KR101788963B1

KR101788963B1 - Microchip for Cell Spheroid Fabrication, Cell Spheroid Fabrication Apparatus Including the Same, and Cell Spheroid Fabrication Method

Info

Publication number: KR101788963B1
Application number: KR1020160030545A
Authority: KR
Inventors: 이정찬; 박지흠; 김희찬; 박중열; 이기훈
Original assignee: 서울대학교산학협력단; 중앙대학교 산학협력단
Priority date: 2016-03-14
Filing date: 2016-03-14
Publication date: 2017-10-24
Also published as: KR20170107145A

Abstract

The present invention relates to a microchip for forming a cell spoiloid, an apparatus for forming a cell spoloid, and a method for forming a cell spoloid, which can increase the efficiency and diversity of single or multicellular spoloid formation using centrifugal force. A microchip for forming a cell spoloid according to an embodiment of the present invention includes: a housing having an internal space; An inlet provided at the center of the housing to allow the cell culture fluid to flow into the inner space; And a plurality of microwells provided around the inner space, wherein the cells contained in the cell culture fluid flowing into the inner space through the inlet are subjected to centrifugal force generated by rotation of the microchip To form a micro-well.

Description

TECHNICAL FIELD [0001] The present invention relates to a microchip for forming a cell spoloid, a device for forming a cell spoloid containing the same, and a method for forming a cell spoloid,

TECHNICAL FIELD The present invention relates to a microchip for forming a cell spoloid, an apparatus for forming a cell spoloid, and a method for forming a cell spoloid. More particularly, the present invention relates to a microchip for forming a cell spoloid using a centrifugal force to increase the efficiency and diversity of single or multi- A cell spoloid forming apparatus comprising the same, and a method of forming a cell spoloid.

In general, it is very difficult to cultivate the cells in three dimensions. Therefore, the cells are cultured two-dimensionally to be used for drug screening and various experiments. In the case of two-dimensional culture, however, It is very difficult to obtain desired experimental results as a result of losing the characteristics of the cell itself or the tissue specificity of the cells used in the cell.

On the other hand, the three-dimensional cell spoil is obtained by gathering cells to form a three-dimensional aggregate, and the experimental results can be obtained under substantially the same conditions as in vivo. Therefore, 3-dimensional cell spoiloid plays a very important role in the study of cells constituting general tissues, cells constituting organs, cancer cells and stem cells in clinical studies for the development of new drugs or stem cell differentiation .

On the other hand, this technique of forming three-dimensional cell spoilids is basically based on preventing cells from adhering to the outer wall. Representative conventional techniques for this purpose include Hanging Drop Technique, a method using microcellular structures, a liquid-overlay culture method using non-adhered surface grating, A method using a spinner culture flask, a microchip-based method, and the like are used.

In the case of the most commonly used air drop method, cell cultivation is carried out in a water droplet suspended in a micro-hole, which makes it very difficult to handle the droplet even in a very small external shake. Other methods have disadvantages such as non-uniformity of spore size, excessive time required, and difficulty in extracting formed spheroids. Particularly, in the above-described conventional methods, formation of multicellular spheroids through co-culture of different cells is difficult and formation of three-dimensional spheroids capable of properly reflecting various types of cell-cell interactions There is a limit to In addition, due to inherent characteristics of each cell, there is a difference in the efficiency of spoloid formation of each of the above technologies, and in some cases, there is a problem that addition of a substance for enhancing cohesion of cells is separately required.

Korean Patent Laid-Open Publication No. 2015-0035957 (published on April 5, 2015)

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a microsphere for forming a cell spoloid capable of forming uniform three-dimensional cell spoils efficiently in a short time, The present invention has been made in view of the above problems.

A microchip for forming a cell spoloid according to an embodiment of the present invention includes: a housing having an internal space; An inlet provided at the center of the housing to allow the cell culture fluid to flow into the inner space; And a plurality of microwells provided around the inner space, wherein the cells contained in the cell culture fluid flowing into the inner space through the inlet are subjected to centrifugal force generated by rotation of the microchip To form a micro-well.

