KR100734584B1 - Stamp for cell dissociation, method for cell dissociation using the same, and apparatus for manual/automatical cell dissociation using the same - Google Patents

Abstract

A stamp for cell dissociation, a method for cell dissociation by using the same stamp, and an apparatus for manual/automatic cell dissociation by using the same stamp are provided to dissociate the human and animal cells into the cell units having a certain cell number at one time and divide them into a certain pattern according to a purpose. The stamp(100) for dissociation of human and animal cells comprises a body(110) having an upper side and a lower side, and a micro-pattern portion(120) which is located at any one of the upper and lower sides, and has at least one repeated micro-pattern(130) with a certain height and width, wherein the micro-pattern portion is made of polydimethylsiloxane(PDMS), polymethylmethacrylate(PMMA), polyacrylates, polycarbonates, polycyclic olefins, polyimides, polyurethanes or polyester. The method for cell dissociation comprises the steps of: (a) preparing cell population in a culture dish; (b) placing the stamp on the culture dish in parallel; (c) pressing the culture dish with the stamp for a certain time to dissociate the cell population; and (d) removing the stamp.

Description

Stamp for cell dissociation, Method for cell dissociation using the same, and Apparatus for manual / automatical cell dissociation using the same}

1 is a schematic perspective view of a stamp having a square fine pattern according to an embodiment of the present invention;

Figure 2 is a schematic perspective view of a stamp with a hexagonal fine pattern according to another embodiment of the present invention,

Figure 3 is a schematic diagram showing the process of dividing the cell colonies cultured with the stamp of Figure 1,

Figure 4 is a schematic diagram showing the process of dividing the cell colonies cultured with the stamp of Figure 2,

5 is a flowchart illustrating a cell division method using a stamp according to an embodiment of the present invention;

6 is a schematic diagram after division of cell colonies in FIG. 6,

7 is a photograph of human embryonic stem cells cultured for applying a stamp according to an embodiment of the present invention,

8 is a photograph showing embryonic stem cells divided by the stamp of FIG.

Figure 9 is a photograph of the separated embryonic stem cells of Figure 8 separated from the culture dish bottom,

10 to 12 is a process chart for explaining a mold manufacturing process of the stamp according to an embodiment of the present invention,

13 is an enlarged pattern photo of a completed stamp mold,

14 is a photograph of a stamp made of a completed stamp mold,

15 is a schematic perspective view of a manual cell division apparatus according to an embodiment of the present invention,

16 is a schematic perspective view of an automatic cell division apparatus according to an embodiment of the present invention,

17 is a schematic perspective view of an automatic cell division apparatus according to another embodiment of the present invention.

<Description of Major Symbols in Drawing>

10. Petri dishes 100, 100 '. stamp

110, 110 '. Body 120, 120 '. Fine pattern part

130, 130 '. Fine Pattern 140. Wall

300. Manual cell division device 310. Housing

320. Canister guide 330. Stamp canister

400, 500. Automatic cell division device 450, 560. Cylinder

The present invention relates to a cell division stamp, a cell division method and apparatus using a stamp, and more particularly, cell populations that grow in two or three dimensions in the in vitro culture of human and animal cells. The present invention relates to a stamp for human and animal cell division, a cell division method using a stamp, and manual and automatic division apparatus using a stamp, for dissociation, division or patterning.

In order to sustain culture while maintaining the characteristics of human and animal cells, it is important to subculture at a suitable time and conditions depending on the cells. However, the use of enzymes or compounds for subculture is often very cytotoxic, which is a major obstacle to cell culture. If this is replaced by a mechanical method or by hand, it takes a lot of time and there is a problem such as not being able to culture by maintaining a constant number of cells. Human embryonic stem cells are a typical example.

Human embryonic stem cells are self-renewal and have been isolated for the first time in 1998 due to their pluripotency, which can differentiate into all tissue cells of the human body (Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM., Science. 282 (5391), 1145-1147, 1998), The importance of development as a cell therapy for the treatment of intractable disease is emphasized, and the economic and industrial utility is highlighted. Human embryonic stem cells can be cultured in vitro by co-culture with feeder-cells or by single culture in a culture medium containing appropriate growth factors. In particular, embryonic stem cells are characterized by growing with cells having the same characteristics, forming a cell colony.

In the current state of the art, it is impossible to subculture by dissociating human embryonic stem cells into single cells, and for subculture, the cells growing on the culture dish are enzymes (eg, collagenase IV, GIBCO-BRL / Invitrogen, Carlsbad, Calif.) Can be removed and finely divided into mechanical plates, transferred to a new petri dish, and the culture can be continued (enzymatic method, enzymatic method), or the whole process can be relyed on by hand using a cell scraper or a sharp device. (Mechanical method) (Schatten G, Smith J, Navara C, Park JH, Pedersen R., Nature Methods, 2 (6): 455-463, 2005). ).

