WO2018048209A1

WO2018048209A1 - Apparatus for ultrathin-cutting cell colonies or tissues and a fragmenting method using the same

Info

Publication number: WO2018048209A1
Application number: PCT/KR2017/009794
Authority: WO
Inventors: Myung Soo Cho; Mi Sun Lim; So Yeon Ji
Original assignee: Jeil Pharmaceutical Co., Ltd.
Priority date: 2016-09-08
Filing date: 2017-09-07
Publication date: 2018-03-15

Abstract

The present invention relates to an apparatus for ultrathin-cutting cell colonies or tissues and a fragmenting method using the same. According to the present invention, the apparatus for ultrathin cutting cell colonies or tissues can easily fragment various cell colonies or tissues including spherical neural masses and the like under an stereoscopic microscope and can be usefully used for pure separation, subculture, cryopreservation, differentiation, transplantation, and the like.

Description

APPARATUS FOR ULTRATHIN-CUTTING CELL COLONIES OR TISSUES AND A FRAGMENTING METHOD USING THE SAME

The present invention relates to an apparatus for ultrathin-cutting cell colonies or tissues and a fragmenting method using the same.

Since adult stem cells including mescenchymal stem cells (MSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and somatic cell nuclear transfer cells (SCNTs) that can be collected from the bone marrow, the umbilical cord blood, and fat, and the like have the ability to differentiate into various kinds of cells and a proliferation capacity, the cells have been proposed as a supply source of cell therapy which can treat degenerative diseases and cell damage occurring in various organs. In particular, since human embryonic stem cells can accurately define the shapes and characterize ability to differentiate and infinite proliferation, the human embryonic stem cells have been considered as a powerful candidate group capable of abundantly supplying cells for clinical treatment to replace dysfunctional cells with new cells.

It is expected that differentiated cells constituting various organs derived from the human ESCs have the same shapes and functions as the corresponding cells formed by a normal developmental process. Based on this possibility, differentiation methods providing an environment similar to a developmental stage have been attempted in pancreatic, neuron, muscular cell differentiation, and the like. However, the conventional differentiation method has a low differentiation rate and a limitation that the total differentiation time is too long for clinical application.

As a result, the clinical application is almost actually impossible, but recently, the present inventors have developed a method for effectively inducing differentiation into neural precursor cells and dopaminergic neural cells from the human embryonic stem cells to maintain the neural precursor cells in a form of spherical neural masses and use the neural precursor cells for other brain, neurological diseases, and solved a quantitative problem that are clinically useful and a temporal problem that can be used immediately when needed (KR 10-0838013).

In another method, the present inventors invented a method for inducing differentiation to retinal pigment epithelial cells by obtaining spherical neural masses from pluripotent stem cells and using a cystic structure formed from the spherical neural masses and succeeded obtainment of the retinal pigment epithelial cells for a short time using the method of inducing differentiation to retinal pigment epithelial cells (KR 10-1357851), and improved a human cell therapy using stem cells to a clinically and industrially usable level.

Meanwhile, in the methods proposed by the present inventors of the present invention, the process of fragmenting tissues and three-dimensional cell aggregation structures (cell colonies), for example, spherical neural masses into fine pieces is required, and conventional methods such as a chemical method and a physical method have been used. In the case of the chemical method, mostly, decomposing enzymes such as collagenase and trypsin are used and in the physical method, methods of using a surgical knife or a thinly drawn glass pipette or a laser is mainly used. However, in the case of the chemical method, there is a disadvantage that a long processing time is required and a specific protein and the like are decomposed to cause cell damage. In the case of the physical method, there is a disadvantage that the glass pipette is easily fragile and can not be reused and cell necrosis around the cut surface occurs by the laser, and the equipment is expensive to purchase. Thus, it is evaluated that the conventional fragmenting methods have difficulties in industrialization and clinical application. For this reason, it is required to develop a fragmenting method that enables standardized operating processes and stable quantity supply, and industrialization and clinical application.

As a result, the present inventors have developed an apparatus for ultrathin cutting cells including a fine metal wire or a fine metal mesh while making an effort to develop an efficient fragmenting method capable of overcoming the problems of the conventional fragmenting method. In the case of using a ultrathin cutting apparatus including a fine metal wire or a fine metal mesh according to the present invention, cell colonization or tissue fragmentation can be achieved in a large amount for a short time without any special skilled process, and an efficient fragment production is possible by comparing the conventional fragment methods. Also, the present invention has been completed by confirming that it can be usefully used as a method applicable to clinical application and industrialization without damaging cells.

Therefore, an object of the present invention is to provide an apparatus for ultrathin cutting cell colonies or tissues including a fine metal wire; and a supporter supporting a part or the whole of the fine metal wire.

Further, another object of the present invention is to provide an apparatus for ultrathin cutting cell colonies or tissues including: a fine metal mesh in which lattice patterns are repeated; and a supporter supporting a part or the whole of the fine metal mesh.

Yet another object of the present invention is to provide a method for ultrathin cutting cell colonies, a method for freeze-preserving cut cells, and a method for inducing cell differentiation using the ultrathin cutting apparatus.

To achieve the objects, the present invention provides an apparatus for ultrathin cutting cell colonies or tissues including a fine metal wire; and a supporter supporting a part or the whole of the fine metal wire.

Further, the present invention provides an apparatus for ultrathin cutting cell colonies or tissues including: a fine metal mesh in which lattice patterns are repeated; and a supporter for supporting a part or the whole of the fine metal mesh.

Further, the present invention provides a method for ultrathin cutting cell colonies including: ultrathin cutting cell colonies using an apparatus for ultrathin cutting cell colonies or tissues including a fine metal wire; and a supporter supporting a part or overall of the fine metal wire.

Further, the present invention provides a method for ultrathin cutting cell colonies including: ultrathin cutting cell colonies using an apparatus for ultrathin cutting cell colonies or tissues including a fine metal mesh in which lattice patterns are repeated; and a supporter supporting a part or overall of the fine metal mesh.

Further, the present invention provides a method for cryopreservation of cut cells including: 1) ultrathin cutting cell colonies using an apparatus for ultrathin cutting cell colonies or tissues including a fine metal mesh in which lattice patterns are repeated; and a supporter supporting a part or overall of the fine metal mesh; and 2) freezing cut cells.

Further, the present invention provides a method for inducing cell differentiation including: 1) ultrathin cutting cell colonies using an apparatus for ultrathin cutting cell colonies or tissues including a fine metal mesh in which lattice patterns are repeated; and a supporter supporting a part or overall of the fine metal mesh; and 2) co-culturing a differentiation substrate by obtaining the cut cells.

According to the present invention, the apparatus for ultrathin cutting cell colonies or tissues can easily fragment various cell colonies or tissues including spherical neural masses and the like under an stereoscopic microscope and can be usefully used for pure separation, subculture, cryopreservation, differentiation, transplantation, and the like. In particular, the ultrathin cutting apparatus according to the present invention can easily fragment the cells into the desired number of pieces without damaging the cells, and sterilization and reuse are possible. In addition, it is possible to obtain a large amount of fragments with desired sizes by fragmenting cell colonies or tissues for a short time as compared with conventional methods to rapidly and stably supply cells and tissues useful for various neural / sensory diseases such as Parkinson's disease, dementia, stroke, and retinal disease and other regeneration treatments.

FIG. 1 is a schematic diagram summarizing a method of fragmenting spherical neural masses using a fine metal wire or a fine metal mesh.

FIG. 2 is a diagram illustrating a mounting state and characteristics of fragmenting apparatuses, in which FIG. 2A is a diagram illustrating that fine metal wires are mounted on a platinum loop (left) and a pipette tip (right) and FIG. 2B is a diagram illustrating various sizes of fine metal meshes.

FIG. 3 is a diagram illustrating a state in which cell colonies are fragmented using a fine metal wire. FIG. 3A is a photograph obtained by taking an actual state in which cell colonies are fragmented using a fine metal wire by using a camera mounted on a stereoscopic microscope, FIG. 3B is a schematic diagram of a method for fragmenting cell colonies, and FIG. 3C is a lateral schematic diagram of a method for fragmenting cell colonies.

FIG. 4 is a diagram illustrated by photographing a state in which cell colonies are fragmented using a fine metal mesh by using a camera mounted on a stereoscopic microscope. FIG. 4A is a diagram illustrated by photographing cell colonies before fragmentation. FIG. 4B is a diagram illustrating that a fine metal mesh is placed on the cell colonies, and FIGS. 4C to 4E are diagrams illustrating a fragmenting process in sequence. FIG. 4F is a diagram illustrated by photographing fragments of the fragmented cell colonies.

FIG. 5 is a diagram illustrated by verifying that characteristics of the fragmented cell colonies are maintained by immunohistologic staining. FIG. 5A is a diagram illustrating a blue nucleus stained with DAPI, FIG. 5B is a diagram illustrating nestin as a marker of neural precursor cells stained by a green color, and FIG. 5C is a diagram illustrating beta III tubulin as a marker of neural cells stained by a red color. FIG. 5D is a diagram illustrating overlapping of FIGS. 5A, 5B, and 5C.

FIG. 6 is a schematic diagram of a ultrathin cutting apparatus including a fine metal wire according to the present invention.

FIG. 7 is a schematic diagram of a ultrathin cutting apparatus including a fine metal wire according to the present invention.

FIG. 8 is a diagram illustrating an example of a ultrathin cutting apparatus of the present invention, as a ultrathin cutting apparatus having a fine metal mesh consisting of fine metal wires and a supporter.

FIG. 9A is a photograph of a ultrathin cutting apparatus in Example 1 of the present invention and FIGS. 9B to 9G are photographs of ultrathin cutting apparatuses in Comparative Examples 1 to 6.

FIG. 10 is a diagram illustrated by verifying results of ultrathin cutting a spherical neural mass (SNM) of about 500 μm³ by a ultrathin cutting apparatus including fine metal wires made of respective materials: (a) Tungsten (Example 1), (b) Copper (Comparative Example 1), (c) Nickel (Comparative Example 2), (d) Stainless (Comparative Example 3), (e) Aluminum (Comparative Example 4), (F) Iron (Comparative Example 5) and (g) Titanium (Comparative Example 6).

FIG. 11 is a diagram illustrating results obtained by verifying shape changes of ultrathin cutting apparatuses after flame-disinfected with an alcohol lamp in Comparative Examples 1, 4, 5, and 6 of the present invention.

FIG. 12 is a schematic diagram illustrating a suitable mesh hole size in the case of performing fragmentation for subculture using a ultrathin cutting apparatus (a metal mesh) of the present invention.

FIG. 13 is a schematic diagram illustrating a suitable mesh hole size in the case of performing fragmentation for differentiation or cryopreservation using a ultrathin cutting apparatus (a metal mesh) of the present invention.

FIG. 14 is a diagram illustrating results obtained by comparing differentiation shapes of cut fragments according to a lattice size of a fine metal mesh. FIG. 14A illustrates a differentiation pattern of cell fragments cut with a size suitable for differentiation. FIG. 14B is a diagram illustrating a state in which fragments in cell colonies are very largely formed and maintained as non-differentiated cell colonies when the lattice size of the fine metal mesh is large.

FIG. 15 is a diagram illustrating results obtained by verifying maintenance of shapes according to a thickness of a tungsten fine metal wire and a shape after flame-disinfecting.

FIG. 16 is a diagram illustrating results obtained by verifying whether there is blocking of vision when a spherical neural mass is cut according to a thickness of a tungsten fine metal wire.

FIG. 17 is a diagram illustrating results obtained by verifying the degree of medium bottom scratch according to a thickness of a tungsten fine metal wire.

The present invention provides an apparatus for ultrathin cutting cell colonies or tissues.

More particularly, the apparatus for ultrathin cutting cell colonies or tissues of the present invention includes a fine metal wire and a supporter supporting a part or overall of the fine metal wire.

The apparatus for ultrathin cutting of the present invention will be described with reference to the accompanying drawings for helping understanding.

As illustrated in FIGS. 6 and 7, apparatuses (11 and 21) for ultrathin cutting cell colonies or tissues according to a first exemplary embodiment of the present invention include a fine metal wire (1).

In addition, the fine metal wire (1) may be used singly, but preferably, a supporter (2) capable of supporting at least a part of the fine metal wire (1) is further included.

Particularly, FIG. 6 illustrates the ultrathin cutting apparatus (11) of the present invention configured by the supporter (2) and the fine metal wire (1) and may verify that the fine metal wire (1) is fixed to one supporter (2). As an example of such a shape, like Example 2 of the present invention below, it may be understood that the fine metal wire is connected to a tip portion of a pipette, or it may be understood as a ultrathin cutting apparatus which is integrally fabricated.

In this case, the supporter (2) may use all materials as long as the material has physical properties capable of supporting the fine metal wire (1), but preferably, metals or polymers may be used.

Meanwhile, the supporter (2) may be fabricated integrally with the fine metal wire (1) or the supporter (2) as a separation type may be fabricated so that the fine metal wire (1) is detachable.

For example, after at least a part of the fine metal wire (1) is fixed and used to the supporter (2), the fine metal wire (1) may be separated and washed or reused, and then fixed to the supporter (2) again and reused, or the fine metal wire may be replaced and used.

For example, the fine metal wire (1) may be fixed and used to a rod of a platinum loop, or the fine metal wire (1) may be fixed and used to a pipette.

FIG. 7 illustrates a ultrathin cutting apparatus of the present invention configured by a grip portion (3), a supporter (2), and a fine metal wire (1), and the fine metal wire (1) is fixed to fractioned or divided supporter (2). As an example of such a shape, like Example 1 of the present invention, the fine metal wire may be fixed with a metal rod of the platinum loop.

The supporter of the ultrathin cutting apparatus of the present invention may be divided into three portions and configured to have an open / close structure.

That is, as illustrated in FIG. 7, the three supporters (2) may be divided to be opened or closed, respectively, and after a predetermined portion of the fine metal wire (1) is inserted while the supporters (2) are opened, the fine metal wire (1) may be fixed by fixing the three supporters (2).

In the present invention, the supporters may use any material having a physical property capable of supporting the fine metal wire, and preferably, the material may be a metal or a polymer.

The ultrathin cutting apparatus of the present invention may further include a grip portion (3) for fixing the supporters.

The grip portion may be understood as a portion provided for user's convenience, and the supporter (2) of the ultrathin cutting apparatus of FIG. 6 may be understood as a grip portion for user's convenience in addition to a support role.

In addition, the ultrathin cutting apparatuses (11 and 21) having the shapes of FIGS. 6 and 7 may be modified for convenient use or include additional components, and a fixing portion that introduces modification such as forming teeth of the fine metal wire (1) for easily ultrathin cutting the cell colonies or the tissues or fixes the cell colonies or the tissues not to be moved may be considered.

It is preferable that the fine metal wire of the ultrathin cutting apparatus of the present invention is made of a material which is suitable for ultrathin cutting cells, is easy to sterilization, and does not generate unnecessary foreign substances when ultrathin cutting cells. In order to achieve such an object, the fine metal wire may be made of at least one metal selected from the group consisting of tungsten, stainless steel, and titanium, or an alloy including the same. Most preferably, the fine metal wire may be made of tungsten or an alloy including the same.

The fine metal wire (1) may have a thickness of 50 μm to 800 μm and it is preferred that the fine metal wire has rigidity enough to cut cell colonies or tissues to maintain the shape even after ultrathin cutting. For example, when tungsten is used as the fine metal wire, if tungsten having a thickness of less than 50 μm is used, the shape of the wire may not be properly maintained as a needle shape, and may be melted after flame disinfection and may not be reused. Further, when the thickness of tungsten wire exceeds 800 μm, the thickness of the tungsten wire is too thick to cover the cell colonies and block the field of view, and thus, the cell ultrathin cutting may not be easily achieved. In addition, since the tungsten wire having a thickness of more than 800 μm is too elastic, scratches on the bottom of the culture medium are caused and foreign substances are generated from the bottom of the damaged medium, and thus the tungsten wire is not suitable for cell ultrathin cutting. Accordingly, the fine metal wire of the present invention preferably has a thickness of 50 μm to 800 μm, and more preferably 50 μm to 200 μm. Particularly, the tungsten wire having the thickness of 50 μm to 200 μm is suitable for reuse by maintaining the shape even after flame disinfection as well as maintaining the shape well and may easily cut the cells and the tissues without view blocking and medium scratches.

As illustrated in FIG. 8, an apparatus (31) for ultrathin cutting cell colonies or tissues according to a second exemplary embodiment of the present invention include a mesh made of the fine metal wires (1).

Particularly, as the ultrathin cutting apparatus (31) of the present invention configured by the fine metal mesh made of fine metal wires and the supporter (2), it can be seen that the fine metal wires (1) cross each other to form the fine metal mesh and the supporter (2) supporting the fine metal mesh are included.

As an example of such a shape, fine metal meshes having the same standards as those of Examples 3 to 8 of the present invention may be considered.

In this case, the supporter (2) may maintain the standard (that is, the pattern or lattice pattern) of the fine metal mesh well to produce fragments having predetermined sizes.

Further, the supporter (2) may be extended to form a grip portion for the convenience of the user, or a separate grip portion may be connected and used to the supporter.

In addition, the ultrathin cutting apparatus 31 illustrated in FIG. 8 may be mounted on the end of the pipette to form fragments at the same time with the pipetting, and this is included in the scope of the present invention.

Meanwhile, the fine metal mesh is configured by the fine metal wires (1).

The fine metal wire may be made of at least one metal selected from the group consisting of tungsten, stainless steel, and titanium, or an alloy including the same, preferably. Most preferably, the fine metal wire may be made of tungsten or an alloy including the same.

The fine metal wire configuring the fine metal mesh may have a thickness of 50 μm to 800 μm, and it is preferred that the fine metal wire has rigidity enough to cut cell colonies or tissues to maintain the shape even after ultrathin cutting. The fine metal wire may have most preferably a thickness of 50 μm to 200 μm.

The fine metal mesh is configured by two or more fine metal wires (1) and may be used irregularly by forming a mesh, but preferably, may be used by forming a predetermined pattern. For example, the fine metal mesh may be a mesh in which square holes as a lattice structure are repeatedly formed, but may be a mesh having holes having triangular, pentagonal, hexagonal, circular shapes, and the like, and may be fabricated regardless of the shapes of the holes. In the case of using a regular pattern shape, fragments of cut cell colonies or tissues may be predicted, and thus, a cut product having a desired size can be obtained.

When the fine metal mesh has square holes, the area of the square hole may be appropriately adjusted according to the purpose. The purpose means a process of using the obtained cut cells and for example, may include purposes for subculture, cryopreservation or freeze preservation, differentiation, and the like.

For example, in order to cut spherical neural masses for using subculture, the spherical neural masses having about 200 μm³ to 1 μm³, preferably about 500 μm³ may be cut and used to preferably 10 pieces or less, more preferably 4 to 7 pieces, and much more preferably 4 to 5 pieces. If the spherical neural masses are cut to more than 10 pieces having about 200 μm³ to 1 mm³, preferably about 500 μm³, there is a problem in that very small fragments are produced to be differentiated because a long proliferation period is taken or the fragments are attached to a culture container. In addition, when the spherical neural masses are cut into less than 4 pieces, there is a disadvantage that an object to be cut is passed, or substantial subculture is difficult because the sizes of the cut fragments are not constant (see FIG. 12).

For example, in order to cut the spherical neural masses for differentiation, the hole of the fine metal mesh may be used to have a hole having an area of preferably (50 to 250 μm) x (50 to 250 μm), and more preferably (75 to 230 μm) x (75 to 230 μm). This area is a range that is considered to be efficient in performing differentiation and allows differentiation to occur well. In the case of using the fine metal mesh having holes below the lower limit at the time of differentiation, there is a problem that the cell colonies or the tissues are not cut but pressed with a lump or the physical damage of the cut fragments is large, and thus the amount of loss due to apoptosis is large. In addition, in the case of using a fine metal mesh having holes above the upper limit, there is a problem that the size of the fragment is so large that the adherent culture for differentiation is difficult and the undifferentiated mass is maintained and then the cells are killed at the time of differentiation culture (see FIGS. 13 and 14).

Meanwhile, the fine metal mesh may be used singly, but preferably, a supporter (2) capable of supporting at least a part of the fine metal mesh or at least a part or overall of the edge of the fine metal mesh is further included.

In this case, the supporter (2) may use all materials as long as the material has physical properties capable of supporting the fine metal wires (1), but preferably, metals or polymers may be used.

Further, the supporter (2) may be fabricated integrally with the fine metal mesh, or the supporter (2) as a separation type may be fabricated so that the fine metal mesh is detachable. For example, after at least a part of the fine metal mesh is fixed and used to the supporter (2), the fine metal mesh may be separated from the supporter (2) and washed or reused, and then fixed to the supporter (2) again and reused, or the fine metal mesh (1) may be replaced and used.

FIGS. 6, 7, and 8 are only examples for implementing the present invention, and the present invention is not limited thereto. As described above, the present invention includes modifications commonly known for the convenience of the user.

Further, the present invention provides a method for ultrathin cutting cell colonies using the ultrathin cutting apparatus of the present invention.

More particularly, the present invention provides a method for ultrathin cutting cell colonies including: ultrathin cutting cell colonies using an apparatus for ultrathin cutting cell colonies or tissues including a fine metal wire; and a supporter supporting a part or overall of the fine metal wire.

In the present invention, the cell colonies mean that one or more cells are formed collectively, and may be used interchangeably with cell aggregates. The cell colonies or aggregates may have a volume of preferably 200 μm³ to 2 mm³ and more preferably 500 μm³ to 1 mm³. The tissue derived from the cell colonies may include all tissues of the human body, but preferably, may be brain tissue and a nerve tissue.

In the present invention, the cell colonies may be cell colonies consisting of one or more cells selected from adult stem cells including mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer cells (SCNTs), and the like which can be collected from the bone marrow, the umbilical cord blood, fat, and the like, and preferably, cell colonies or aggregates derived from the stem cells or adult cells. . In the present invention, the cell colonies or aggregates may be cell aggregates of one or more selected from the group consisting of retinal precursor cells, neural retinal precursor cells, spherical neural masses, photoreceptor precursor cells, photoreceptor cells, hepatic photoreceptor cells, conical photoreceptor cells, horizontal cells, amacrine cells, interneurons, ganglion cells, retinal pigment epithelial cells, and ciliary edge cells, and preferably spherical neural masses (SNMs). The SNMs may be formed from the embryoid body (EB).

The method for ultrathin cutting the cell colonies may be performed as follows. First, cell colonies or tissues to be cut may be positioned on a predetermined substrate. The predetermined substrate means a glass substrate or the like, and may include a culture dish commonly used in the art.

Thereafter, the cell colonies or tissues positioned on the substrate are cut using the ultrathin cutting apparatus according to the present invention. A fine metal wire included in the ultrathin cutting apparatus of the present invention cuts the cell colonies. The fine metal wire used to cut the cell colonies may be sterilized before or after use. The sterilization may be used without limitation by a sterilization method widely used in the art, and preferably sterilized by EO gas sterilization or high pressure steam sterilization.

When performing the ultrathin cutting of the cell colonies, generally, it can be understood that the cell colonies or tissues are pressed and cut with the fine metal wire, and preferably, the ultrathin cutting of the cell colonies may be performed by fixing the cell colonies or tissues by preventing the cell colonies or tissues from being slid or departing from the fine metal wire with elastic. At this time, an additional support may be used as far as possible, and a support capable of being integrally or detachably attached to the predetermined substrate may be included to facilitate ultrathin cutting. For example, the ultrathin cutting of the cell colonies may be performed like the following exemplary embodiment of the present invention, but the present invention is not limited thereto.

Further, the present invention provides a method for freeze preservation of cut cells including: ultrathin cutting cell colonies using an apparatus for ultrathin cutting cell colonies or tissues including a fine metal mesh in which lattice patterns are repeated; and a supporter supporting a part or overall of the fine metal mesh, and freezing cut cells.

For example, for cryopreservation or freeze preservation by ultrathin cutting the spherical neural masses, the holes of the fine metal mesh may be fabricated to have a hole area of preferably 50 μm² to 500 μm² and more preferably 75 μm² to 480 μm². In the case of using a fine metal mesh having holes below the lower limit, there is a problem that the fragment is too small, so that there is almost no remaining fragments in the process of removing dead cells due to freezing damage when thawing after freezing and there is a problem that it is almost differentiated rather than proliferated when culturing. On the other hand, in the case of using a fine metal mesh having the holes exceeding the upper limit, there is a problem that it is difficult to penetrate a frozen liquid because the size of the fragment is larger than a proper size, and the cells inside the fragment are killed during thawing, and thus it is impossible to maintain cell aggregates or tissues (see FIG. 13).

The freeze preservation may be used interchangeably with cryopreservation, and may be performed using a freeze preservative or a cryopreservative commonly used in the art.

Further, the present invention provides a method for inducing cell differentiation including: ultrathin cutting cell colonies using an apparatus for ultrathin cutting cell colonies or tissues including a fine metal mesh in which lattice patterns are repeated; and a supporter supporting a part or overall of the fine metal mesh; and co-culturing a differentiation substrate by obtaining the cut cells.

The cell colonies may preferably be spherical neural masses and may be cultured with the differentiation substrate after being cut by the ultrathin cutting apparatus according to the present invention to be effectively differentiated into nerves. The differentiation method used in the present invention may use differentiation methods used in the art without limitation, and use methods for growing fragments of the cut cell colonies to differentiate the structure or function without limitation.

In the present invention, the apparatus for ultrathin cutting the cell colonies or tissues used in the method for inducing cell differentiation may include a fine metal mesh having square holes, and the square hole may have an area of 75 μm² to 480 μm².

When the area of the square hole is less than 75 μm², the cell colonies or the tissues are not cut but pressed with a lump or the physical damage of the cut fragments is large, and thus the amount of loss due to apoptosis is large. When the area exceeds 480 μm², the size of the fragment is so large that the adherent culture for differentiation is difficult and the undifferentiated mass is maintained and then the cells are killed at the time of differentiation culture.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples just exemplify the present invention, and the contents of the present invention are not limited to the following Examples and Experimental Examples.

<Example 1> Fabrication of Ultrathin cutting Apparatus

As illustrated in a left photograph of FIG. 2A and FIG. 9a, when a fine metal wire (a diameter of 100 μm) made of tungsten was used as a metal rod of a platinum loop, a front side screw was loosened and inserted or wound around a groove, and then the screw was wound again and firmly fixed. If the size of the groove winding the screw was larger than the diameter of the metal wire, the metal wire was folded or folded several times, and then the screw was wound and fixed. The fine metal wire was cut at a portion about 3 cm from the front end and used after sterilization.

<Example 2> Fabrication of Ultrathin cutting Apparatus

As illustrated in the right photograph of FIG. 2A, when a fine metal wire (a diameter of 100 μm) made of tungsten was used as a pipette tip, the fine metal wire was inserted to a hole at the tip end and pressed and fixed by applying heat, and then, the metal wire was cut from a portion of about 2 to 3 cm from the front end portion and used after sterilization.

<Comparative Examples 1 to 6> Fabrication of Ultrathin cutting Apparatus

In order to compare with the fine metal wire made of tungsten fabricated in Example 1, the ultrathin cutting apparatus was fabricated by varying a material of the metal rod of the platinum loop as copper, nickel, stainless steel, aluminum, iron and titanium, respectively. Each ultrathin cutting apparatus was fabricated in the same manner except that the fine metal wires were made of copper, nickel, stainless steel, aluminum, iron, and titanium, and the ultrathin cutting apparatuses fabricated in Comparative Examples were illustrated in FIGS. 9B to 9G.

<Examples 3 to 8> Fabrication of ultrathin cutting mesh

A ultrathin cutting mesh consisting of a fine metal wire was fabricated by varying a thickness of the fine metal wire made of tungsten and an area of a square hole. The conditions of the ultrathin cutting mesh to be fabricated were listed in Table 1, and the ultrathin cutting mesh was illustrated in FIG. 2B, respectively. Each ultrathin cutting mesh was fabricated and then used after sterilization.

	Thickness of tungsten fine metal wire	Area of square hole	Figure
Example 3	30 μm	230 μm²	Upper left side of FIG. 2B
Example 4	30 μm	333 μm²	Upper middle side of FIG. 2B
Example 5	30 μm	480 μm²	Upper right side of FIG. 2B
Example 6	50 μm	75 μm²	Lower left side of FIG. 2B
Example 7	50 μm	115 μm²	Lower middle side of FIG. 2B
Example 8	50 μm	164 μm²	Lower right side of FIG. 2B

<Experimental Example 1> Fragmenting Method for Subculture

An experiment was performed, in which cell colonies or soft tissues, such as spherical neural masses, having a diameter of 500 μm³ to 1 mm³ contained in a culture dish were cut into 10 pieces or less using a ultrathin cutting apparatus connected with the fine metal wire fabricated in Example 1 or Example 2. First, a middle portion of a fragmented object was first dissected and then the cut fragment was dissected again to be cut with a desired size and number. When the end of the metal wire was first contacted with the bottom of the culture dish and then pressed and cut from the front of the center of the fragmented object. In addition, when the surface was slippery, one portion was fixed and then cut using two ultrathin cutting apparatuses or first, the surface of the object was slightly grooved and cut. The cut fragments were first divided and subcultured into two culture dishes and then divided and subcultured into four culture dishes when a diameter was 250 micrometers or more. The fragmenting process was photographed and illustrated in FIG. 3A. Further, the order of the fragmenting process was illustrated in FIG. 3B and a lateral schematic diagram of the fragmenting process was illustrated in FIG. 3C.

As illustrated in FIG. 3A, a process of fragmenting spherical neural masses for subculture using the ultrathin cutting apparatus of Example 1 or 2 was confirmed in order of the right photograph from the left photograph. First, it can be seen that the fine metal wire of the ultrathin cutting apparatus of Example 1 or 2 of the present invention was positioned at the spherical neural masses, and the spherical neural masses were grooved by pressing, and then dissected into two pieces, and as a result, the fabricated fragments can be seen by a photograph. After the fragmenting process of Experimental Example 1, the photograph of the fabricated fragments was illustrated in FIG. 1.

<Experimental Example 2> Fragmenting method for differentiation and cryopreservation

For differentiation, it is preferred that an apparatus having holes of 75 μm² to 230 μm² is used, and in Experimental Example 2, the ultrathin cutting apparatus of Example 3, 6, 7 or 8 was used. For cryopreservation, it is preferred that a mesh having a larger hole area than that of a differentiation mesh is used not to exceed 480 μm² or more, and in Example 2, the ultrathin cutting apparatus of Example 4 or 5 was used. The fragmented object in the culture dish was collected in the center, and the fine metal mesh cut according to the size of the culture dish was placed on the object. To prevent scattering of the object and formation of air bubbles, the fine metal mesh was sufficiently soaked in the same culture medium before use. The mesh placed on the object was lightly tapped with 1 mm micropipette having a tip. The naturally cut fragments were transferred to a new culture dish including the medium, and all the remaining object are pipetted until being fragmented and transferred to the same culture medium. The fragments were counted under a dissecting microscope, fragments to be used for differentiation were transferred to a culture dish coated with a differentiation substrate at an appropriate concentration and cultured in a CO₂ incubator, the fragments for cryopreservation were transferred to a freezer container, and then cryopreserved in a frozen preserve.

The fragmenting process for differentiation or cryopreservation performed in Experimental Example 2 was illustrated in FIG. 4. Referring to FIG. 4, fragments fabricated in the order of FIG. 4A to FIG. 4F, respectively, can be confirmed. Specifically, in FIG. 4A, about 30 spherical neural masses located on the culture dish may be confirmed. As illustrated in FIG. 4B, the ultrathin cutting apparatus fabricated by the fine metal mesh of the present invention (all of Examples 3 to 8 are performed in the same manner) are placed on the spherical neural masses. Next, as illustrated in FIGS. 4C to 4E, tapping with two 1 mm micropipettes was performed, and one micropipette serves to fix the fine metal mesh to be positioned on the spherical neural masses well and the fragments was fabricated by tapping or pipetting with the other micropipette. After the above process, a photograph of the fabricated fragments can be seen in FIG. 4F.

Also, it can be seen that different sizes of fragments are fabricated as illustrated in FIG. 1 for fragmentation for cryopreservation or differentiation, when the fragmenting is performed in the same manner using the ultrathin cutting apparatuses having different specifications. Among such fabricated fragments, the fragment for differentiation may be differentiated into the nerve as illustrated in FIG. 1.

<Experimental Example 3> Treating method for reusing fragmentation tool

In the case of the ultrathin cutting apparatus of Example 1 used in Experimental Example 1 or 2, the ultrathin cutting apparatus can be used after washing and sterilization processes, and EO gas sterilization or high pressure steam sterilization is possible. In the case of the ultrathin cutting apparatus of Example 1, the gas sterilization is further recommended because of the corrosion of platinum loop itself, and the ultrathin cutting apparatus of Example 2 can be put in a tip case or in a suitable container sterilized by high pressure steam or sterilized by high pressure steam sterilization, or can be put in an EO gas sterilizing film and sterilized by gas. In the case of the high pressure sterilization, the ultrathin cutting apparatus is dried well in a dry oven and used after irradiating radiation for 20 minutes in a sterile table before use. It was confirmed that Examples 1 to 8 of the ultrathin cutting apparatus made of tungsten according to the present invention were reusable after the treatment for reuse in Experimental Example 3.

<Experimental Example 4> Analysis of characteristics of produced fragments after fragmenting process ( immunohistological method)

After the fragmenting process according to the present invention, immunohistological staining was performed to confirm whether the produced fragments kept the characteristics of a specific cell group or other cartilage tissues. In Experimental Example 4, an immunohistochemical method for confirming whether to maintain the nature of spherical neural masses is described and various antibodies can be used according to a desired cell type.

It was confirmed that the spherical neural mass fragments obtained by using the ultrathin cutting apparatus of the present invention were adhered and cultured to a glass cover glass coated with a differentiation substrate, and then cells expressing a marker of neural precursor cells (nestin) and neural cells (betaIII-tubulin) were observed. Samples were treated with a 3% formalin solution for 20 minutes and stored in a phosphate buffer. The samples were treated with blocking solution (2% bovine serum albumin, Sigma) for 1 day before treating antibodies, reacted with a primary antibody for 12 hours and with a secondary antibody for 1 hour, sealed with a fluorescence preservation solution, and then observed under a cofocal fluorescence microscope (Nikon, Japan). In this case, for immunological staining, a mouse anti-human nestin antibody (1 : 200, Chemicon, USA) and a rabbit anti-beta tubulin antibody (1 : 100, Chemicon) were used as the primary antibody and Alexa Fluor 488 donkey antimouse IgG and Alexa Fluor594 donkey anti-rabbit IgG (1 : 200, Molecular Probes, USA) were used as the secondary antibody, and the results of the above experiment were illustrated in FIG. 5.

As illustrated in FIG. 5, it was confirmed that the cell colonies fragmented by the apparatuses of Examples 1 and 2 maintained the characteristics of neural precursor cells and neural cells. Specifically, in FIG. 5A, blue nuclei stained with DAPI were observed, and in FIG. 5B, nestin, a marker of neural precursor cells, was observed in green. In FIG. 5C, beta III tubulin, a marker for neural cells, was observed in red. FIG. 5D is a diagram illustrating overlapping of FIGS. 5A, 5B, and 5C.

Accordingly, the apparatus for ultrathin cutting cell colonies or tissues according to the present invention may be used for a major production process such as 3-dimensional microcellular colonies or micro tissues required for the development of a functional cell therapeutic agent or a biomimetic tissue. In addition, when the apparatus of the present invention is used, the microcellular colonies or tissues are easily cut into desired sizes, and at the same time, various cell colonies may be fragmented, thereby mass-producing the fragmented tissues and cells while reducing manpower, times, and costs.

Furthermore, since the fragmenting method using the ultrathin cutting apparatus of the present invention can be applied to various micro cell colonies and tissues, it is expected that clinical and commercialization will be possible in various disease fields. Since any more expensive equipment is not required in addition to metal materials and commonly used experimental tools, the fragmenting method can also have the effect of lowering the price of the produced product.

<Experimental Example 5> Selection of material of ultrathin cutting apparatus

In order to select the most ideal material for fabricating the ultrathin cutting apparatus according to the present invention, the following experiment was conducted.

<Experimental Example 5-1> Evaluation of Elasticity

In order to evaluate whether the ultrathin cutting apparatus according to the present invention had a suitable degree of elasticity when ultrathin cutting cell colonies or tissues, spherical neural masses (SNMs) of about 500 μm³ were cut by using ultrathin cutting apparatuses made of tungsten (Example 1), copper (Comparative Example 1), nickel (Comparative Example 2), stainless (Comparative Example 3), aluminum (Comparative Example 4), iron (Comparative Example 5), and titanium (Comparative Example 6) fabricated in Example 1 and Comparative Examples 1 to 6, and then the elasticity of each material was evaluated and whether to efficiently cut cell colonies or tissues by the ultrathin cutting apparatus and whether to reuse were evaluated, and the results were illustrated in FIG. 10.

FIG. 10 is a photograph illustrating an experiment for evaluating elasticity for ultrathin cutting spherical neural masses (SNMs) of about 500 μm³ made of materials (tungsten (Example 1), copper (Comparative Example 1), nickel (Comparative Example 2), stainless (Comparative Example 3), aluminum (Comparative Example 4), iron (Comparative Example 5), and titanium (Comparative Example 6)) using a ultrathin cutting apparatus according to the present invention.

FIG. 10A is a photograph illustrating fragments obtained by ultrathin cutting the SNM by the ultrathin cutting apparatus of the present invention in Example 1, and FIGS. 10B and 10C are photographs illustrating fragments obtained by ultrathin cutting the SNM in Comparative Examples 1 and 2 and a bending apparatus, respectively. FIGS. 10D to 10G are photographs illustrating fragments obtained by ultrathin cutting the SNM by the apparatus made of the materials of Comparative Examples 3 to 6.

As illustrated in FIG. 10, it was confirmed to have elasticity in order of tungsten (Example 1) > titanium (Comparative Example 6) >> copper (comparative Example 1), nickel (Comparative Example 2), stainless steel (Comparative Example 3), iron (Comparative Example 5) > aluminum (Comparative Example 4). In addition, all ultrathin cutting devices made of the materials except for aluminum can cut spherical neural masses (SNMs), but the apparatuses made of copper, nickel, stainless steel, and iron can not be reused due to bending or weak elasticity. It is exhibited that the ultrathin cutting apparatuses made of tungsten and titanium in Example 1 and Comparative Example 6 can be reused while maintaining a suitable shape even after use.

When considering the above results, it was confirmed that aluminum is not suitable for the apparatus of the present invention for performing ultrathin cutting of cell colonies or tissues, and it is preferable to use an alloy including tungsten or titanium.

<Experimental Example 5-2> Evaluation of Sterilization Easiness and Heat Resistance

In order to reuse the ultrathin cutting apparatus, sterilization after use is essential. Accordingly, in order to evaluate easiness of sterilization and heat resistance according to each material (Example 1 and Comparative Examples 1 to 6) of the ultrathin cutting apparatus according to the present invention, the following experiment was conducted.

After alcohol lamp disinfection (flame disinfection), high pressure steam sterilization disinfection and gas sterilization disinfection were performed using the ultrathin cutting apparatus made of each material fabricated in Example 1 and Comparative Examples 1 to 6, the shape and the change of the ultrathin cutting apparatus were observed, and then easiness of sterilization and heat resistance were evaluated, and the results were illustrated in FIG. 11.

FIG. 11 is a photograph illustrating the results of observing the shape of the ultrathin cutting apparatus after flame disinfection of each apparatus. Example 1, Comparative Example 2 and Comparative Example 3 were not deformed even after flame disinfection and it was found that alcohol lamp disinfection, high pressure steam sterilization disinfection and gas sterilization disinfection were all possible. However, as illustrated in FIG. 11, copper in Comparative Example 1 and aluminum, iron and titanium in Comparative Examples 4 to 6 were not suitable for flame disinfection because the shapes were changed during flame disinfection using an alcohol lamp, and only high pressure steam sterilization or gas sterilization disinfection was possible.

Therefore, the ultrathin cutting apparatus according to the present invention can be used in various disinfection methods depending on the material. In particular, in the case of the ultrathin cutting apparatus made of a material including tungsten, nickel or stainless steel, flame disinfection, high pressure steam sterilization disinfection, and gas sterilization disinfection all can be used, and thus it was confirmed that the apparatus may be usefully used as an apparatus for ultrathin cutting cell colonies or tissues of the present invention.

Through Experimental Examples 5-1 and 5-2, it was confirmed that the most suitable material as the apparatus for ultrathin cutting cell colonies or tissues according to the present invention was tungsten having elasticity suitable for cell or tissue ultrathin cutting and easiness of sterilization or an alloy including tungsten.

<Experimental Example 6> Comparison of Cell differentiation effect according to lattice area of fine metal mesh

The fine metal mesh for ultrathin cutting cell colonies may be fabricated to have regular lattices, and the area of the lattice may be adjusted according to the purpose. Experiments were performed as follows to compare production of cell colony fragment according to a lattice size and differentiation effects thereof. First, spherical neural masses (SNMs) of 500 mm were placed in a culture dish, and then a fine metal mesh having square holes having an area of 75 to 230 μm² ((75 to 230 μm) x (75 to 230 μm)) was placed on the cell colonies to cut the cell colonies (see FIG. 13). As Comparative Example, a fine metal mesh having larger square holes was used, and the cell differentiation pattern after ultrathin cutting was confirmed by a microscope, and the results was illustrated in FIG. 14.

As illustrated in FIG. 14, when a fine metal mesh having square holes having an area of 75 to 230 μm² was used, it was confirmed that fragments having a size suitable for differentiation were formed and the differentiation was smoothly performed (a). On the other hand, in the case of using a fine metal mesh having a larger area, it was confirmed that the fragments of cell colonies were too large to be maintained as undifferentiated cell colonies (b).

<Experimental Example 7> Comparison of Suitability according to thickness of tungsten fine metal wire

It was confirmed that the tungsten material was most suitable for ultrathin cutting the cells or colonies of the present invention. In Experimental Example 7, the suitability of the apparatus according to the thickness of the tungsten material was further compared.

<Experimental Example 7-1> Comparison of shapes according to thickness

The apparatus was fabricated by the method of Example 1 and the shapes were compared while modifying only the thickness of the tungsten material to 50 to 800 μm. It was also confirmed whether the shape was remained after flame disinfection. The shape and the change of the fabricated apparatus were illustrated in FIG. 15 and listed in Table 2.

Thickness (μm) of tungsten	Wire form
Thickness (μm) of tungsten	Normal state	State after flame disinfection
20	Amorphous form(unsuitable)	Melt and removed(unsuitable)
50	Needle shape(suitable)	No change(suitable)
100	Needle shape(suitable)	No change(suitable)
200	Needle shape(suitable)	No change(suitable)
400	Needle shape(suitable)	No change(suitable)
800	Needle shape(suitable)	No change(suitable)

As illustrated in FIG. 15 and listed in Table 2, the apparatus having a thickness of the fine metal wire of 50 μm to 800 μm normally maintained the needle shape maintained its shape even after flame disinfection. On the other hand, it was confirmed that the apparatus fabricated with a thickness of the fine metal wire of 20 μm did not maintain the needle shape and melted and removed after flame disinfection, and as a result, the apparatus was not suitable for the apparatus for ultrathin cutting cells or colonies.

<Experimental Example 7-2> Comparison of easiness of ultrathin cutting of spherical neural masses

The apparatus was fabricated by the method of Example 1 and fabricated by modifying only the thickness of the tungsten material to 50 to 800 μm. The ultrathin cutting of the SNMs was performed using the fabricated apparatus and suitability was evaluated according to a degree of view blocking when the SNMs were cut. The view blocking test procedure and the results were illustrated in FIG. 16 and listed in Table 3.

Thickness ( μm ) of tungsten	Degree of view blocking
Thickness ( μm ) of tungsten	View blocking	Suitability
50	None	Suitable
100	None	Suitable
200	None	Suitable
400	Little presence	Suitable
800	Overall blocking	Unsuitable

As illustrated in FIG. 16 and listed in Table 3, if the thickness of the tungsten fine metal wire was thicker than 400 μm to 800 μm, the SNM as a cut object was covered and the view of a person performing ultrathin cutting was disturbed, and as a result, the ultrathin cutting cannot be smoothly performed. Therefore, it was confirmed that it was suitable that the thickness of the tungsten fine metal wire was 50 to 400 μm or less.

<Experimental Example 7-3> Comparison of medium bottom scratching degree according to difference in tungsten elasticity

The apparatus was fabricated by the method of Example 1 and fabricated by modifying only the thickness of the tungsten material to 50 to 800 μm. The ultrathin cutting of the SNMs was performed using the fabricated apparatus and a scratch degree of a medium bottom was confirmed when the SNMs were cut. The scratch degree of the medium bottom may vary depending on the degree of elasticity, and the more bottom scratches, the more foreign substances were generated, and thus, the apparatus was not suitable for ultrathin cutting cells or tissues. The test results of the scratch degree of the medium bottom and the suitability according to the respective thicknesses were illustrated in FIG. 17 and listed in Table 4.

Thickness ( μm ) of tungsten	Bottom scratch (elasticity) of culture medium
Thickness ( μm ) of tungsten	Bottom scratch	Suitability
50	None	Suitable
100	None	Suitable
200	None	Suitable
400	Presence	Unsuitable
800	Worse	Unsuitable

As illustrated in FIG. 17 and listed in Table 4, it was confirmed that when the thickness of the tungsten fine metal wire was 400 μm or more, the scratch degree of the medium bottom became worse, and when the thickness of the tungsten fine metal wire was 800 μm or more, the scratch degree of the bottom is very worse and a large amount of foreign material was generated. Therefore, it was confirmed that it was suitable that the thickness of the tungsten fine metal wire was 50 μm or more and less than 400 μm.

The results of Experimental Examples 7-1 to 7-3 were summarized in Table 5.

Thickness of tungsten ( μm )	Shape of fine metal wire		Degree of view blocking		Bottom scratch (elasticity) of culture medium		Final
Thickness of tungsten ( μm )	Normal state	State after flame disinfection	View blocking	Suitability	Bottom scratch	Suitability
20	Amorphous form (unsuitable)	Melt and removed(unsuitable)	-	-	-	-	Unsuitable
50	Needle shape(suitable)	No change(suitable)	None	Suitable	None	Suitable	Suitable
100	Needle shape(suitable)	No change(suitable)	None	Suitable	None	Suitable	Suitable
200	Needle shape(suitable)	No change(suitable)	None	Suitable	None	Suitable	Suitable
400	Needle shape(suitable)	No change(suitable)	Little presence	Suitable	Presence	Unsuitable	Unsuitable
800	Needle shape(suitable)	No change(suitable)	Overall blocking	Unsuitable	Worse	Unsuitable	Unsuitable

When considering the results of sterility suitability, view blocking, and the scratching of the medium bottom, it was confirmed that it was most preferable that the thickness of the tungsten fine metal wire used for the apparatus for ultrathin cutting cells or tissues had a thickness of 50 to 200 μm. When the thickness of the tungsten fine metal wire was 20 μm, it is difficult to maintain the shape such as melting and removing after flame disinfection without maintaining the needle shape. When the thickness of the tungsten fine metal wire was 400 μm or more, it is difficult to fragment the SNM due to view blocking and a large amount of foreign substances was generated by scratching the medium bottom due to strong elasticity. The tungsten wire having the thickness of 50 μm to 200 μm is suitable for reuse by maintaining the shape even after flame disinfection as well as maintaining the shape well and may easily cut the cells and the tissues without view blocking and medium scratches.

Claims

An apparatus for ultrathin cutting cell colonies or tissues, the apparatus comprising:

a fine metal wire; and

a supporter supporting a part or overall of the fine metal wire.
The apparatus of claim 1, wherein the supporter is divided into three portions to have an open and close structure.
The apparatus of claim 1, wherein the supporter is made of metals or polymers.
The apparatus of claim 1, further comprising:

a grip portion fixing the supporter.
The apparatus of claim 1, wherein the fine metal wire is made of at least one metal selected from the group consisting of tungsten, stainless steel, and titanium, or an alloy including the same.
The apparatus of claim 1, wherein the fine metal wire has a thickness of 50 μm to 200 μm.
An apparatus for ultrathin cutting cell colonies or tissues, the apparatus comprising:

a fine metal mesh in which lattice patterns are repeated; and

a supporter supporting a part or overall of the fine metal mesh.
The apparatus of claim 7, further comprising:

a grip portion fixing the supporter.
The apparatus of claim 7, wherein the fine metal mesh is made of at least one metal selected from the group consisting of tungsten, stainless steel, and titanium, or an alloy including the same.
The apparatus of claim 7, wherein the fine metal mesh is formed by fine metal wires having a thickness of 50 μm to 200 μm.
The apparatus of claim 7, wherein the fine metal mesh has square holes and the square holes have areas of 75 μm² to 480 μm².
A method for ultrathin cutting cell colonies, the method comprising:

ultrathin cutting cell colonies using the ultrathin cutting apparatus of claim 1.
The method of claim 12, wherein the cell colonies are cell aggregates derived from stem cells or adult cells.
The method of claim 13, wherein the cell colonies or aggregates are cell aggregates of one or more selected from the group consisting of retinal precursor cells, neural retinal precursor cells, spherical neural masses, photoreceptor precursor cells, photoreceptor cells, hepatic photoreceptor cells, conical photoreceptor cells, horizontal cells, amacrine cells, interneurons, ganglion cells, retinal pigment epithelial cells, and ciliary edge cells.
A method for ultrathin cutting cell colonies, the method comprising:

ultrathin cutting cell colonies using the ultrathin cutting apparatus of claim 7.
A method for freeze-preserving cut cells, the method comprising:

1) ultrathin cutting cell colonies using the ultrathin cutting apparatus of claim 7; and

2) freezing the cut cells.
A method for inducing cell differentiation, the method comprising:

1) ultrathin cutting cell colonies using the ultrathin cutting apparatus of claim 7; and

2) co-culturing a differentiation substrate by obtaining the cut cells.
The method of claim 17, wherein the cell colonies are spherical neural masses.
The method of claim 17, wherein the fine metal mesh for the ultrathin cutting apparatus has square holes and the square holes have areas of 75 μm² to 230 μm².