Also, in the microchip for forming a cell spoloid according to an embodiment of the present invention, the plurality of microwells may be arranged radially around the inlet.

Also, the micro-chip for forming a cell spoloid according to an embodiment of the present invention may include a plurality of grooves formed at predetermined intervals along the circumferential direction of the plurality of microwells.

Also, in the microchip for forming a cell spoloid according to an embodiment of the present invention, a guide part having a shape narrowing inward in the radial direction may be provided between each of the plurality of microwells.

In addition, the microchip for forming a cell spoloid according to an embodiment of the present invention may have an increased width as the inlet is moved downward.

In addition, the microchip for forming a cell spoloid according to an embodiment of the present invention may include a barrier protruding downwardly from a lower portion of the inlet.

In addition, the microchip for forming a cell spoloid according to an embodiment of the present invention may be provided with an inclined portion at an edge of the inner space to reduce the height of the inner space.

In addition, the microchip for forming a cell spoloid according to an embodiment of the present invention may include an outlet for discharging a cell culture fluid flowing into the inner space at an upper portion of the housing.

According to another aspect of the present invention, there is provided a microchip for forming a cell spoloid, wherein the housing comprises an upper housing having the inlet and a lower housing having the microwell, And the lower housing.

Meanwhile, the apparatus for forming a cell spoloid according to an embodiment of the present invention includes: a microchip for forming the cell spoloid; A rotating device for rotating the microspectrosphere-forming microchip; And a control unit for controlling a rotating speed of the rotating device.

According to another aspect of the present invention, there is provided an apparatus for forming a cell spoloid, the apparatus further comprising: a pair of fixed plates disposed on upper and lower sides of the cell spoloid forming microchip, As shown in Fig.

Meanwhile, a method of forming a cell spoloid according to an embodiment of the present invention includes: a step of injecting a cell culture fluid into an inner space of a microchip for cell spoloid formation; And rotating the microchip to allow the cell culture fluid to flow into a microwell formed around the inner space, wherein the microwell is placed under an over gravity condition by centrifugal force, To form a cell spoloid. &Lt; Desc / Clms Page number 7 >

According to another aspect of the present invention, there is provided a method of forming a cell spoloid, the method comprising: injecting a surface coating solution through the inlet; A surface coating step of rotating the microchip to cause the coating solution to flow into the microwell; And an inner cleaning step of cleaning the interior of the microchip, wherein the surface coating solution injection step, the surface coating step, and the inner cleaning step may be sequentially performed before the cell culture liquid injection step.

The method of forming a cell spoloid according to an embodiment of the present invention may further include an incubator storing step of storing the microspheres for forming the cell spheroids in an incubator for a predetermined time after the surface coating step.

The method of forming a cell spoloid according to an embodiment of the present invention may further include a step of pipetting a cell culture liquid into which the cell culture liquid injected into the inlet is pipetted after the cell culture liquid injection step .

According to an embodiment of the present invention, a microchip for forming a cell spoloid, an apparatus for forming a cell spheroid comprising the same, and a method for forming a cell spoloid can efficiently form a uniform three-dimensional cell spoloid within a short time And it is possible to culture for a long time, and it is easy to collect the formed three-dimensional cell spoloids.

The effects according to the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the description of the claims and the detailed description Be able to

FIG. 1 is a perspective view of a device for forming a cell sphere according to an embodiment of the present invention,
2 is a perspective view of a microchip for forming a cell spoloid according to an embodiment of the present invention,
FIG. 3 is an exploded perspective view of a microchip for forming a cell spoloid according to FIG. 2,
4 is a cross-sectional view taken along the line A-A 'in Fig. 2,
5 (a) is a plan view of the upper mold,
5 (b) is a cross-sectional view taken along line B-B 'of FIG. 5 (a)
6 is a plan view of the lower mold,
FIG. 7 is an exploded perspective view illustrating a coupling between a microchip for forming a cell spoloid and a pair of fixed plates according to an embodiment of the present invention,
8 is a flowchart of a method of forming a cell spoloid according to an embodiment of the present invention,
FIG. 9 is a photograph showing changes in single-cell spoloids formed in the cell spoloid-forming step with time,
10 is an example of formation of a three-dimensional cell spoloid formed by the method of forming a cell spoloid according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. Rather, the intention is not to limit the invention to the particular forms disclosed, but rather, the invention includes all modifications, equivalents and substitutions that are consistent with the spirit of the invention as defined by the claims.

Also, in the accompanying drawings, thickness and size are exaggerated for the sake of clarity of the description, and thus the present invention is not limited by the relative size or thickness shown in the accompanying drawings.

Relative terms such as "axial direction", "radial direction", "circumferential direction" and the like can be used herein to describe the relationship between the structures on the basis of the directions shown in the drawings, .

1 is a perspective view of a device for forming a cell sphere according to an embodiment of the present invention.

1, a three-dimensional cell spoloid forming apparatus 10 according to an embodiment of the present invention includes a microchip 100 for forming a cell spoloid in which a cell spoil is formed, a microchip 100 A fastening means 13 for fastening the pair of fixing plates 14 so as to be detachable, a rotating device 11 for rotating the microchip 100, And a control unit 12 for adjusting the rotational speed of the device 11. [

FIG. 2 is a perspective view of a microchip for forming a cell spoloid according to an embodiment of the present invention, FIG. 3 is an exploded perspective view of a microchip for forming a cell spoloid according to FIG. 2, Fig.

4, the axial direction refers to the upward and downward directions, that is, the direction from the bottom to the top of the housing 110, or from the top to the bottom of the housing 110, The radial direction refers to the left or right direction in Fig. 4, that is, the direction toward the inlet 110 from the outer peripheral surface of the housing 110, or the direction from the inlet 110 to the outer peripheral surface of the housing 110. [

The circumferential direction means a direction of rotation along the outer circumferential surface of the housing 110.

2 through 4, the microchip 100 for forming a spoloid according to an embodiment of the present invention includes a housing 110 having an inner space 111 in a substantially disc shape, The inlet 120 may include a plurality of microwells 130 radially disposed about the inlet 120.

The housing 110 may include an upper housing 110a constituting an upper portion and a lower housing 110b constituting a lower portion. In other words, the housing 110 can be made by the engagement of the upper housing 110a and the lower housing 110b so that the inner space 111, the inlet 120 and the microwell 130 are connected to the upper housing 110a ), A lower housing 110b or a combination of the upper housing 110a and the lower housing 110b.

For example, the inlet 120 is provided in the upper housing 110a, the microwell 130 is provided in the lower housing 110b, and the inner space 111 is formed between the upper housing 110a and the lower housing 110b, As shown in FIG. The upper portion of the microwell 130 may be covered by the upper housing 110a when the upper housing 110a and the lower housing 110b are coupled.

The inlet 120 may be formed at the center of the housing 110 so as to communicate with the outside of the upper side. In addition, the inlet 120 may have a shape increasing in width as it goes downward. For example, the inlet 120 may be a truncated cone having a larger diameter toward the lower axially.

The microwell 130 may be formed in the form of a plurality of grooves formed around the inner space 111 as a space in which the three-dimensional cell spoloid is formed. Further, they may be arranged radially around the inlet 120, and may be spaced apart from one another along the circumferential direction. Accordingly, as shown in FIG. 3, a plurality of microwells 130 may be arranged in a circular shape on the outer side in the radial direction of the inlet 120.

Meanwhile, a ramp 130a for guiding the cell culture liquid and the cells contained therein to flow into the microwell 130 may be formed on the inner side of the microwell 130 in the radial direction. At this time, the entrance road 120a may be shaped so as to be radially outward, that is, to become narrower toward the microwell 120 side.

In other words, between the microwell 130 and another microwell 130 adjacent to the microwell 130, that is, between the plurality of microwelles 130, a guide portion 112 having a shape narrower inward in the radial direction, May be provided.

Accordingly, when the centrifugal force acts radially outward due to the rotation of the microparticle 100 for forming a cell spoloid, the cell culture liquid and the cells contained therein can be smoothly introduced into the microwell 130 side. In addition, it is possible to prevent the cell culture fluid and cells contained in the microwell 130 from flowing into another microwell 130 adjacent to the microwell 130.

On the other hand, by controlling the size, number, and distance from the center of rotation of the microwell 130, it is possible to control the size of the produced cell spleoid and the throughput of the spore.

Since the space of the microwell 130 is formed by the coupling of the upper housing 110a and the lower housing 110b, the upper housing 110a, the lower housing 110b, And the lower housing 110b are separated from each other, it is easy to collect the formed three-dimensional cell spoil.

The inner space 111 connects the inlet 120 and the microwell 130 so that the cell culture liquid flowing into the inlet 120 and the cells contained therein are mixed with the centrifugal force due to the rotation of the microchip 100 It is possible to easily move the microwell 130 to the microwell 130 located around the inner space 111. At this time, the inner space 111 may have a substantially disc shape.

An inclined portion 113 may be provided at an edge of the inner space 111 to reduce the height of the inner space 111. For example, the inclined portion 113 may be formed along the circumferential direction so that the height of the inclined portion 113 decreases toward the outer side in the radial direction, so that the inclined portion 113 has a ring shape formed along the outer peripheral surface of the inner space 111 . The inclined portion 113 narrows the cross-sectional area of the inner space 111 toward the plurality of microwells 120 so that the cell culture fluid and the cells contained therein can flow smoothly into the microwell 130 do.

In addition, the upper portion of the housing 110 may have a discharge port 114 through which the cell culture fluid flowing into the inner space 111 is discharged. The discharge port 114 may be formed in the upper housing 110a so that the inner space 111 communicates with the outside and a plurality of the discharge ports 114 may be formed along the circumferential direction on the upper side of the inner space 111 in the axial direction. Accordingly, the microparticle 100 for forming a cell spoloid according to an embodiment of the present invention can exchange an old cell culture liquid with a new cell culture liquid through the discharge port 114, Dimensional cell spoloids can be cultured for a long time.

In addition, a barrier 115 protruding downward may be provided at a lower portion of the inlet 120. The barrier 115 may be formed to extend axially downward from the lower surface of the upper housing 110a so as to minimize the communication space between the inlet 120 and the inner space 111. [ For example, the barrier 115 may protrude axially downward along the circumferential direction on the radially outer side of the inlet 120. Accordingly, the barrier 115 may be provided in a ring shape at the edge of the inlet 120. This is to prevent the cell culture fluid containing cells from spreading toward the microwell 130 when the cell suspension initially containing the cells is injected into the inlet 120. In other words, when the cell spoloid forming microchip 100 is not rotating, the barrier 115 does not move the cell culture liquid injected into the inlet port 120 to the microwell 130 due to surface tension, (120). &Lt; / RTI >

Alternatively, in the absence of the barrier 115, the cell culture fluid injected into the inlet 120 can be transferred to the microwell 130 without rotation of the microparticle 100 for cell spoloid formation, The amount of the cell culture fluid flowing into each microwell 130 and the amount of the cells contained therein are different, and thus it is difficult to form uniform three-dimensional cell spoloids. Thus, the barrier 115 allows the cell culture fluid injected into the inlet 120 to move to the microwell 130 only by rotation, such that each cell uniformly forms in the microwell 130.

In addition, prior to rotating the cell spoloid-forming microchip 100, the cell culture liquid injected into the inlet 120 is subjected to additional pipetting to prevent precipitation of cells contained in the cell culture liquid, The barrier 140 may allow the cell culture fluid to remain in the inlet 120 and not spread out into the microwell 130 during an additional pipetting operation so that the pipetting operation So that it can be efficiently performed.

In addition, a surface coating layer (not shown) may be formed on the surface of the inlet 120, the microwell 130, and the inner space 111 to prevent cells from adhering to the surface. At this time, the surface coating layer may be formed by Pluronic Coating or Agarose Coating.

