |
