KR20230035838A - Microfluid chip for spheroid or organoid and method for producing spheroid or organoid model using the same - Google Patents

Abstract

In order to achieve one object of the present invention, a microfluidic chip for three-dimensionally simulating a spheroid or organoid in vitro model according to the present invention comprises a supply unit, a forming unit formed under the supply unit, and a culture unit formed below the formation unit and receiving cells for culture or materials for a vascular composition from the supply unit, wherein the fluid supplied through the supply unit may be condensed and formed inside the forming unit to maintain a spaced apart state from the lower portion of the culturing unit.

Description

Spheroid or organoid microfluidic chip and method for forming a spheroid or organoid in vitro model using the same

The present invention relates to a microfluidic chip for 3-dimensionally simulating a spheroid or organoid in vitro model.

Numerous drugs are being developed and evaluated for the treatment of human diseases, and in vitro human tissue and organ mimic model studies are being conducted for drug development. Among the models, there are spheroids and organoids that collect cells to form a three-dimensional shape, which has the advantage of providing a more complex and colorful microenvironment than cells cultured in two dimensions. However, general spheroids and organoids have problems in that oxygen and nutrient supply is not smooth due to the absence of perfusable three-dimensional blood vessels, and it is difficult to create an internal environment in the model.

To overcome these limitations, models co-cultured with spheroids and organoids and vascular networks in microfluidic chips have been developed. However, in the case of previous co-culture models, the process of injecting spheroids and organoids into the chip is quite complicated and difficult.

Specifically, in the preceding co-culture models, spheroids and organoids must first be formed in advance on another platform (U-shaped 96 well plate), and the formed spheroids and organoids are placed in a well plate using a pipette. After extracting and injecting it into the chip, considerable skill is required. In addition, reproducibility is not secured, and convenience and efficiency are low because there may be differences in experimental results depending on the individual ability of the experimenter.

One object of the present invention is to provide a microfluidic chip capable of producing a three-dimensional vascularized spheroid or organoid in vitro model on a single chip.

Another object of the present invention is to provide a microfluidic chip to which hanging drop and 3D bioprinting are applicable.

In order to achieve one object of the present invention, a microfluidic chip for three-dimensionally simulating a spheroid or organoid in vitro model according to the present invention includes a supply unit, a formation unit formed under the supply unit, and a lower portion of the formation unit. and may include a culture unit receiving culture cells or blood vessel composition materials from the supply unit, and the fluid supplied through the supply unit may be condensed and formed inside the formation unit to be maintained in a state of being spaced apart from the lower portion of the culture unit. there is.

In one embodiment, the supply unit may be formed in a tapering shape that becomes narrower toward the bottom.

In one embodiment, the supply unit may include a first supply unit for supplying the cells for culture and a second supply unit for supplying the material for forming blood vessels.

In one embodiment, the first supply part is formed on the upper part of the second supply part so that the cells for culture are supplied along the side of the supply part, and the second supply part is formed on the upper part of the formation part to supply the material for forming blood vessels. This may be directly supplied to the forming section.

In one embodiment, a step may be formed in the first supply unit.

In one embodiment, the forming portion may include a tapering shape that becomes narrower toward the top.

In one embodiment, one or more of the three-dimensional vascularized spheroids and organoids may be formed therein.

In one embodiment, the microfluidic chip may further include a reservoir that is formed in contact with the upper portion of the culture unit and is partitioned from the supply unit and the formation unit, and capable of supplying the culture medium or test material to the culture unit.

In one embodiment, the microfluidic chip may further include an injection part capable of supplying vascular endothelial cells to an edge of the culture part and a channel connecting the injection part and the culture part.

In one embodiment, the microfluidic chip may further include a base part formed below the culture part.

The method for forming a spheroid or organoid in vitro model using a microfluidic chip according to the present invention includes the steps of supplying cells for culture to the supply unit of the microfluidic chip, and supplying the supplied cells for culture to the supply unit and the forming unit. It may include forming spheroids or organoids by condensing and forming spheroids or organoids, supplying a material for forming blood vessels to the supply unit, and forming three-dimensional vascularized spheroids or organoids to the culture unit.

In one embodiment, the step of supplying a culture solution, a test substance, or vascular endothelial cells to the culture unit may be further included.

The above-described means for solving the problems is only illustrative and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments described in the drawings and detailed description.

The microfluidic chip according to the present invention is equipped with an inlet that meets the pipette tip specifications, and enables the formation of spheroids or organoids by various 3D bioprinting and hanging drop methods. Through this, the existing process of forming spheroids or organoids externally and transferring them to a microfluidic chip through co-culture with blood vessels can be integrated and conveniently and quickly performed on one chip.

A vascularized spheroid or organoid model may be obtained by co-cultivating a material for forming blood vessels with the formed spheroid or organoid. This is an in vitro model with higher in vivo similarity than existing models, and can be used as a model for drug development.

In addition, since the microfluidic chip of the present invention can be manufactured through a 3D printer and injection molding, high efficiency can be secured.

FIG. 1A is a plan view of a microfluidic chip according to an embodiment of the present invention, and FIG. 1B is a perspective view.
2A is a view showing a well, which is a culture unit of a microfluidic chip, according to an embodiment of the present invention, and FIG. 2B is a cross-sectional view taken along line AA' of FIG. 2A.
Figure 3a is a cross-sectional view taken along A-A' of Figure 2a, Figure 3b is the interface between the supply unit and the formation unit of the microfluidic chip according to one embodiment of the present invention, Figure 3c is a microfluidic chip according to one embodiment of the present invention It is a drawing showing the surface where the forming part and the culturing part of the fluid chip are in contact.
FIG. 4A is a plan view of a microfluidic chip according to an embodiment of the present invention, FIG. 4B is a bottom view, and FIG. 4C is a cross-sectional view along line AA′ of FIG. 2A.
5 is a diagram illustrating a process of forming a three-dimensional vascularized spheroid or organoid in vitro model as an example of a microfluidic chip according to an embodiment of the present invention.
6A to 6C are diagrams illustrating optimal conditions for performing hanging drop and 3D bioprinting according to an embodiment of a microfluidic chip according to an embodiment of the present invention.
FIG. 7a shows 3D modeling when hanging drop and 3D bioprinting by an embodiment of a microfluidic chip according to an embodiment of the present invention succeeds (pinning) or fails (bulging), and FIG. 7b shows when it succeeds. It is a drawing showing a photograph taken with a fluorescence microscope.
8 is a diagram showing the correlation between spheroid diameter and cell number according to an example of a microfluidic chip according to an embodiment of the present invention.
9 is a diagram showing the change over time or the number of cells of spheroids or organoids according to an example of a microfluidic chip according to an embodiment of the present invention.
10 is a diagram showing photographs of patterning spheroids or organoids using normal cell lines (HCT116, U87MG, HEPG2, SW620) and cancer patient cells (Patient #1, Patient #2).
11 is a diagram showing a fluorescence micrograph of a three-dimensional vascularized spheroid or organoid in vitro model manufactured according to an example of a microfluidic chip according to one embodiment of the present invention.

Hereinafter, embodiments of the present application will be described in detail so that those skilled in the art can easily practice with reference to the accompanying drawings. However, the present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. And in order to clearly describe the present application in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the present specification, when a member is said to be located “on” another member, this includes not only a case where a member is in contact with another member, but also a case where another member exists between the two members.

Throughout the present specification, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

As used throughout this specification, the terms "about," "substantially," and the like are used at or approximating the value when manufacturing and material tolerances inherent in the stated meaning are given, and do not convey the understanding of this application. Accurate or absolute figures are used to help prevent exploitation by unscrupulous infringers of the disclosed disclosure. The term "step of (doing)" or "step of" as used throughout the present specification does not mean "step for".

Throughout this specification, the term "combination(s) of these" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, It means including one or more selected from the group consisting of the above components.

Throughout this specification, reference to "A and/or B" means "A or B, or A and B".

The term 'spheroid' used throughout the present specification refers to a cell mass formed by cell-to-cell junctions by external forces without natural self-assembly or the presence of a scaffold. In general, spheroids maintain the composition of the natural ECM by secreting ECM on their own without the need to separately add a substance that serves as an ECM (extracellular matrix) from the outside.

Spheroids have been used to reproduce the basic properties of brain tissue, such as the diversity of cellular composition, electrophysiological properties, ECM secretion and physical properties. Dingle made spheroids from newborn rat cerebral cortical cells at four different densities (1K, 2K, 4K, and 8K cells/spheroid). After 14 days, they reached a stable size and were assembled similarly to the biological density (2 ~ 4 x 105 cells/mm3). Immunohistochemical analysis confirmed the presence of not only neurons but also several glial cells, including astrocytes, oligodendrocytes and even microglia. Patch-clamp experiments revealed that the neurons within the spheroids were electrically active and detected the generation of postsynaptic currents (PSCs), demonstrating the formation of synaptic networks. In addition, it secreted laminin and exhibited an elastic modulus similar to that of neonatal rat cortex (Pei et al., 2017).

The term 'organoid' used throughout the present specification is a large cell aggregate resembling the cell composition, structure, and function of an actual organ. Organoids using PSCs and adult stem cells (ASCs) can represent some of the basic functions of human tissue. In many ways the organism can be described as an advanced spheroid. This is because in organoids, stem cells self-organize spatially differentiated differentiation and cell alignment, whereas in spheroid models, stem cells or progenitor cells are lacking, resulting in insufficient self-renewal and differentiation. As a result, organoids produce more stable 3D cell structures that can be maintained for longer periods of time than spheroids.

In general, organoids are more suitable than other methods for preserving the intrinsic capabilities of various types of cells and reproducing detailed parts of the developmental process. In particular, many achievements have been made in discovering the basic structure and physiological characteristics of real tissues. Nevertheless, models made through such self-assembly have some limitations. First, they vary greatly in size and shape. In addition, as it was revealed that Matrigel can play the role of a niche (microenvironment) in which PSCs are sensed and grown, Matrigel is mostly used to form neural organoids. Since it has not been analyzed, many organoids involving the use of Matrigel cannot avoid the problem of batch to batch inconsistency, i.e., heterogeneity in the composition of Matrigel produced, which is why organoids It is difficult to use for high throughput screening. In addition, organoid models are generally larger in size than spheroid models. Because of this, necrosis of cells due to lack of nutrients and oxygen in the center is inevitable. Therefore, it is necessary to form a capillary network within the organoid structure to promote gas and nutrient exchange (Pei et al., 2017).

The term 'hanging drop' used throughout the present specification means that 10 to 50ul of the culture medium is hung and fixed on the lid of the culture vessel, and the cells gathered at the lower part of the droplet due to gravity form one lump. It means a model for 3-dimensional cell culture in the form (spheroid).

The term '3D bio printing' used throughout the present specification means a concept in which 3D printing technology and biotechnology are converged. Using this, living cells can be piled up in a desired shape to produce tissues and organs. The basic output method is the same as general 3D printing, but 3D bioprinting has a difference in that biocompatible polymers and biomaterials are used as basic materials to output living cells. 3D bioprinting techniques include an inkjet method that sprays and prints materials in small-sized droplets, a micro-extrusion method that pushes materials with a certain viscosity or higher with pneumatic pressure or a piston, and a light source on the surface of a photocurable resin. There is a stereolithography method that investigates. The inkjet method has advantages of high printing speed and low cost, but there is a risk of cell damage due to thermal stress. The microextrusion method is the most widely used because it has the advantage of being able to manufacture various shapes by stacking cells at a high density, but it is necessary to further improve the printing precision and speed. The stereolithography method has the advantage that there is no clogging of cells in the nozzle and precise printing is possible, but it is necessary to improve the slow output speed and improve the compatibility between the material and the cells for stereolithography (Kim Dong-rip, Jeon Yale. 2018).

The term 'cells for culture' used throughout the present specification refers to cells intended to create a 3D vascularized spheroid or organoid in vitro model using the microfluidic chip according to the present invention, and its types include vascular endothelial cells, fibroblasts, etc. Including, but not limited to.

The term 'material for forming blood vessels' used throughout the present specification is a material that is injected to form spheroids or organoids by methods such as hanging drop or 3D bioprinting in the forming unit and then 3-dimensionally vascularize them. It includes, but is not limited to, extracellular matrix, fibroblasts, vascular cells, culture media, and the like.

The term 'constant fluid' used throughout the present specification is a material for moving cells for culture or materials for vascular composition, and may include all types of cell culture media commonly used, but is not limited thereto.

Hereinafter, embodiments and embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. However, the disclosure may not be limited to these embodiments and examples and drawings.

implementation. Example of a microfluidic chip 1000 according to the present invention

1A to 3C, the microfluidic chip 1000 for three-dimensionally simulating a spheroid or organoid in vitro model according to the present invention is formed in a supply unit 200 and a lower portion of the supply unit 200. It may include a formation unit 320 and a culture unit 340 formed below the formation unit 320 and receiving a supply of cells for culture or materials for forming blood vessels from the supply unit 200 . Hereinafter, the structure of the microfluidic chip 1000 according to an embodiment of the present invention will be described with reference to each drawing.

Referring to the plan view of FIG. 1A , the microfluidic chip 1000 according to the present invention may include a plurality of wells 100 that are one or more culture units.

Referring to FIG. 2A , the well 100 may include a supply unit 200 into which a supply member (hereinafter, a supply member) such as a pipette or a syringe may be inserted. Referring to FIG. 2B , the supply unit 200 may be formed in a tapering shape that becomes narrower toward the bottom. In addition, the supply unit 200 includes a first supply unit 210 for supplying cells for culture and certain fluids (hereinafter, cells for culture) and a second supply unit for supplying materials for forming blood vessels and certain fluids (hereinafter, materials for forming blood vessels). 220, and a step 230 may be formed in the first supply unit 210.

The supply unit 200 may be formed in a tapering shape that becomes narrower toward the bottom so that cells for culture supplied to the first supply unit 210 through the supply member flow along the side of the supply unit 200 . Specifically, when cells for culture are supplied by bringing the tip of the supply member in contact with the side surface of the first supply unit 210, an effect of minimizing the inflow of air into the cells for culture can be obtained.

In addition, a step 230 may be formed in the first supply unit 210 for convenience of supply to the first supply unit 210 . The step 230 may be formed as a flat surface, and more specifically, may be formed to have the same or similar length as the outer diameter of the supply member. Through this, the tip of the supply member can be mounted on the step 230, making supply convenient, and since the length of the step 230 corresponds to the length of the outer diameter of the supply member, the gap between the supply member and the first supply unit 210 is reduced. The effect of reducing air inflow can be obtained.

Referring to FIGS. 2B and 3A , the second supply unit 220 is formed below the first supply unit 210 and above the forming unit 320 so that the material for forming blood vessels can be directly supplied to the forming unit 320. This will be described together with the formation part 320 .

Referring to FIGS. 3A to 3C , a forming unit 320 may be formed under the supplying unit 200 , and the forming unit 320 may include an interface 322 in contact with the supplying unit 200 . The lower portion of the forming unit 320 may be formed in a cylindrical shape from a surface in contact with the culturing unit 340 to a certain height, and then in a tapering shape that becomes narrower toward the upper portion. The interface 322 and the surface where the forming unit and the culturing unit come into contact may be formed in a circular shape.

As the forming unit 320 takes the above-described shape, the cells for culture supplied to the first supplying unit 210 and flowing into the forming unit 320 along the side of the supplying unit 200 span the upper and lower portions of the forming unit 320. be able to bind More specifically, the first supplying part 210 and the forming part 320 may be formed in an hourglass shape based on the interface 322, and through this shape, the cells for culture are formed at the upper and lower parts of the forming part by surface tension. It is condensed and formed so that any one or more of spheroids and organoids can be formed. The formation process of any one or more of the spheroids and organoids may be performed through a hanging drop or 3D bioprinting method, but is not limited to the above method.

A tip of the supply member inserted into the second supply unit 220 may come into contact with the interface 322 . More preferably, the shape and size of the interface 322 is formed to correspond to the shape and size of the tip of the supply member, so that no gap is created when the supply member comes into contact with the interface 322 . Unlike supplying the cells for culture to the side of the supply unit 200 , the cells for forming blood vessels in the second supply unit 220 may be supplied after placing the tip of the supply member so as to come into contact with the interface 322 . Through this, the cells for forming blood vessels can pass through the formation unit 320 and move directly to the culture unit 340 under the formation unit 320 without being affected by surface tension. In this process, the spheroids or organoids formed in the forming unit 320 may move to the culturing unit 340 together with the supplied cells for forming blood vessels.

Referring to FIG. 3A , a culturing unit 340 may be formed under the forming unit 320 . In the culturing unit 340, any one or more of the above spheroids and organoids and supplied cells for forming blood vessels may be located to form one or more of three-dimensional vascularized spheroids and organoids. In addition, one or more of the three-dimensional vascularized spheroids and organoids are formed in the culture unit 340, and may be formed larger than the formation unit 320 for subsequent experiments.