As described above, the microparticle-forming microchip 100 according to an embodiment of the present invention introduces the cell culture fluid and the cells contained therein into the microwell 130 disposed radially through centrifugal force by rotation By applying the gravitational force to the microwell 130 by continuous rotation, the cell cohesion can be enhanced without additional additive material, thereby increasing the efficiency of the three-dimensional cell spoloid formation.

Also, by providing a plurality of microwells 130 in the same size in a radial manner and applying a centrifugal force, the cells can be uniformly distributed, and a three-dimensional cell spoloid of uniform size can be formed.

In addition, the microchip 100 for forming a cell spoloid according to an embodiment of the present invention can be used for various types of multicellular steroids by controlling the number of cells contained in the cell culture fluid and sequentially injecting the cell culture fluid. And it is possible to continuously supply and supply the cell culture liquid through the outlet 114, so that long-term culture is possible.

Meanwhile, the microchip 100 for forming a cell spoloid according to an embodiment of the present invention can be manufactured by applying a replica molding technique using a mold.

In other words, the method for fabricating the three-dimensional cell spoloid-forming microchip 100 according to an embodiment of the present invention includes steps of fabricating a top plate mold 110a ', fabricating a bottom mold 101b' Fabricating an upper housing 110a through the upper mold 110a 'by applying a replica molding technique, fabricating a lower housing 110b through the lower mold 110b' by applying a duplicate molding technique, And coupling the upper housing 110a and the lower housing 110b.

FIG. 5A is a plan view of the upper mold, FIG. 5B is a cross-sectional view taken along line B-B 'of FIG. 5A, and FIG. 6 is a plan view of the lower mold.

5A and 5B, the top plate mold 100a 'has a disk-shaped base portion 111a', a base portion 111a ', and a base portion 111a' The inner space forming portion 111 'having the small disk-like shape and the inflow opening forming portion 120' having the cut-out conical shape formed in the upper part of the center of the inner space forming portion 111 'may be provided. In other words, the top plate mold 110a 'may have a shape corresponding to the upper housing 110a. Through this, the upper housing 110a can be manufactured by applying the replica molding technique.

Referring to FIG. 6, the lower plate mold 110b 'may be in the form of a disk having a microwell forming part 130' formed by being disposed in a radial direction. In other words, the lower plate mold 110b 'may have a shape corresponding to the lower housing 110'. Through this, the lower housing 110b can be manufactured by applying the replica molding technique.

At this time, the top plate mold 110a 'and the bottom plate mold 110b' can be manufactured using a 3D printer. Also, the top plate mold 110a 'and the bottom plate mold 110b' may be fabricated using micro-processing technology.

Meanwhile, PDMS (Polydimethylsiloxane), which is a biocompatible material, can be used for manufacturing the upper plate 100a and the lower plate 100b by using the upper plate mold 110a 'and the lower plate mold 110b'. That is, the upper housing 110a and the lower housing 110b may be made of polydimethylsiloxane (PDMS), which is a biocompatible material.

FIG. 7 is an exploded perspective view illustrating a coupling between a microchip for forming a cell spoloid and a pair of fixing plates according to an embodiment of the present invention. Referring to FIG.

Referring to FIG. 7, a pair of fixing plates 13 may be positioned on the upper and lower sides of the microchip 100, respectively. In other words, the upper fixing plate 13a may be located on the upper side in the axial direction of the upper housing 110a, and the lower fixing plate 13b may be located on the lower side of the lower housing 110b.

In addition, the pair of fixing plates 13 can be detachably coupled by the fastening means 14 located at the edges. That is, the microchip 100 may be positioned between the pair of fixing plates 13 so that the upper housing 110a and the lower housing 110b can be closely contacted with each other.

Here, the fastening means 14 may be a fastening bolt 14a and a fastening nut 14b. 7, a plurality of fastening bolts 14a and fastening nuts 14b are arranged along the circumferential direction on the radial outline of the pair of fastening plates 13, So that the upper housing 110a and the lower housing 110b can be brought into close contact with each other. At this time, the upper housing 110a and the lower housing 110b can be separated by unscrewing the fastening bolt 14a and the fastening nut 14b, and the three-dimensional cell spacer Can be collected.

The rotating device 11 can rotate the microchip 100. [ Since the microchip 100 is located between the pair of fixing plates 13, the rotating unit 11 is configured to couple the rotation axis to the lower fixing plate 13b of the pair of fixing plates 13, , The microchip 100 can be rotated.

The control unit 12 may be connected to the rotating device 11 to control the rotating direction and the rotating speed of the rotating device 11. [

For example, when the surface coating is performed to prevent cells from adhering to the surfaces of the inlet 120, the microwell 130, and the inner space 111, the control unit 12 controls the surface coating solution from the inlet 120 to the inner space The speed of the rotating device 11 can be adjusted to a level of 0 to 1,000 RPM so that the microwell 130 can be easily introduced into the microwell 130 through the microwell 111. In addition, the controller 12 may control the rotation speed of the rotating device 11 to apply various over gravity conditions to the cell spleot formed in the microwell 120.

Hereinafter, a method of forming a cell spoloid according to an embodiment of the present invention will be described in detail.

8 is a flowchart illustrating a method of forming a three-dimensional cell spoiloid according to an embodiment of the present invention.

8, a method (S100) for forming three-dimensional cell spoils according to an embodiment of the present invention includes a step of coating a surface coating solution for injecting a surface coating solution through an inlet 120 of a microsphere 100 for forming a cell spoloid A surface coating step S120 for rotating the microchip 100 to allow the surface coating solution to flow into the microwell 130, the microchip 100 being stored in the incubator for a predetermined period of time (S140) for washing the interior of the microchip (100), a cell culture fluid injection step (S150) for injecting a cell culture fluid containing cells through the inlet port (120), an inlet port A cell culture liquid pipetting step (S160) of pipetting the cell culture fluid injected into the microwell (120) at least once, and a step (S160) of rotating the microchip (100) To be And a cell spoloid formation step (S170).

The surface coating solution injecting step S110 may include an internal space 111 formed in the housing 110 to prevent the cells from adhering to the surface of the inlet 120, the plurality of microwells 130 and the internal space 111, The inlet 120 and the surface of the microwell 130. In this case, At this time, the surface coating solution may be a Pluronic or Agarose coating solution.

The surface coating step S120 may be a step of rotating the microchip 100 so that the surface coating solution injected into the inlet 120 can be easily moved to the microwell 130 arranged in a radial direction. At this time, the controller 12 can maintain the rotation speed of the rotating device 11 at a level of 0 to 1000 RPM.

The incubator storage step (S130) may be a step of storing the surface coating solution in the incubator with the surface coating solution contained in the microchip (100) It can be stored for a period of time.

The inner cleaning step S140 may be a step of removing the coating solution contained in the microchip 100. At this time, phosphate buffered saline (PBS) may be used for internal cleaning of the microchip 100.

The step of injecting the cell culture solution (S150) may be a step of injecting a cell culture solution containing cells into the inlet 120. At this time, the cell culture liquid injected into the inlet 120 can not be spread due to the barrier 115 provided in the housing 110, and can be stagnated inside the inlet 120 by surface tension.

The cell culture liquid pipetting step (S160) may be a step of pipetting the cell culture drug stagnant at the inlet 120 at least once, thereby preventing deposition of cells contained in the cell culture liquid, It can be evenly distributed inside the cell culture fluid.

The cell spoiloid formation step S170 is performed by rotating the microchip S100 through the rotating device 11 so that the cell culture fluid injected into the inlet port 120 flows into the microwell 130 through the inner space 111 And allowing the cells to agglomerate in the microwell 130 to form a three-dimensional cell spoloid by causing the cells to migrate and undergo gravity conditions through continuous rotation.

FIG. 9 is a photograph showing a time-dependent change in 3-dimensional single-cell spoloid formed in the cell spoloid forming step. FIG. 9 shows the results of a three-dimensional single cell speckle using Adipose stem cell (ASC) and Lung fibroblast (MRC- Lloyd.

