KR101750416B1 - Microfluidic chip for HET-CAM test - Google Patents
Microfluidic chip for HET-CAM test Download PDFInfo
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- KR101750416B1 KR101750416B1 KR1020150144473A KR20150144473A KR101750416B1 KR 101750416 B1 KR101750416 B1 KR 101750416B1 KR 1020150144473 A KR1020150144473 A KR 1020150144473A KR 20150144473 A KR20150144473 A KR 20150144473A KR 101750416 B1 KR101750416 B1 KR 101750416B1
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Abstract
The microfluidic chip for HET-CAM test of the present invention provides a microfluidic chip for HET-CAM test capable of material transportation, reaction, signal generation, signal recognition, and signal analysis by constructing an integrated skin-vascular system imitating system having high physiological similarity , It is possible to control the signal transmission process between epithelial cells cultured in each compartment, and it has an advantage that it can be monitored in real time.
Description
The present invention relates to an apparatus for a HET-CAM (Hen's Egg Test - Chorioallantoic Membrane) test.
Since external skin preparations such as cosmetics may cause skin irritation due to skin contact, safety evaluation is essential. In the safety evaluation, the safety evaluation items for the conventional external skin preparations are 8 items such as hair loss, eye irritation, oral / inhalation toxicity, oral mucosa, skin contact, clothing damage, environmental hormones and regulated substances , And each manufacturer and each country has established a standard evaluation method to conduct strict safety evaluation.
In recent years, the ethical concept of the sacrifice of experimental animals has been strengthened, and in the safety evaluation part of the external medicine for skin, the regulations for the abolition or prohibition of animal experiments have been enacted centering on the EU Union (7th Amendment to the EU Cosmetics Directive) The development of alternative animal testing methods is underway worldwide. In this way, the awareness of the welfare of experimental animals is strengthened and alternative methods such as replacement (replacement), reduction of the number of experimental animals (reduction), and refinement of the pain of experimental animals Is being developed. In addition, since March 2013, all animal experiments on European cosmetics are banned, necessitating the development of alternative testing methods for animals.
Currently, Europe has developed a new alternative test method based on ECVAM (European Center for the Validation of Alternative Methods), and has been registering validation and test methods. An alternative test method that is useful is the OECD Guideline (TG 420, 423, 425, 428-432, 435, 437-439, 442, etc.) Likewise, the United States has been working with the NTP for the Evaluation of Alternative Toxicological Methods (NICEATM) and Interagency Coordinating Committee on the In Japan, alternative testing methods have been developed focusing on the JACVAM (Japanese Center for the Validation of Alternative Methods) centering on the Validation of Alternative Methods. In Japan, an internationally recognized animal replacement And submit the data according to the test method. China also allows phototoxicity alternatives to be used for safety assessments. In addition, Korea has established KoCVAM (Korean Center for the Validation of Alternative Methods) on November 3, 2009, and is working to register the research, confirmation and test methods of the new alternative test method. The Korea Food & Drug Administration Guidelines for Alternative Test Animals for Test Animals "are issued and recommended for alternative testing.
In recent years, as an alternative test method that does not use an experimental animal directly, a safety evaluation method by in vitro culturing of a living body tissue or a cell extracted from an experimental animal is emerging. Various studies have been conducted on the stability evaluation method through culture such as tissue culture and cell culture through primary culture and subculture and the culture method for various kinds of cells is well established have. Therefore, experiments using cells and tissues cultured in vitro have the advantage of obtaining useful experimental results without using experimental animals. A representative example of an alternative test method of the skin primary stimulation test is an artificial skin model (RhE, Reconstituted human) which reconstructs a three-dimensional human skin by differentiating keratinocytes, which are the main cells constituting the epidermis of human skin, skin equivalent model). In ECVAM, various alternative validation studies on artificial skin models such as EPISKIN, EpiDerm, SkinEthic, etc. have been added to the OECD Guideline to replace skin irritation. In the United States and Japan, many artificial skin models recognize its usefulness through various validation. In Korea, commercialization of KeraSkin, an artificial skin model using normal human epidermal keratinocytes for an animal replacement test method, has already been carried out NeoDerm, an experimental 3D artificial skin model that reproduces all layers of skin from the human dermis to the epidermis, is being developed and commercialized. The NeoDerm is used to evaluate morphological and physiological skin studies and toxicity, efficacy, and permeability of cosmetics and drugs.
However, until now, in the case of an extracellular cell culture based on a well-plate as an alternative test method and a technology for evaluating toxicity of a substance to a cultured cell, the initial concentration of the injected substance is defined, but a disadvantage .
Acute mucosal irritation test methods for chemicals such as cosmetics, which are likely to come into contact with human eyes, have been made using the Draize rabbit eye irritation test method proposed in 1944 (JH Draize et al., J. Pharmacol. Exp. Therapeut., 1944, 82, 377-390.). However, this method has an advantage of providing a very useful result for the mucosal test, but various alternative test methods are required for animal welfare because a lot of animals are sacrificed and a lot of pain is given to experimental animals such as rabbits. Therefore, the HET-CAM test method using the chorioallantoic membrane (CAM) of the embryo has emerged as a very simple and useful method which is highly correlated with the Draize rabbit eye irritation test in the animal substitution test for the mucous membrane stimulation .
Meanwhile, the conventional microfluidic chip has advantages of culturing the cells in each channel and continuously injecting the substance or solution with the syringe pump to keep the concentration constant. However, in the case of applying the HET-CAM test method to the conventional microfluidic chip And there are various problems with respect to efficient culturing of epithelial cells and fibroblasts and mass transfer characteristics even when applied to conventional microfluidic chips.
In addition, the HET-CAM test method is a test method for observing the phenomenon that occurs when a test substance is applied to the embryo endothelial cells cultured in an incubator for 10 days by visual observation and giving a score (normal: 0 point, bleeding: 1 point, : 2 points, coagulation: 3 points), the subjective evaluation of the observer is an important factor, and even though the skilled observer evaluates, there is still a problem that objectivity is lacking and quantitative evaluation is impossible.
It is an object of the present invention to provide a three-dimensional HET-CAM microfluidic chip that can replace the HET-CAM test method in order to solve the conventional problem that requires high objectivity and can not be quantitatively evaluated.
It is also an object of the present invention to provide a microfluidic chip for HET-CAM testing capable of co-culturing and controlling epithelial cells and fibroblasts.
It is also an object of the present invention to provide a microfluidic chip for HET-CAM testing capable of carrying out substance transport, reaction, signal generation, signal recognition, signal analysis, and controllability by constructing a skin-blood vessel mimic integration system having high physiological similarity will be.
It is also an object of the present invention to provide a microfluidic chip for HET-CAM testing capable of qualitative / quantitative analysis of the mucosal membrane stimulation of a test substance.
The present invention relates to an epithelial cell culture section; A fibroblast culture unit provided at a position higher than the epithelial cell culture unit and having a channel portion including a plurality of channels communicating with the epithelial cell culture unit; An epithelial cell supply unit communicating with the epithelial cell culture unit and supplied with epithelial cells; And a fibroblast supply part communicating with the fibroblast culture part and supplied with fibroblasts. The present invention also provides a microfluidic chip for HET-CAM testing.
In one embodiment of the present invention, the width of the channel is such that the epithelial cells and the fibroblasts are present in the epithelial cell culture portion and the fibroblast culture portion, respectively, while the test substance is transferred to the epithelial cell culture portion, ≪ / RTI >
In one example of the present invention, the microfluidic chip for HET-CAM test may satisfy the following formula (1).
[Equation 1]
C W ≤ F W
(Wherein W is the width C of the channel, the W F is the magnitude of the fibroblasts.)
In one embodiment of the present invention, the width of the channel is not limited as long as the fibroblasts are not transferred to the epithelial cell culture portion but the test substance is delivered, but may be, for example, 3 to 100 탆.
In one embodiment of the present invention, the test substance supply unit may be disposed at an upper portion of the fibroblast culture unit and communicated with the channel unit.
In one example of the present invention, the test substance supply part may be separated and assembled from the fibroblast culture part.
In one example, the present invention can provide an immunochemical staining method comprising a step of separating a test substance supply portion of a microfluidic chip for HET-CAM test and then dyeing.
In one example, the present invention can provide a method for co-culturing epithelial cells and fibroblast cells using a microfluidic chip for HET-CAM test.
The microfluidic chip for HET-CAM test of the present invention provides a microfluidic chip for HET-CAM test capable of material transportation, reaction, signal generation, signal recognition, and signal analysis by constructing an integrated skin-vascular system mimetic system having high physiological similarity , It is possible to control the signal transmission process between epithelial cells cultured in each compartment, and it has an advantage that it can be monitored in real time.
Specifically, the microfluidic chip for HET-CAM test of the present invention can control the mass transfer process through the membrane formed by the polymer membrane or the fibroblast, and has an advantage that it can be monitored in real time.
Further, the microfluidic chip for HET-CAM test of the present invention can be recognized as individual cells of each compartment by signals generated from environmental changes around the cells due to stimulation such as chemical, optical, magnetic, There is an advantage.
Further, the microfluid chip for HET-CAM test of the present invention is advantageous in co-culturing epithelial cells and fibroblasts.
In addition, the microfluidic chip for HET-CAM test of the present invention has an advantage that it can qualitatively / quantitatively analyze the mucosal membrane stimulation of the test substance.
In addition, the microfluidic chip for HET-CAM test of the present invention can be manufactured in a small scale and can effectively replace the HET-CAM test method using embryo.
1 is a schematic diagram of a microfluidic chip of the present invention.
2 is a diagram of a cell culture module according to
3 is a view of a test substance supply module according to
4 is a schematic view of a mask for fabricating the cell culture module and the test substance supply module according to the first embodiment of the present invention.
5 is an image showing a microfluidic chip manufactured by using a mask for fabricating the cell culture module and the test substance supply module according to the first embodiment of the present invention.
FIG. 6 is an image obtained by inserting and culturing fibroblasts into a microfluidic chip according to Example 1 of the present invention, and observing using a microscope. FIG.
7 is an image obtained by observing the degree of test substance delivery over time after injecting a fluorescent solution into a microfluidic chip according to Example 1 of the present invention using a microscope.
Hereinafter, a microfluid chip for HET-CAM testing according to the present invention will be described in detail with reference to the accompanying drawings.
The drawings described in the present invention are provided by way of example so that a person skilled in the art can sufficiently convey the idea of the present invention. Therefore, the present invention is not limited to the illustrated drawings, but may be embodied in other forms, and the drawings may be exaggerated in order to clarify the spirit of the present invention.
In addition, unless otherwise defined, technical terms and scientific terms used in the present invention have the same meanings as those of ordinary skill in the art to which the present invention belongs. In the following description and the accompanying drawings, Description of known functions and configurations that may unnecessarily obscure the subject matter will be omitted.
Also, units of% used unclearly in the present invention means weight percent.
The term " compartment " used in the present invention refers to both connected and unconnected regions of an epithelial cell culture, a fibroblast culture, an epithelial cell supply, a fibroblast supply, a channel, and a test material supply unit.
The term " epithelial cell " and " fibroblast " used in the present invention may mean a culture medium which is a mixture containing a medium.
The present invention relates to an epithelial cell culture section; The fibroblasts injected into the fibroblast culture section are not transferred to the epithelial cell culture section, and the test substance is not transferred to the cultured section of the epithelial cell, A fibroblast culture unit in which a channel part including a plurality of channels communicating with the epithelial cell culture unit is formed so as to be delivered; An epithelial cell supply unit communicating with the epithelial cell culture unit and supplied with epithelial cells; And a fibroblast supply part communicating with the fibroblast culture part and supplied with fibroblasts. The present invention also provides a microfluidic chip for HET-CAM testing.
Specifically, the present invention relates to an epithelial cell culture unit; A channel part provided at a position higher than the epithelial cell culture part and including a plurality of channels communicating with the epithelial cell culture part; A fibroblast culture section located above the channel section and communicating with the plurality of channels; An epithelial cell supply unit communicating with the epithelial cell culture unit and supplied with epithelial cells; And a fibroblast supply part communicating with the fibroblast culture part and supplied with fibroblasts, and a grooved test material supply module, wherein the cell culture module and the test material supply module are combined Wherein the fibroblast culture section is formed as a space in which the upper surface of the channel section of the cell culture module and the surface of the groove of the test substance supply module are coupled to each other to form the fibroblast culture section. A microfluidic chip for testing can be provided.
Therefore, the HET-CAM microfluidic chip of the present invention can arrange and arrange the divided areas in a three-dimensional structure to efficiently perform material transport, reaction, signal generation, signal recognition, and signal analysis. Specifically, depending on the width, length, and height of the epithelial cell culture unit, the fibroblast culture unit, the channel-containing epithelial cell supply unit, the fibroblast supply unit, etc., the cell movement preferably depends on the channel width, It is possible to inhibit and transport the substance. In each compartment, the cells can be injected, adhered, and grown, and each compartment is fluidically connected and the culture conditions can be leveled through the flow of the fluid.
Accordingly, the microfluidic chip for HET-CAM test of the present invention can quantitatively determine environmental variables such as flow rate, concentration, temperature, and humidity of cell culture fluids, test substances and the like through respective compartments such as the epithelial cell culture section using an optical microscope or the like And more precise control and analysis can be performed through, for example, fluorescence staining analysis method stained by immunochemical analysis.
In one example of the present invention, the microfluidic chip for HET-CAM test may satisfy the following formula (1).
[Equation 1]
C W ≤ F W
(Wherein W is the width C of the channel, the W F is the magnitude of the fibroblasts.)
Further, the size of the fibroblast may mean the smallest element among the length, width, and height elements of the fibroblast, and may mean, for example, the diameter of a cell that refers to the size of a conventional cell.
That is, the width of the channel may be less than the size of the fibroblasts such that the epithelial cells and the fibroblasts are separately present in the epithelial cell culture unit and the fibroblast culture unit, respectively, and the test substance is delivered to the epithelial cell culture unit. If the width of the channel is less than the fibroblast size (diameter), the fibroblasts will not enter the channel. Therefore, fibroblasts are cultured in fibroblast culture to reach confluent state, and the membrane formed by fibroblasts plays the same role as the polymer membrane, thereby controlling the process in which the injected test substance is transferred to the epithelial cells present in the epithelial cell culture part can do.
In one embodiment of the present invention, the width of the channel is not limited as long as the fibroblasts are not transferred to the epithelial cell culture portion but the test substance is delivered, but may be, for example, 3 to 100 탆. When the width of the channel satisfies the above range, the test material flows into the channel without the fibroblasts flowing into the channel of the epithelial cell culture section. Therefore, the epithelial cell culture and the respective epithelial cells injected into the fibroblast culture section, Fibroblasts are present in fractions. That is, when the width of the channel does not satisfy the range of 3 to 100 탆, the fibroblasts flow into the epithelial cell culture section, the cells can not be fractionated, and a cell membrane can not be formed.
In one embodiment of the present invention, the width of the interval of the channels is not limited. For example, the width of the channels may be equal to or the same as the width of the channel, For example, 3 to 100 mu m. The spacing between the channels may mean concave and convex portions formed between the channel and the channel so as to form a repetitive pattern of the channel and the channel.
In one example of the present invention, the length of the channel is not limited within a range that can achieve the object of the present invention, but may be, for example, 10 to 2,000 mu m. When the length of the channel satisfies the above range, it is preferable that the test substance can be introduced into the channel more efficiently without the fibroblasts flowing into the channel of the epithelial cell culture section, but this is a preferable example only. But is not limited to.
In one example of the present invention, the height of the channel is not limited within a range that can achieve the object of the present invention, but may be, for example, 1 to 20 탆. When the height of the channel satisfies the above range, the fibroblasts can be prevented from flowing into the channel of the epithelial cell culture section more efficiently, but this is a preferable example, but the present invention is not limited thereto.
In one embodiment of the present invention, the epithelial cell culture unit includes a first epithelial cell culture unit and a second epithelial cell culture unit formed on both sides of the fibroblast culture unit, And the second epithelial cell culture section, respectively.
In one embodiment of the present invention, the epithelial cell supply unit may be formed in the first epithelial cell culture unit and the second epithelial cell culture unit, respectively. In one specific example, the number of the epithelial cell supply units is not limited, but may be 1 to 10.
In one example of the present invention, the width of the epithelial cell culture portion is not limited, but may be 100 to 2,000 mu m. In one embodiment of the present invention, the length of the epithelial cell culture section is not limited, but may be 1 to 20 mm. In one embodiment of the present invention, the height of the epithelial cell culture unit is not limited, but may be 50 to 500 탆. When the above range is satisfied, epithelial cells and test substances can be efficiently delivered, which is preferable, but the present invention is not limited thereto.
In one example of the present invention, the number of the fibroblast supply parts is not limited, but may be, for example, one to two. Specifically, the fibroblast supply part may be formed on one side or both sides of the fibroblast culture part.
In an exemplary embodiment of the present invention, the path of the fibroblast culture portion communicating with the fibroblast culture portion in the fibroblast supply portion is not limited, but may be in the range of 70 to 120 degrees, It is preferable to be formed as shown in Fig. 1, for example, to be positioned in a direction close to 90 degrees and to be communicated. If this is the case, fibroblasts can be injected efficiently and the number of compartments per area (epithelial cell supply, fibroblast supply, etc.) can be increased without overlapping with other compartments.
In one example of the present invention, the width (diameter) of the compartment of the epithelial cell culture unit, the epithelial cell supply unit, the fibroblast supply unit, the test substance supply unit and the like is not limited, but may be, for example, 100 to 2,000 탆, But may be, for example, 50 to 500 mu m. When the above range is satisfied, epithelial cells, fibroblasts, test substances and the like can be efficiently introduced, which is preferable, but the present invention is not limited thereto.
In one embodiment of the present invention, the test substance supply unit may be disposed at an upper portion of the fibroblast culture unit and communicated with the channel unit. Therefore, the HET-CAM microfluidic chip of the present invention can arrange and arrange the divided areas in a three-dimensional structure to efficiently perform material transport, reaction, signal generation, signal recognition, and signal analysis.
In one example of the present invention, the test substance supply part may be separated and assembled from the fibroblast culture part. Therefore, the test substance supply part can be reversibly assembled and separated from the fibroblast culture part, so that it can be controlled and analyzed more easily if necessary.
In one example of the present invention, the test material supply portion, that is, the groove of the test material supply module may include a test material supply hole. The test material supply hole has a role to supply the test material directly to the fibroblast culture unit using a device such as a syringe or a syringe. As a specific example, the size (diameter) of the test material supply hole is not limited as long as the test substance can be supplied, but may be, for example, 10 to 2,000 mu m.
In one example of the present invention, the microfluidic chip for HET-CAM test may include a polymer membrane located on the upper portion of the fibroblast culture unit. As a specific example, the polymer membrane may be laminated or covered or formed over a region including a fibroblast culture portion or an area including another compartment, or may be coated on a region including a fibroblast culture portion (for example, a channel portion including a channel or a channel) . Therefore, it is advantageous to monitor the mass transfer process through the polymer membrane in real time, and it is preferable from the viewpoint of real-time monitoring to use a transparent polymer membrane. The thickness of the polymer membrane and the size and frequency of the holes can be exemplified as important parameters to be considered at this time. The size of the holes is not limited within the scope of achieving the object of the present invention, . Therefore, the polymer membrane can perform the same function as the fibroblast membrane formed in the fibroblast culture section, and can control the process of transferring the injected test substance to the cells.
In one embodiment of the present invention, the microfluidic chip for HET-CAM testing may include a polymer membrane positioned between the fibroblast culture section and the test substance supply section. Therefore, fibroblasts can be cultured on the polymer membrane to control the transfer of the injected material to the epithelial cells. That is, it is possible to control the mass transfer process through the membrane formed of the polymer membrane and the fibroblast, and there is an advantage that it can be monitored in real time.
In an exemplary embodiment of the present invention, the microfluid chip for HET-CAM test may be made of various materials such as polymers, metals, and the like. For example, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA) Polyurethane (PU), and the like.
In one example of the present invention, the inner and outer surfaces of the microfluidic chip for HET-CAM testing may be treated with one or more of oxygen plasma treatment, extracellular matrix application treatment, and self-assembled monolayer treatment using a silane compound .
In one example, the present invention can provide an immunochemical staining method comprising a step of separating a test substance supply portion of a microfluidic chip for HET-CAM test and then dyeing. Accordingly, the present invention provides a system capable of analyzing a signal or the like obtained by a reaction of a cell after an injected test substance is transferred to a cell with an image of an optical microscope, and obtaining and analyzing a fluorescence microscope image of cells stained by immunochemical analysis Can be provided. Specifically, when the microfluidic chip for HET-CAM test of the present invention is used, objectivity and quantitative evaluation can be performed as compared with the conventional HET-CAM test method. However, when cells are treated with Trypan blue or the like, It is preferable because more improved objectivity and quantitative evaluation are possible.
The microfluid chip for HET-CAM test of the present invention has an effect of monitoring environment changes around cells cultured in an incubator (temperature, humidity, gas can be controlled) in real time.
In one example, the present invention can provide a method for co-culturing epithelial cells and fibroblast cells using a microfluidic chip for HET-CAM test.
In an embodiment of the present invention, a microfluidic chip for HET-CAM test of the present invention is applied to a signal analyzer or the like to generate and analyze a signal that a cell recognizes and reacts to a signal formed from an environmental change, Can provide a smart chip.
Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is described in more detail with reference to the following Examples. However, the scope of the present invention is not limited by the following Examples.
In order to manufacture a mask for fabricating a microfluidic chip for HET-CAM test, as shown in FIGS. 4A, 4B, 4C and 4D, a mask for fabricating a microfluidic chip was designed using a CAD and printed on a transparent paper To prepare a mask. Each of the above masks was placed on a silicon wafer coated with a photosensitizer, followed by exposure to photoreaction. After the photoreaction is completed, the polymer is developed in a solvent to prepare a master for a duplicate casting. A polymer chip is prepared by pouring a mixture of a thermosetting polymer (PDMS) before curing into a master and curing the polymer chip. The cell culture module was drilled. FIG. 2B is an image of the cell culture module, and FIG. 2C is an enlarged view of a portion indicated by a rectangle in FIG. 2B. In addition, a test substance supply module was fabricated in the same manner as the cell culture module, which is shown in FIG. 3B. Specifically, a fibroblast culture section is prepared by using FIG. 4A, a cell culture module is prepared by using FIG. 4B, a test substance supply module is prepared by using FIG. 4C, Was assembled to the cell culture module as shown in Fig. 4D, and the microfluidic chip for HET-CAM test was prepared by aligning and assembling the test substance supply module.
The microfluidic chip for HET-CAM test thus manufactured is shown in Figs. 5A and 5B. The surface of the microfluidic chip for HET-CAM test was activated by oxygen plasma treatment.
More specifically, FIG. 4A shows a mask for producing a channel portion including a channel of a fibroblast culture portion. The channel width and the channel spacing of the fibroblast culture portion thus produced are both 20 μm. FIG. 4B is a mask for preparing a cell culture module having a first epithelial cell culture section, a fibroblast culture section and a second epithelial cell culture section, an epithelial cell supply section, a fibroblast supply section, and the like, The width of each compartment of the cell culture module is 2 mm, the length is 7 mm, and the height is 100 탆. Diameter and height of the epithelial cell supply part were 6 mm and 100 ㎛, respectively. Diameter and height of the fibroblast supply part were 4 mm and 100 ㎛, respectively. Fig. 4C is a mask for preparing a test material supply part. The test material supply part made therefrom has a width of 2 mm, a length of 7 mm, and a height of 100 m.
FIG. 6 is an optical microscope image obtained by injecting fibroblast (NHDF) into a microfluid chip for HET-CAM test according to Example 1. FIG. Specifically, FIGS. 6A and 6C are images obtained by inverted-phase microscopy after culturing the fibroblast culture with fibroblasts for 36 hours, and FIGS. 6B and 6B show staining with a calcein AM reagent (0.04 mM, 30 Min). Observation revealed that the cells were growing very well during the time set in the culture channel, and were formed without fractionation in each compartment. This mimics the HET-CAM film.
FIG. 7 is an image of a fluorescence microscope obtained at intervals of time with a solution of a fluorescent substance (0.1%, fluorescein sodium salt) corresponding to a test substance in a test substance supply portion of a microfluidic chip for HET-CAM test according to Example 1. FIG. Specifically, it can be seen that the test substance injected from FIGS. 7A, 7B and 7C is present only in the test substance supply portion at first, and from the D, E and F of FIG. 7, 7 (D) to (F)). This is a simulation of the kinetic process of the drug delivery to the blood vessels where the epithelial cells are growing through the HET-CAM membrane.
It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims and their equivalents.
1: epithelial cell supply part
2: fibroblast supply part
3: Epithelial cell culture section
4: Fibroblast culture section
Claims (13)
A channel part provided at a position higher than the epithelial cell culture part and including a plurality of channels communicating with the epithelial cell culture part;
A fibroblast culture section located above the channel section and communicating with the plurality of channels;
An epithelial cell supply unit communicating with the epithelial cell culture unit and supplied with epithelial cells; And
A cell culture module including a fibroblast supply part communicating with the fibroblast culture part and supplied with fibroblasts;
And a groove-formed test substance supply module,
Wherein the cell culture module and the test substance supply module are combined to form the fibroblast culture section, wherein the upper surface of the channel section of the cell culture module and the surface of the groove of the test substance supply module are coupled to each other, Wherein the fibroblast culture portion is formed on the surface of the microfluidic chip.
A microfluidic chip for HET-CAM testing satisfying the following formula (1).
[Equation 1]
C W ≤ F W
(Wherein W is the width C of the channel, the W F is the magnitude of the fibroblasts.)
The microfluidic chip for HET-CAM testing has a width of 3 to 100 mu m.
Wherein the epithelial cell culture unit comprises a first epithelial cell culture unit and a second epithelial cell culture unit formed on both sides of the fibroblast culture unit and the channel is formed in the first epithelial cell culture unit and the second epithelial cell culture unit And a microfluidic chip for HET-CAM testing.
The microfluidic chip for HET-CAM testing according to claim 1, wherein the epithelial cell supply unit is formed in the first epithelial cell culture unit and the second epithelial cell culture unit, respectively.
The microfluidic chip for HET-CAM test wherein the epitaxial cell culture section has a width of 100 to 2,000 mu m.
The test substance supply module is separated and assembled with the cell culture module, and a microfluid chip for HET-CAM test.
The microfluidic chip for HET-CAM test in which the test material supply hole is formed in the groove of the test material supply module.
Further comprising a polymer membrane located above the fibroblast culture section or a polymer membrane positioned between the fibroblast culture section and the test substance supply module.
Wherein the inner and outer surfaces of the microfluidic chip are treated with one or more of oxygen plasma treatment, extracellular substrate coating treatment, and self-assembled monolayer treatment using a silane compound.
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| JP5815643B2 (en) | 2008-07-16 | 2015-11-17 | チルドレンズ メディカル センター コーポレーション | Organ mimic device having microchannel and method of using and manufacturing the same |
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