KR100740228B1 - A novel electrohydrodynamic printing technique for cell patterning and its application as cell chips - Google Patents

Abstract

The present invention relates to an apparatus and method for patterning cell-fixing biomaterials, and a cell chip manufactured by the method. Specifically, collagen for fixing animal cells using an electrostatic hydrodynamic printing method An apparatus and method for precisely patterning a biopolymer material such as (collagen) and a cell chip manufactured by the method.

In order to achieve the above object, a method for patterning a cell-fixing biomaterial on a substrate, comprising a nozzle that sprays a solution containing the cell-fixing biomaterial at a constant hydraulic pressure, and a position corresponding to the nozzle Providing a pin-shaped ground electrode installed at a distance spaced from a predetermined distance; Positioning the substrate between the nozzle and the ground electrode; Converting the spray form by applying a voltage between the nozzle and the ground electrode when the solution is sprayed from the nozzle; And attaching the solution to the substrate while moving the substrate in a predetermined pattern shape while being sprayed in the converted spray form.

Biomaterial for cell fixation, collagen, electro-hydraulic spraying method, cell chip

Description

A novel electrohydrodynamic printing technique for cell patterning and its application as cell chips}

1 shows various spray modes generated by a change in flow rate under a potential difference or an electric field,

2 and 3 show explanatory diagrams for comparing the electro-hydraulic spray method and the inkjet injection method,

4 shows an electro-hydraulic spraying device for fine cell patterning on a substrate according to the invention,

5 is an explanatory diagram for explaining a patterning process with respect to a patterning substrate,

Figure 6 shows a photograph of the collagen solution patterned by various modes of electro-hydraulic spraying method according to the present invention,

Figure 7 shows a photograph taken after staining the collagen patterned by the electro-hydraulic spray method according to the present invention,

8 and 9 show photographs of HeLa cells cultured on collagen patterned by the electro-hydraulic spray method according to the present invention.

The present invention relates to an apparatus and method for patterning cell-fixing biomaterials, and a cell chip manufactured by the method. Specifically, collagen for fixing animal cells using an electrostatic hydrodynamic printing method An apparatus and method for precisely patterning a biopolymer material such as (collagen) and a cell chip manufactured by the method.

Recently, as the researches of cell biology, tissue engineering, bio-MEMS, and biosensor have been actively conducted, studies on the physiological activity of single cells or cell populations have become important. Cell patterning technology is required for growth control and precise manipulation of culture of animal cells for tissue culture. In particular, the collagen patterning technique, which is widely used as a biomaterial for tissue culture, is an innovative technology that can contribute to the nano bio industry by applying to cell biology and tissue engineering.

For biopolymers such as gelatin, collagen, poly-L-lysine, etc., by using cell patterning, cells that grow flat simply can be controlled and cultured to the desired population and desired form. Physiologically important cell populations can be specifically arranged.

Representative cell patterning methods studied so far include classical softlithography using a mask, photolithographic protein patterning, and laser-directed cell writing using a laser. Inkjet Printing Technique Using Cell Writing, Dip-pen Nanolithography Using AFM, and Inkjet Spraying Method [EA Roth, T. Xu, M. Das, C. Gregory, J.J. Hickman, T. Boland., “Inkjet printing for high-throughput cell patterning” Biomaterials, pp. 3707-3715, 2004], and Pattern Techniques Using MEMS Processes [Kang, Hyun et al. 4, “Manufacturing gelatin-patterned glass substrates for cell fixation for single cell manipulation”, 6th Korea MEMS Conference Abstract pp. 225-229, 2004; Choi, Yo-Han, Seung-Seop Lee, “Control of Cell Proliferation Patterns by Surface Treatment”, 6th MEMS Conference 367-371, 2004].

The present invention is a technique of primarily patterning cell polymers for cell fixation using electro-hydraulic spraying techniques and ultimately cell patterning through cell culture. Here, the electro-hydraulic nebulization technique, as will be described later, refers to a method capable of spraying very fine droplets using electrostatic charge charging.

According to the electro-hydraulic spraying method adopted in the present invention, since it is possible to spray an aqueous solution on a glass plate with an accuracy of several micrometers or hundreds of nanometers, compared with conventional patterning methods, The aim is to enable more sophisticated and finer cell patterning.

In addition, by the electro-hydraulic nebulization method according to the present invention, cell patterning is possible in an easier manner compared with the conventional art such as a complicated lithography process using a mask or AFM methods using expensive equipment. It aims to be able to pattern biomaterials quickly and inexpensively.

In addition, the cell patterning method using the electro-hydraulic spraying method according to the present invention enables single cell culture and cell co-culture in a limited substrate space, thereby reducing the costs associated with drug development and basic research and the accompanying material costs. It is aimed at reducing.