Referring to FIG. 9, it can be seen that the color inside the microwell becomes thicker with time, which indicates the process of forming the three-dimensional cell spoloid (S) with time.

As described above, according to the three-dimensional cell spoloid forming apparatus 10 and the method (S100) for forming a cell spoloid using the same according to an embodiment of the present invention, the microwell disposed through the centrifugal force by rotation 130 and the cells contained therein are introduced into the microwell 130 and the microwell 130 is subjected to an over gravity condition by continuous rotation to enhance the cohesion of cells without any additional additive material, The efficiency can be increased.

In addition, a plurality of microwells 130 are radially arranged in the same size, and the centrifugal force is applied so that the cells can be uniformly distributed, and a plurality of three-dimensional cell spoils of uniform size can be formed.

In addition, according to the apparatus 10 for forming a cell spoloid and the method for forming a cell spolide using the same (S100) according to an embodiment of the present invention, it is possible to perform various forms of symbiotic culturing, It is possible to form a concentric or Janus-type three-dimensional cell spoloid as well as a simple mixed-type symbiotic culture.

10 is an example of formation of a three-dimensional cell spoloid (S) formed by the cell spoloid forming apparatus 10 according to an embodiment of the present invention and the cell spoloid forming method using the same (S100) And the Janus form.

As described above, the present invention provides a microchip for forming a cell spoloid which can increase the efficiency and diversity of single or multicellular spoloid formation by using centrifugal force, a device for forming a cell spoloid containing the same, and a method for forming a cell spoloid And the embodiments may be modified in various forms. Accordingly, the present invention is not limited to the embodiments disclosed herein, and all changes which can be made by those skilled in the art are also within the scope of the present invention.

10: Cell spoloid forming device
11: Rotating device 12:
13: a pair of fixing plates 14: fastening means
100: Microchip for cell spoiloid formation
110: housing 110a: upper housing
110b: lower housing 111: inner space
112: guide portion 113: inclined portion
114: outlet 115: barrier
120: inlet 130: microwell
110a ': Upper plate mold 110b': Lower plate mold
S100: Method of forming cell sphere
S110: Coating solution injection step S120: Surface coating step
S130: Incubator storage step S140: Internal washing step
S150: cell culture fluid injection step S160: cell culture fluid pipetting step
S170: step of forming a cell sphere

Claims

In a microchip for forming a cell spoloid,
A housing having an internal space;
An inlet provided at the center of the housing to allow the cell culture fluid to flow into the inner space;
A plurality of microwells provided around the inner space; And
And a barrier protruding downward from a lower portion of the inlet port,
Wherein the cells contained in the cell culture fluid flowing into the inner space through the inlet are formed by agglomeration in the microwells by centrifugal force generated by the rotation of the microchip, Microchip for.

The method according to claim 1,
Wherein the plurality of microwells are radially disposed about the inlet. &Lt; RTI ID = 0.0 > 11. < / RTI >

3. The method of claim 2,
Wherein the plurality of microwells comprise a plurality of grooves spaced apart from one another along a circumferential direction.

The method of claim 3,
Wherein a guide portion having a shape narrowing inward radially inward is provided between each of the plurality of microwells.

The method according to claim 1,
And the width of the inlet is increased toward the lower side.

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The method according to claim 1,
Wherein the inner space is provided with an inclined portion at an edge thereof to reduce the height of the inner space.

The method according to claim 1,
Wherein the upper portion of the housing is provided with a discharge port through which the cell culture fluid flowing into the inner space is discharged.

The method according to claim 1,
Wherein the housing comprises an upper housing having the inlet and a lower housing having the microwell,
Wherein the inner space is formed by a combination of the upper housing and the lower housing.

A microchip for forming a cell spoloid of claim 1;
A rotating device for rotating the microspectrosphere-forming microchip; And
And a controller for controlling the rotation speed of the rotating device.

11. The method of claim 10,
Further comprising: a pair of fixing plates positioned respectively above and below the microspheres for forming a cell spoloid,
Wherein the pair of fixed plates is detachable by a fastening means.

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