Referring to FIG. 4A , the well 100 is formed in contact with the top of the culture unit 340 in addition to the supply unit 200 and is partitioned from the supply unit 200 and the formation unit 320, and a culture medium or test material is supplied to the culture unit 340. A reservoir 420 capable of supplying may be included. Specifically, a barrier rib exists between the reservoir 420, the forming unit 320, and the supply unit 200, and a lower portion of the reservoir 420 may be formed to be connected to an upper portion of the culturing unit 340. A culture medium or test substance may be supplied to the reservoir 420 when an experiment is performed using the microfluidic chip 1000 on which one or more of 3D vascularized spheroids and organoids are formed.

Referring to FIGS. 4A and 4B , the well 100 may include an injection unit 440 and a channel 460 connecting the injection unit 440 and the culture unit 340 . The injection unit 440 may supply vascular endothelial cells to be located at the edge of the culture unit 340 . The supplied vascular endothelial cells may move to the culture unit 340 along the channel 460 . Specifically, the injection unit 440 is not connected to the supply unit 200, the forming unit 320, or the reservoir 420, and is connected to the culturing unit 340 only through the channel 460. Accordingly, the channel 460 may be formed in an 'L' shape inside the microfluidic chip.

The vascular endothelial cells supplied to the injection unit 440 move to the culture unit 340 through the channel 460 and are located at the edge of one or more of the 3D vascularized spheroids and organoids formed in the culture unit 340. can do. Thereafter, the vascular endothelial cells form a vascular endothelial tissue and serve as a passage through which materials can be smoothly exchanged in and out of any one or more of the three-dimensional vascularized spheroids and organoids, or the cells can move within the culture unit 340. can be performed.

Referring to FIG. 4C , a base part 480 may be further provided under the culture part 340 . The microfluidic chip 1000 of the present invention may be manufactured or sold without the base part 480, but it is preferable to additionally include the base part 480 when in use. Materials that can be used for the base portion 480 include, but are not limited to, polycarbonate film (PC flim) or glass.

Example 1. Formation of a three-dimensional vascularized spheroid or organoid in vitro model

Referring to FIG. 5, the in vitro model formation process according to Example 1 will be described.

The following is only one process of forming an in vitro model, and is presented only to aid understanding of the present invention, and does not mean that the present invention is limited to the following examples.

Step 1. Hanging drop and 3D bioprinting (Day 0)

A pipette is inserted into the first supply unit 210 to supply cells for culture to the side of the first supply unit 210 . It is more convenient to use the step 230 formed in the first supply unit 210. The supplied cells for culture flow along the side of the supply unit 200 and are condensed in the formation unit 320 to form. The ligated cells are used to form one or more of spheroids and organoids using hanging drops and 3D bioprinting.

Step 2. Vessel Patterning (Day 1)

A pipette is inserted into the second supply unit 220 to supply the material for blood vessel formation to the second supply unit 220 . At this time, the tip of the pipette is brought into contact with the interface 322 of the forming unit 320 without gaps. Accordingly, the material for forming blood vessels does not form inside the forming unit, and passes through the forming unit 320 and moves to the bottom surface of the culturing unit 340 . At the same time, any one or more of the spheroids and organoids already formed in the forming unit 320 moves to the culturing unit 340 together with the supplied material for forming blood vessels.

Step 3. 3D vascularization (Day 7~)

In the culture unit 340, one or more in vitro models of 3D vascularized spheroids and organoids are formed. Additionally, vascular endothelial cells may be supplied to the edge of the model through the injection unit 440 and the channel 460 so that material exchange between the culture medium and the model is smoothly performed. After the model is completed, culture medium and test materials may be supplied through the reservoir 420 .

Example 2. Optimal conditions for hanging drop and 3D bioprinting

As an embodiment of the present invention, the optimal conditions for hanging drops will be described.

Referring to FIG. 6A, the volume of the fluid material accommodated in the supply unit 200 and the forming unit 320 is V (μl), the diameter of the interface at the bottom of the forming unit 320 is D (mm), and the culture unit 340 from the interface ) The distance between the bottom surfaces is H (mm), and the angle between the boundary surface of the upper portion of the forming part 320 and the tapering shape is α.

At this time, the conditions between the volume and the diameter of the interface for effectively forming cells on the interface 322 of the forming unit 320 are the same as those in FIG. 6B. (α = 30°, 45°, 60°, 64°, 75°, 90° from the bottom)

Referring to FIG. 6B, it was confirmed that hanging drop and 3D bioprinting of cells for culture were stably possible when the following mathematical condition was satisfied in the area below the linear function expressed as a straight line for each value of α (pinning; 7a and 7b), and in other cases, cell droplets for culture may burst (bulging; see FIG. 7a).

In addition, for the above embodiment, the condition between the height H of the lower portion of the forming portion 320 and the diameter of the interface between the upper and lower portions of the forming portion is as follows. (see Fig. 6c).

[Equation] H > = 0.35D (regardless of α value)

Separately, as it was confirmed that spheroids or organoids are not properly formed when D is less than 1 mm, D is preferably at least 1 mm or more.

8 is a diagram showing the correlation between the diameter (μm) of a spheroid and the number of cells.

Example 3. Production of spheroid or organoid in vitro models

Using an embodiment of the microfluidic chip 1000 of the present invention, a spheroid or organoid in vitro model of a normal cell line or cancer patient cell was constructed.

9 is a diagram showing the process of manufacturing an in vitro model. Referring to this diagram, it can be seen that (1) as the number of cells inside the model increases (top), and (2) as culture progresses (bottom), cell aggregation is better. there is.

10 is a photograph of patterning spheroids or organoids using normal cell lines (HCT116, U87MG, HEPG2, SW620) and cancer patient cells (Patient #1, Patient #2). Specifically, a total of 20,000 cells, 10,000 each of cancer cells and fibroblasts, were diluted in 12ul of the culture medium, and then injected into the microfluidic chip 1000 to proceed with patterning.

When a specific time elapsed, spheroids or organoids with a specific diameter were formed, and it was confirmed that spheroids or organoids with a diameter between 200 μm and 300 μm were formed after 24 hours from the start of the experiment.

11 shows a fluorescence micrograph of a 3-dimensional vascularized spheroid or organoid prepared using the microfluidic chip 1000 according to the present invention.

1000: microfluidic chip
100: well
200: supply unit
210: first supply unit
220: second supply unit
230: step
320: forming part
322: interface
340: culture unit
420: reservoir
440: inlet
460: channel
480: base part

Claims (12)

In the microfluidic chip,
supply unit;
a forming unit formed under the supply unit; and
A culture unit formed below the formation unit and receiving a supply of cells for culture or materials for forming blood vessels from the supply unit;
The microfluidic chip, wherein the fluid supplied through the supply unit is condensed and formed inside the forming unit to be maintained at a distance from the lower portion of the culturing unit. According to claim 1,
the supply department
It is formed in a tapering shape that narrows toward the bottom,
microfluidic chip. According to claim 1,
The supply unit includes a first supply unit for supplying the cells for culture; and
A second supply unit for supplying the material for forming blood vessels
Including, microfluidic chip. According to claim 3,
The first supply part is formed above the second supply part so that the cells for culture are supplied along the side of the supply part;
The second supply unit is formed above the forming unit so that the material for forming blood vessels is directly supplied to the forming unit.
microfluidic chip. According to claim 3,
A step is formed in the first supply unit,
microfluidic chip. According to claim 1,
The forming portion comprising a tapering shape that becomes narrower toward the top,
microfluidic chip. According to claim 1,
The culture department
It is configured to form one or more of the three-dimensional vascularized spheroids and organoids therein,
microfluidic chip. According to claim 1,
In contact with the upper part of the culture unit,
It is formed to be partitioned from the supply unit and the forming unit,
Further comprising a reservoir capable of supplying the culture medium or the experimental material to the culture unit,
microfluidic chip. According to claim 1,
an injection unit capable of supplying vascular endothelial cells to the edge of the culture unit; and
Further comprising a channel connecting the infusion unit and the culture unit,
microfluidic chip. According to claim 1,
Further comprising a base portion formed below the culture portion,
microfluidic chip. In the method of forming a spheroid or organoid in vitro model using a microfluidic chip,
supplying cells for culture to the supply unit of the microfluidic chip;
Forming spheroids or organoids by condensing the supplied cells for culture between the supplying unit and the forming unit;
supplying a material for forming blood vessels to the supply unit; and
Forming a three-dimensional vascularized spheroid or organoid in the culture part
including,
Methods for Forming Spheroid or Organoid In Vitro Models. According to claim 11,
Further comprising supplying a culture medium, a test material, or vascular endothelial cells to the culture unit,
Methods for Forming Spheroid or Organoid In Vitro Models.

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