In addition, by the cell patterning method using the electro-hydraulic nebulization method according to the present invention, in recent years in connection with the field of research of nanobiotechnology, smaller and more precise cell chips using fine cell patterning (Cell Patterning) ( The purpose is to enable the production (cell chip).

In order to achieve the above object, the present invention provides a method for patterning a cell-fixing biomaterial on a substrate, comprising: a nozzle in which a solution containing the cell-fixing biomaterial is sprayed at a constant hydraulic pressure, and corresponding to the nozzle Providing a pin-shaped ground electrode installed at a distance spaced a predetermined distance as a position to be formed; Positioning the substrate between the nozzle and the ground electrode; Converting the spray form by applying a voltage between the nozzle and the ground electrode when the solution is sprayed from the nozzle; And attaching the solution to the substrate while moving the substrate in a predetermined pattern shape while being sprayed in the converted spray form.

In addition, the step of converting the spray form by applying the voltage, characterized in that for converting the spray form in the conical liquid injection mode.

In addition, the patterning method, characterized in that to provide collagen (collagen) as the cell-fixing biomaterials.

In addition, in the patterning method, the substrate is coated with agar medium (agarose) solution, characterized in that the surface of the substrate and dried for a predetermined time at room temperature and then sterilized with ultraviolet rays.

In addition, the present invention provides a patterning device for patterning a cell-fixing biomaterial on a substrate, in order to achieve the above object, a nozzle for spraying a solution containing the cell-fixing biomaterial at a constant hydraulic pressure; A pin-shaped ground electrode installed at a distance spaced a predetermined distance as a position corresponding to the nozzle; A stage positioned between the nozzle and the ground electrode to move the substrate while holding the substrate; And a power supply device for converting the spray form by applying a voltage between the nozzle and the ground electrode, and attaching the solution to the substrate according to a pattern shape preset in the converted spray form. .

On the other hand, the converted spray form is characterized in that the conical liquid injection mode.

In addition, the cell-fixing biomaterial is characterized in that the collagen (collagen).

In addition, the substrate is characterized in that the surface is coated with agar medium (agarose) solution.

On the other hand, the present invention is characterized in that the cell chip is formed by culturing predetermined cells on the patterned cell-fixing biomaterial on the patterned substrate by the patterning method described above as a cell chip.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, embodiments of the present invention may be modified in many different forms, and the scope of the present invention should not be construed as being limited by the embodiments described below. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Accordingly, the shape and the like of the elements in the drawings are exaggerated to emphasize a more clear description, and the elements denoted by the same reference numerals in the drawings means the same elements.

First, the electro-hydraulic spraying principle employed in the present invention will be described in comparison with the conventional inkjet spraying method, prior to the detailed description of the adhesion and cell culture of the biomaterial by the electro-hydraulic spraying method.

Electro-hydraulic spraying is a spraying method capable of spraying fine droplets, spraying particles composed of monodispersed particles, and having excellent surface adhesion compared to conventional spraying methods. Electro-hydraulic spraying refers to a series of processes in which a high voltage is applied to a conductive capillary to spray a target fluid toward ground. In other words, electro-hydraulic spraying is a principle of making unipolar charged liquid using the conductivity of a liquid and spraying by an electric field formed by a potential difference.

1 illustrates various modes caused by changes in flow rate under a potential difference or an electric field. In the absence of an electric field (state ①), the hydrostatic area of the nozzle outlet creates an equilibrium of hydrostatic pressure and capillary pressure, but in the presence of an electric field (state ②), electrostatic pressure from the surface charge on the surface is added and a general drop is achieved. Droplets smaller than the size of the droplets generated by the mode are generated. When the potential on the surface increases beyond the critical potential after the stable pressure equilibrium on the surface, a column of fluid is ejected in the form of a liquid column on the shaft, and since the fluid is charged, to compensate for the loss of charge corresponding to the amount of ejected Conducting current flows. At this time, state ③ is called a cone-jet mode in a fairly stable state, and compared with other modes, the droplet size distribution is sprayed closer to monodisperse. State ④ is an unstable conical liquid column mode, which is caused by the instability of the external electric field or the supply flow rate. State ⑤ is a mode that occurs in a very high voltage state, the conical shape disappears near the nozzle, the spray material is divided into several jet state and sprayed, and the charge caused by the large magnetic field applied to the droplet The strong coulomb repulsion results in a spray mode in which the liquid is atomized.

That is, in the electro-hydraulic spraying, the spraying pattern is changed according to the magnitude of the applied voltage, so that a series of spraying modes such as dropping mode, micro dropping mode, conical liquid pouring mode, unstable liquid pouring mode, and spray mode can be controlled. Will be. This spray mode can be variously applied depending on the application.