In the mechanical method, by selectively taking only undifferentiated cells, the cell quality can be maintained stably, and the cell populations of relatively similar size can be passaged, but the time-consuming and very small amount of cells can be handled in a limited time. The disadvantage is the lack of efficiency. Enzymatic methods have the advantage that they can handle large amounts in a shorter time than mechanical methods, but differentiated cells can be passaged together with undifferentiated cells, the size of the cells is not constant, and as the passage is repeated Problems have been pointed out that are likely to cause chromosomal abnormalities (Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, Sui G, Cutler DJ, Liu Y, Brimble SN, Noaksson K, Hyllner J, Schulz TC , Zeng X, Freed WJ, Crook J, Abraham S, Colman A, Sartipy P, Matsui S, Carpenter M, Gazdar AF, Rao M, Chakravarti A., Nature Genetics, 37 (10): 1099-1103, 2005 Mitalipova MM , Rao RR, Hoyer DM, Johnson JA, Meisner LF, Jones KL, Dalton S, Stice SL. Nature Biotechnology, 23 (1): 19-20, 2005). New culture methods are being developed to improve this but are still in the early stages (Joannides A, Fiore-Heriche C, Westmore K, Caldwell M, Compston A, Allen N, Chandran S. Stem Cells, 24 (2): 230-235 , 2006).

Due to the importance of research and development of human embryonic stem cells, efficient mass culture is indispensable and leading research and development are urgently needed.

Cell patterning methods have been actively studied and various proposals have been reported. An energy irradiation method for forming the cell adhesion part and the cell adhesion inhibition part on a pattern by the action of a photocatalyst (Japanese Patent Publication No. 2005-261432, Japanese Patent No. 2005-270055), and using a stamp to form a single layer film, Patterns of hydroxy groups and methods of patterning or arraying cells using hydroxyl-reactive bifunctional molecules (US Pat. No. 6,548,263) have been proposed. However, these methods are surface processing in the previous stage of cell culture, and thus are not suitable for application to cells cultured for clinical use such as human embryonic stem cells. Therefore, there is an urgent need for the development of new devices for efficiently patterning and isolating human embryonic stem cells.

The present invention has been made in view of the above, the present invention is easy to separate the human and animal cell population difficult to pass passage by conventional enzymatic method or mechanically (cell unit) including a certain number of cells ( It is an object of the present invention to provide a stamp for splitting human and animal cells and a cell division method using a stamp that can be efficiently sub-cultured by dissociation, division, and patterning.

 In addition, an object of the present invention is to provide a manual cell splitting device using a stamp and an automatic cell splitting device using a stamp to divide human and animal cell groups by manually or automatically applying appropriate pressure to the stamp.

Human and animal cell division stamp according to the present invention for achieving the above object, the body having a top and bottom; And a fine pattern part disposed on any one surface of the upper or lower surface of the body and repeating at least one fine pattern having a predetermined height and width.

The micropattern includes a wall defining the height and width and the shape of the micropattern, and the end portion of the wall is preferably smaller in thickness than other portions.

The body may have a polygon according to the shape of the culture plate in which human and animal cells are cultured, and in particular, the body may have a shape of any one of a triangle, a square, a pentagon, a hexagon, and a circle.

Each of the micropatterns may have a polygonal shape, and in particular, the micropattern may have a shape of any one of a triangle, a square, a pentagon, a hexagon, and a circle.

In addition, the height and width of the fine pattern is preferably 1μm or more.

In addition, the material of the body and the fine pattern portion is polydimethylsiloxane (PDMS), polymethylmethacrylate (polymethylmethacrylate (PMMA), polyacrylates (polyacrylates), polycarbonates (polycarbonates), polysilicon ( Polycyclic olefins, polyimides (polyimides), polyurethane (polyurethans), it is preferably formed of any one material of polyester (polyester).

In order to increase the hydrophilicity, the fine pattern portion is preferably subjected to a coating treatment using a plasma surface treatment or a chemical substance that enhances hydrophilicity such as polyethylene glycol (PEG).

On the other hand, the above object can be achieved by the cell division method according to the present invention using the stamp. Cell division method using a stamp according to the present invention, a) preparing a cell colony in a culture dish; b) placing the stamp on top of the petri dish so as to be horizontal with the petri dish; c) dividing the cell colonies by applying a uniform pressure to the culture dish using the stamp for a predetermined time; And, d) removing the stamp.

The cells to be divided are preferably stem cells.

On the other hand, the present invention discloses a manual cell division apparatus using the stamp. Manual cell division apparatus according to the present invention, the culture plate containing the cells in the inner bottom is fixed, the housing is formed with an opening on the upper surface; A fixing guide configured to penetrate the opening of the housing, the lower end of which is positioned above the culture dish, and the upper end of which is exposed to the outside of the housing through the opening; A stamp fixing cylinder inserted into the fixing cylinder guide and installed to be movable, and having a wire installed on an upper surface thereof and a stamp attached to the lower surface thereof; And a weight which is placed on the stamp holder to the stamp holder to press the culture dish to the stamp holder at a desired pressure.

In addition, the present invention discloses an automatic cell division apparatus using the stamp. Automatic cell division device according to an embodiment of the present invention, the lower plate is fixed culture plate; A side plate installed to protrude upward from one end of the lower plate; An upper plate connected to an upper end of the side plate to be parallel to the lower plate; A cylinder guide having one end fixed to a lower surface of the upper plate and installed to protrude toward the lower plate; And a cylinder which is installed to be movable up and down through the cylinder guide, and has a stamp fixed to a lower surface to pressurize the cells contained in the culture dish at a predetermined pressure.

Automatic cell splitting apparatus using the stamp according to another embodiment of the present invention, the lower plate; A side plate installed to protrude upward from one end of the lower plate; An upper plate connected to an upper end of the side plate to be parallel to the lower plate; A stamp holder fixed to the lower surface of the upper plate, installed to protrude in the lower plate direction, and fixed to the stamp on the lower surface; A cylinder stage having one end fixed to an upper surface of the lower plate and installed to protrude in the upper plate direction; And a cylinder which is installed to be movable up and down through the cylinder stage, wherein the stamp is fixed to an upper surface to press the cells contained in the culture dish at a predetermined pressure.

The above objects, features and other advantages of the present invention will become more apparent by describing the preferred embodiments of the present invention in detail with reference to the accompanying drawings. Hereinafter, a human and animal cell division stamp, a cell division method using a stamp, and a manual and automatic cell division apparatus using a stamp according to embodiments of the present invention will be described in detail with reference to the accompanying drawings.

<Stamp for splitting human and animal cells>

1 is a stamp for human and animal cell division according to an embodiment of the present invention, Figure 2 is a schematic perspective view of a stamp for human and animal cell division according to another embodiment of the present invention.

The stamp 100, 100 ′ is composed of a body 110, 110 ′ and a fine pattern portion 120, 120 ′. Body (110, 110 ') has a variety of forms depending on the shape and purpose of the culture dish in which the cells are cultured. That is, in the embodiment of the present invention, the body (110, 100 ') is disclosed that the rectangular, circular, but the scope of the present invention is not limited to this, it can be produced in a variety of forms such as a polygon, such as a triangle, pentagon, hexagon Do.

The fine patterns 120 and 120 'are positioned on the upper surfaces of the bodies 110 and 100', and the plurality of fine patterns 130 and 130 'are repeatedly formed. Each fine pattern 130 has a height H and a width W, and each height H and width W is defined by the wall 140. Thus, each fine pattern 130 in FIG. 1 is formed by four walls 140, and each fine pattern 130 ′ in FIG. 2 is formed by six walls 140 ′.

The height H of the micropattern 130 refers to a step of the groove, that is, the height of the wall 140, and the width W of the micropattern 130 is between the facing walls 140 of each micropattern 130. Say the distance. The height H and the width W of each of the fine patterns 130 and 130 ′ are preferably 1 μm or more.

As shown in FIG. 1, the end portion 141 of the wall 140 of the fine pattern 130 is preferably smaller in thickness than other portions. When the cells are divided by the stamp 100, the end portions of the micropatterns 130 come into contact with the cell colonies, thereby dividing the cell colonies. Thus, by thinning the end portions 141 of the wall 140, the cells are killed. It is possible to reduce the dead zone.

On the other hand, each micropattern 130, 130 'has a variety of forms depending on the purpose or purpose of the cell. That is, in the present embodiment, it is disclosed that the fine patterns 130 and 130 'are rectangular and hexagonal, but the scope of the present invention is not limited thereto and may have various shapes such as polygons or circles such as triangles and pentagons. If the micropattern is circular, the width of the micropattern is a diameter.

On the other hand, since the fine pattern may have a variety of forms, each area is also different. This has the advantage of controlling the size of the cells to be divided. In particular, when the cells to be divided are stem cells to be able to control the size of the stem cells and embryonic body, there is an advantage that can be divided by controlling the desired number of cells constantly. This will be described later.

The material of the stamp 100, that is, the body (110, 100 ') and the material of the fine pattern portion 120, 120', polydimethylsiloxane (polydimethylsiloxane, PDMS), polymethyl methacrylate (polymethylmethacrylate, PMMA) It is preferable to be formed of various polymer materials such as polyacrylates, polycarbonates, polycarbonates, polycyclic olefins, polyimides, polyurethanes, polyesters, and the like.

On the other hand, the fine pattern portion 120, 120 'is preferably subjected to the plasma surface treatment on the surface in order to increase the hydrophilicity. By performing the plasma surface treatment process, the surfaces of the fine pattern portions 120 and 120 'have good hydrophilicity. Alternatively, a method of coating a surface having a hydrophilic compound such as PEG (polyethylene glycol) may be one method. By increasing the hydrophilicity, air bubbles are prevented from being formed when the micropatterns 120 and 120 ', which will be described later, divide the cell colonies.

<Cell division method using stamp>

3 to 5 are diagrams showing a method of dividing cells using a stamp according to an embodiment of the present invention.

First prepare a cell colony 20 in the culture dish (10) (S210). Subculture of human embryonic stem cells and cultured for 4-6 days with daily change of culture medium grow to form cell colony (20).

When the cell colonies 20 are grown to a suitable size, as shown in FIGS. 3 and 4, the stamps 100 and 100 ′ are positioned above the culture dish 10 so as to be horizontal to the culture dish 10 (S220).

Thereafter, the stamps 100 and 100 'are applied in a downward direction for a predetermined time, for example, for about 1-2 seconds to divide the cell colony 20 (S230).

Then, the stamp (100, 100 ') is carefully removed in the horizontal direction so as not to damage the cells (S240).

As shown in FIG. 6, each cell colony 20 is divided into several pieces of cell units 30 to correspond to the shapes of the fine patterns 130 and 130 ′ of the stamps 100 and 100 ′. . The cell unit 30 will contain a constant cell number.

7 to 9 are photographs showing the process of dividing cells by the cell division method using the stamp (100, 100 ') according to an embodiment of the present invention.

The human embryonic stem cells were coated with 0.1% gelatin coated with STO feeder cells (ATCC, Manassas, VA) treated with mitomycin C (Sigma, St. Louis) 24 hours before passage. Seeds in -mm culture dishes followed by feeder cell medium (DMEM (GIBCO-BRL, Gaithersburg, MD), 10% FBS, 2 mM glutamine (Sigma, St. Louis, Missouri, USA), 0.1 mM ß-mercaptoethanol (Sigma) , 1% nonessential amino acid (GIBCO-BRL) was incubated in a 37 ⁰ C, 5% CO ₂ cell incubator. , 20% knockout serum replacement (Invitrogen), 1% non-essential amino acids (Invitrogen), 0.1 mmol / L-mercaptoethanol (Sigma), 100 U / ml penicillinstreptomycin (Invitrogen), 4 ng / ml basic fibroblast growth factor (bFGF (Invitrogen)) and about 50 divided cell colonies per petri dish were placed on the nutrient medium at appropriate intervals. For 2 days to stabilize so that you can grow attached above did not change after the culture medium was changed every day as a new medium. Cells were incubated about 4-6 days, the size of the colony to grow until properly (see Fig. 7).

After subculture, the culture medium was placed in a culture dish of about 1 ml, and the stamps were carefully removed after the cell colonies were divided using the actual manufactured stamp of FIG. It was confirmed that the cell colonies in the culture dish were uniformly divided into the pattern of the stamp (see FIG. 8).

The divided cell colonies can be used for a variety of purposes, including subculture, after separation from the culture dish using a scraper (see FIG. 9).

As described above, according to the stamp for splitting human and animal cells according to the present invention and a cell division method using the same, cells grown in colonies in a culture dish can be easily divided into cell units having a uniform number of cells at a time. There is an effect that can be easily divided into the desired pattern shape. In addition, since the cell colonies can be divided into cell units having a uniform number of cells, there is an advantage that a more reproducible research and experiment can be carried out by reducing the variable according to the variation in the number of cells that can appear in each subculture.

Recently, the importance of research on human embryonic stem cells (embryonic stem cells) has been emphasized at home and abroad, and a large amount of cells are urgently needed, but conventional culture methods have not solved this problem. However, when the present invention is applied in the process of culturing human embryonic stem cells, it is possible to divide the cells in a very short time, thereby dramatically improving the passage speed and reducing the viability of the cells by reducing the time outside the cell incubator. It is possible to provide an optimization condition that increases the efficiency and maintains the integrity of the cell. This will be a significant contribution to the establishment of a mass culture system to expand the range of utilization of human embryonic stem cells.

The biggest obstacle in human embryonic stem cell research is the inability to cultivate a constant cell number. Although it is not possible to make a single cell in an embodiment of the present invention, by using a micropattern having the same size, it is possible to divide into cell units having a certain number of cells according to the purpose. Quantitative and qualitative analysis under experimental conditions becomes possible.

The process of forming embryonic bodies by suspension culture of divided human embryonic stem cells after passage is very important in the method of differentiation of human embryonic stem cells. It is very difficult to standardize differentiation methods because it is impossible to use a certain number of human embryonic stem cells for embryonic body formation using conventional passage methods. However, according to the present invention, when the human embryonic stem cells are divided into units having a predetermined number by making micropatterns having various shapes and various areas, an embryo consisting of a certain number of unit cells can be made. The present invention can be used to standardize the differentiation method using an embryo, it is possible to increase the added value.

In addition, using the stamp according to the present invention will be able to screen and assay for stem cells and embryos, materials using embryos.

Of course, in the embodiment of the present invention mentioned and described the division of human embryonic stem cells, the scope of the present invention is not limited thereto. That is, it is a matter of course that the stamp of the present invention can be used for the separation, patterning, division, etc. of various human and animal cells.

<Stamp manufacturing method>

Stamp for human and animal cell division according to the present invention is produced by stamping the mold through the MEMS (Micro Electro Mechanical System) technology or ultra-precision processing technology, and poured a polymer resin on the stamp mold. Therefore, as described above according to the present invention, the body and the micropattern of the stamp are polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates ), Polycyclic olefins, polyimides, polyurethanes, polyesters, and the like.

10-13 are schematic diagrams illustrating various methods of fabricating molds of stamps 100, 100 'in accordance with embodiments of the present invention using MEMS techniques.

First, referring to FIG. 10, a photosensitive film 54 of a polymer material is coated on a silicon (Si) wafer 50, and a mask 52 having a desired pattern is aligned. At this time, the thickness of the photosensitive film 54 is the height of the desired fine pattern 130 (see FIG. 1). When the patterning operation is performed by irradiation of ultraviolet rays and the developing operation of the photosensitive film 54 is completed, the mold 60 coated with the polymer material is completed.

Referring to FIG. 11, the photosensitive film 54 is applied to the silicon wafer 50 and the mask 52 drawn with the desired pattern is aligned. The photosensitive film 54 is irradiated with ultraviolet rays to perform panning, and the silicon wafer 50 is shaved through dry etching such as RIE. At this time, the height cut by dry etching becomes the height of the desired fine pattern 130 (see FIG. 1). In this way, a mold 70 made of silicon may be manufactured.

FIG. 12 shows a fabrication method that enables the tip portion 141 (see FIG. 1) of the micropattern 130 to be tapered in order to reduce the dead zone of the cell when the cell is divided by the stamp. . A protective layer 56 may be formed on the silicon wafer 50 such as an oxide film or a nitride film to withstand wet etching. The photosensitive film 54 is apply | coated on it, and the mask 52 which painted the desired pattern is aligned. Then, the photosensitive film 54 and the protective layer 56 are patterned. Thereafter, wet etching or dry etching may be performed to form a mold 80 having a desired height and an end shape of the desired fine pattern 130.

In addition, as another method for fabricating molds for mass production, the chromium / gold (Cr / Au) layer is patterned in the manner described above to form an electroplating substrate and a metal such as nickel or copper may be used. Dip the substrate into the dissolved solution. By applying electricity through the electrode pad, a metal such as nickel or copper may be deposited on the patterned portion to a desired height to manufacture a mold.

The process of manufacturing a stamp according to an embodiment of the present invention using a mold made through the MEMS technique as described above is as follows.

It was prepared by a replica molding method that was poured into a completed mold using a transparent and elastic polydimethylsiloxane (PDMS) and cured, and used as a stamp. The curing process was completed by pouring pre-PDMS mixed with a curing agent in a ratio of 10: 1 to the finished mold and curing at 60 ° C. for 1 hour.

Alternatively, a stamp was prepared by applying an anti-stiction layer to the finished mold and squeezing the mold by pressing polymethyl methacrylate (PMMA) with the finished mold.

FIG. 13 is a photograph of a real mold of a stamp manufactured according to the embodiment, and FIG. 14 is a photograph of a real stamp manufactured using the mold. The width of the fine pattern of the stamp is about 200 μm, and the height of the fine pattern is about 40 μm. The stamp is manufactured to fit a 35-mm culture dish size to produce a pattern of cells at a time.

<Cell division device using stamp>

In order to divide all of the cell colonies cultured in the culture dish with the stamp according to the present invention under uniform conditions and to increase the reproducibility in the repeated experiment, it is necessary to maintain a constant pressure and speed of the stamp acting on the cell surface.

To apply the same force to the cells in a passive way, create a stamp approximately 1 mm thicker than the height of the petri dish (which eventually adjusts the height of the body of the stamp) and place the stamp on the cell and press or stamp it with the petri dish lid. There is a method of attaching a support plate and a handle to attach pressure evenly. In addition, when pressure is applied to the surface of the cell with the stamp, the cells may be damaged if there is a movement in the horizontal direction with the bottom of the culture plate, so that the stamp is about the same size and shape as the culture plate area in order to minimize the horizontal movement of the stamp. It is required to minimize the extra space between the stamp and the petri dish side by making it. Fast pressurization and release of the cell surface is also required. Optimization of the shape and size of the stamp is also important in increasing overall cell yield.

Another method is to develop an automated method after optimizing the pressure and speed of the stamp. The stamp is mounted on the device and the stamped area is adjusted vertically by attaching a guide like a rail, and the cell colonies in the culture dish placed on the platform are divided or applied at constant pressure and speed. It is a way to make a pattern. This has the advantage of creating a more precise pattern than the manual method.

Recognizing this problem, the present applicant has developed a manual / automatic cell division apparatus that compresses and divides cells manually or automatically using stamps.

15 is a schematic perspective view of a manual cell division apparatus 300 using the stamp according to an embodiment of the present invention. As shown, the manual cell division apparatus 300 using the stamp includes a housing 310, a fixing cylinder guide 320, and a stamp fixing cylinder 330.

The housing 310 has a box shape of a substantially rectangular shape, and an inner bottom is provided with a culture dish 10 in which cells are cultured. The culture dish 10 is fixed by a holder for fixing not shown. An opening 311 having a predetermined height is formed on an upper surface of the housing 310.

The fixed tube guide 320 is installed in the height direction of the housing 310 to penetrate through the opening 311 of the housing 310. The fixing tube guide 320 is installed to be movable up and down through the opening 311 and is fixed by a screw 312 formed in the opening 311. The inside of the fixing guide 320 is an empty space.

The stamp fixing cylinder 330 is inserted into the fixing cylinder guide 320 so as to be movable upward and downward, and the stamp 100 according to the embodiment of the present invention is fixed to the lower surface. The stamp 100 may be fixed in a manner by adhesive or vacuum compression. On the other hand, a weight 340 for pressing the stamp fixing cylinder 330 in the downward direction is mounted on the stamp fixing cylinder 330. The weight of the weight 340 may vary depending on the required pressure for dividing the cells contained in the culture dish 10 by the stamp 100 attached to the stamp holder 330. The wire 350 is fixed to the upper surface of the stamp fixing cylinder 330. The stamp fixing cylinder 330 can be moved in the lower or upper direction by using the wire 350.

Looking at the operation of the manual cell division apparatus 300 using the stamp configured as described above is as follows.

First, the culture dish 10 containing the cell colonies to be divided is placed on the bottom of the housing 310 and then fixed using a holder (not shown). Then, the fixing cylinder guide 320 is inserted into the opening 311 of the housing 310, and then fixed to an appropriate position using the screw 312. Thereafter, the stamp fixing cylinder 330 to which the stamp 100 is fixed is inserted into the fixing cylinder guide 320. After placing the weight 340 on the upper surface of the stamp holder 330, the wire 350 is placed. Then, the stamp holder 330 is lowered down along the stamp guide 320, and when the end reaches the stamp 100 is bonded to the stamp holder 330 is the cells contained in the culture dish (10) The colony will be divided.

In this case, the force of the stamp 100 applied to the cells varies depending on which position the stamp holder 330 is lowered or the weight of the weight 340, so that the force applied to the cells by appropriately adjusting them Adjust After dividing the cells, pull the wire 350 to lift the stamp holder 330.

16 is a schematic perspective view of an automatic cell division apparatus using a stamp according to an embodiment of the present invention. As shown, the automatic cell division apparatus 400 includes a lower plate 410, a side plate 420, an upper plate 430, a cylinder guide 440, and a cylinder 450.

The lower plate 410 is fixed to the culture dish 10 in which the cell colonies to be divided are cultured by a not shown fixing device. One side end of the lower plate 410 is installed so that the side plate 420 has a predetermined height in the upper direction. In addition, an upper plate 430 is installed at an upper end of the side plate 420 to be parallel to the lower plate 410.

The cylinder guide 440 is fixed to the lower surface of the upper plate 430 and is installed to protrude a predetermined length in the direction of the lower plate 410.

The cylinder 450 is formed to be movable up and down through the cylinder guide 440. Cylinder 450 may be applied to a hydraulic or pneumatic cylinder. The stamp 100 according to the embodiment of the present invention is fixed to the lower end of the cylinder 450. The stamp 100 may be fixed in a manner by adhesive or vacuum compression. Although not shown, a driving unit for driving the cylinder 450 may be formed at the upper plate 430 or the outside.

Looking at the operation of the automatic cell splitting apparatus 400 using the stamp configured as described above is as follows.

 First, the culture dish 10 is fixed to the lower plate 410 using a fixing device not shown. Then, the stamp 100 is adhered to the lower surface of the cylinder 450. Thereafter, when the operation command is applied to the driving unit, the cylinder 450 moves downward to perform cell division by pushing up and down the cell colony contained in the culture dish 10. On the other hand, before operating the cylinder 450, the drive setting value of the cylinder 450 is specified in advance. That is, an appropriate pressing force, pressurization time, etc. are set in advance.

17 is a schematic perspective view of an automatic cell division apparatus 500 using a stamp according to another embodiment of the present invention.

The automatic cell division apparatus 400 of FIG. 16 compresses and divides the cells in the culture dish 10 by descending the stamp 100 fixed to the cylinder 450, but the automatic cell division apparatus disclosed in this embodiment ( 500 is a method in which the stamp 100 is fixed and the culture dish 10 fixed to the cylinder 560 is raised to compress and divide the cells.

In the present embodiment, as in the previous embodiment, the side plate 520 is installed at one side end of the lower plate 510 in the upper direction, and the lower plate 510 is disposed at the upper end of the side plate 520. Top plate 530 is installed to be parallel to the.

The stamp holder 540 is installed on the lower surface of the upper plate 530 to protrude toward the lower plate 510. The stamp 100 is fixed to the lower surface of the stamp holder 540.

A cylinder stage 550 is fixedly installed on an upper surface of the lower plate 510, and a cylinder 560 is installed on the upper surface of the lower plate 510 so as to be movable up and down through the cylinder stage 550. The culture dish 10 is fixed above the cylinder 560. The stamp 100 may be fixed in a manner by adhesive or vacuum compression. Although not shown, a driving part for driving the cylinder 560 may be formed at the lower plate 510 or the outside.

The automatic cell splitting apparatus 500 according to the present embodiment is similar to the operation of the automatic cell splitting apparatus 400 according to the previous embodiment, and thus a detailed description thereof will be omitted.

Although preferred embodiments of the present invention have been described above, the present invention is not limited to the above-described specific embodiments. That is, those skilled in the art to which the present invention pertains can make many changes and modifications to the present invention without departing from the spirit and scope of the appended claims, and all such appropriate changes and modifications are possible. Equivalents should be considered to be within the scope of the present invention.

As described above, according to the stamp for splitting human and animal cells according to the present invention and a cell division method using the same, cells growing in colonies in a culture dish can be easily divided into cell units having a uniform number of cells at a time. In addition, there is an effect that can be easily divided into the desired pattern shape according to the purpose. In addition, since the cell colonies can be divided into cell units having a uniform number of cells, there is an advantage that a more reproducible research and experiment can be carried out by reducing the variable according to the variation in the number of cells that can appear in each subculture.

On the other hand, the conventional patterning method has a variable effect on the activity, shape or function of the cells due to the surface treatment to the culture dish in advance before culturing the cells, according to the present invention in the state of incubating the cells intact Since the patterning or splitting is performed by mechanical force, the effect on the cells is insignificant, and there is a free advantage in the deformation of the cells according to the incubation time. In addition, since it generates a pattern by applying a force instantaneously, the effect on cells is less than that of the conventional cell division method, and there is relatively little functionality to cause genetic modification by using an enzymatic method and mass passage in a very short time. It is possible to incubate.

In particular, according to the present invention, in viable human embryonic stem cell culture, by virtue of dividing a large amount of cells in a very short time, the passage culture rate is dramatically improved, and the viability of the cells is reduced by reducing the time spent outside the cell incubator. It is possible to suggest optimization conditions for increasing the concentration and maintaining the integrity of the cell. Therefore, the present invention can be said to be an important invention that closely addresses the essential requirements required to maximize human embryonic stem cell utilization range.

In addition, according to the present invention, when the human embryonic stem cells are divided into units having a certain number by a micropattern having various shapes and areas, an embryo consisting of a certain number of unit cells can be made. The present invention can be used to standardize differentiation methods using embryos to increase added value.

Furthermore, according to the present invention, it can be applied to the separation, patterning, division, etc. of various human and animal cells that cannot use existing enzymatic and mechanical methods. In addition, the stamp according to the present invention can be produced in a single-use, and economically competitive because it is high in the overall value of the biological experiments using the cell, it is possible to increase the industrial added value.

On the other hand, according to the manual / automatic cell splitting apparatus using a stamp according to the present invention, there is an effect that the stamp can be accurately patterned or divided by applying a constant pressure and speed to the cell colonies in the culture dish.

Claims (14)

A body having an upper surface and a lower surface; Stamps for human and animal cells comprising a; is located on any one of the upper surface or the lower surface of the body, the fine pattern portion is repeated at least one or more fine patterns having a predetermined height and width. The method of claim 1, The micropattern includes a wall defining the height and width and the shape of the micropattern, An end portion of the wall is stamped for the division of human and animal cells, characterized in that its thickness is smaller than other portions. The method of claim 1, The body is a stamp for human and animal cell division, characterized in that it has a shape of any one of a triangle, a square, a pentagon, a hexagon or a circle according to the shape of the culture plate in which human and animal cells are cultured. The method of claim 1, Each micropattern of the fine pattern portion is divided into human, animal cell stamp, characterized in that it has a shape of any one of a triangle, a square, a pentagon, a hexagon or a circle. The method of claim 1, Stamps for dividing human and animal cells, characterized in that the height of the fine pattern is 1μm or more. The method of claim 1, Stamp of the human and animal cell division, characterized in that the width of the fine pattern is more than 1μm. The method of claim 1, The body and the fine pattern portion may be made of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polycyclic olefins ), Polyimide (polyimides), polyurethane (polyurethans), a stamp for human and animal cell division, characterized in that formed of any one material of polyester (polyester). The method of claim 1, The micropattern portion is a stamp for human and animal cell division, characterized in that the surface is subjected to a coating treatment using a plasma surface treatment process, or a hydrophilic chemical such as PEG (polyethylene glycol) to increase the hydrophilicity. In the cell division method using a stamp according to any one of claims 1 to 8, a) preparing a cell colony in a culture dish; b) placing the stamp on top of the petri dish so as to be horizontal with the petri dish; c) dividing the cell colonies by applying a uniform pressure to the culture dish for a predetermined time using the stamp; And, d) removing the stamp; cell division method using a stamp comprising a. The method of claim 9, Cell division method using a stamp, characterized in that the cells to be divided are stem cells. The method of claim 10, The cell to be divided is a cell division method using a stamp, characterized in that the embryonic form of stem cells. In the manual cell division apparatus using a stamp of any one of claims 1 to 8, A housing in which the culture dish containing the cells is fixed to the inner bottom and having an opening formed in the upper surface thereof; A fixing guide configured to penetrate the opening of the housing, the lower end of which is positioned above the culture dish, and the upper end of which is exposed to the outside of the housing through the opening; A stamp fixing cylinder inserted into the fixing cylinder guide and installed to be movable, and having a wire installed on an upper surface thereof and a stamp attached to the lower surface thereof; And, A weight on which the stamp holder is placed on top of the stamp holder such that the stamp holder presses the culture dish at a desired pressure; Manual cell division apparatus using a stamp comprising a. In the automatic cell splitting apparatus using any one of claims 1 to 8, A lower plate to which the culture plate is fixed; A side plate installed to protrude upward from one end of the lower plate; An upper plate connected to an upper end of the side plate to be parallel to the lower plate; A cylinder guide having one end fixed to a lower surface of the upper plate and installed to protrude toward the lower plate; And The cylinder is installed to be movable up and down through the cylinder guide, the stamp is fixed on the lower surface to press the cells contained in the culture dish at a set pressure; Automatic cell division apparatus using a stamp comprising a. In the automatic cell splitting apparatus using any one of claims 1 to 8, Bottom plate; A side plate installed to protrude upward from one end of the lower plate; An upper plate connected to an upper end of the side plate to be parallel to the lower plate; A stamp holder fixed to the lower surface of the upper plate, installed to protrude in the lower plate direction, and fixed to the stamp on the lower surface; And, A cylinder stage having one end fixed to an upper surface of the lower plate and installed to protrude in the upper plate direction; The cylinder is installed to be movable up and down through the cylinder stage, the stamp is fixed on the upper surface to press the cells contained in the culture dish at a predetermined pressure; Automatic cell division apparatus using a stamp comprising a.

Images