As can be seen from the principle of the electro-hydraulic spraying, the electro-hydraulic spraying method, compared with the inkjet spraying, basically obtains a spraying form that produces various types of monodisperse particles according to the voltage control and the change of the ground shape. In particular, in the case of using the conical liquid injection mode, there is an advantage in that a predetermined shape can be patterned at a desired position without a short circuit.

First, as shown in FIG. 2, electro-hydraulic spraying is advantageous over inkjet spraying in terms of fine patterning. That is, in the case of the conventional inkjet spraying, as shown in FIG. 2 (a), the nozzle size has an influence on the size of the generated droplets, and the generated droplet size is about twice the size of the nozzle (d = 1.89 D ₀ ; d is the droplet diameter, D ₀ means the nozzle diameter). Therefore, in order to spray fine droplets, the size of the nozzle must be so small. Therefore, not only high precision processing is required, but also when spraying insoluble particles, nozzle clogging may occur at the nozzle outlet. exist. There is a limit to reducing the size of the resulting droplets. In contrast, the electro-hydraulic spraying method uses an additional electric force, as shown in FIG. 2 (b), so that finer droplets can be obtained by using a nozzle having a relatively large particle size, and a uniform droplet size can be obtained. You can get it. In addition, as shown in FIG. 2 (b), the size of the droplets generated in the conical liquid column mode depends on the diameter of the liquid _column (D _j ) rather than the size of the nozzle. Since it changes according to the electrical conductivity (K) and the flow rate (Q), it is possible to reduce the diameter of the liquid column by adjusting the material properties and flow rate. That is, since the size of the liquid column can be controlled irrespective of the size of the nozzle outlet, it is possible to form and pattern a liquid column having a fine size without the necessity of micromachining or clogging of the nozzle outlet.

Next, as shown in FIG. 3, the electro-hydraulic spraying method is advantageous over inkjet spraying in terms of the followability of the patterning line, that is, the 'break and short circuit prevention side', and the attachment to a desired position, that is, the 'position error prevention side'. In the case of inkjet injection shown in FIG. 3 (a), even if small droplets are generated in a small nozzle, the generated droplets may not be accurately attached to a desired position due to Brownian motion. . That is, when spraying sub-micron droplets in the drop-by-drop mode, droplets that have once escaped from the central axis of the spray trajectory of the droplets cannot be returned to their original center positions due to the Brownian motion. It must be. On the other hand, in the case of the intact jet in the electro-hydraulic spray shown in FIG. 3 (b), since the liquid column is in the form of a liquid column to which the droplets are connected, the liquid column may be deviated from the central axis due to the influence of external force. The tendency to align along the central axis by the surface tension of the column itself can be prevented from deformation of the filament, so that the jet can accurately reach the desired position.

Electro-hydraulic nebulizer for fine cell patterning

4 shows an electro-hydraulic spraying device for fine cell patterning on a substrate according to the present invention.

The device implements a desired electro-hydraulic spray mode by supplying a nozzle 11, a liquid supply unit 1 for supplying a constant flow rate of liquid to the nozzle 11, and a predetermined voltage to the nozzle 11. To control the position and the moving speed of the voltage supply unit 2, the precision movement stage unit 3 for moving the substrate 5 to pattern the circuit in the input shape while maintaining the substrate 5, and the precision movement stage unit. It consists of a control unit 4 for.

The electro-hydraulic spraying device is placed in a cleanbooth environment of class 100 (0.5 mm standard). On the other hand, the electro-hydraulic spraying process according to the configuration was monitored by a CCD camera equipped with a high magnification lens (Hirox, up to 600 times), each spray image is stored in a computer in real time.

First, the liquid supply unit 1, the syringe pump (12; kds-100, KD Scientific Inc.) that can be freely adjusted to the flow rate of the material to be sprayed up to 0.1 μl / min and the syringe pump (12) and a nozzle (11) for spraying a liquid containing such a substance onto a substrate and a ground pin (13) installed at a position opposite to each other in a state where the nozzle (11) and the substrate are placed. . The nozzle 11 is a nozzle made of a conductive material for electro-hydraulic spraying. The nozzle 11 is formed of a stainless material having an inner diameter of 0.14 mm and an outer diameter of 0.32 mm, and the ground pin 13 has the same shape and size as that of the nozzle. It was fixed using a nozzle fixing stage (not shown) 2.7 mm below the position of the nozzle.

The voltage supply unit 2 includes an electrode including a nozzle 11 and a ground pin 13 and a power supply 21 for applying a voltage to the electrode. As the ground electrode, the ground pin 13 having the same shape and size as the nozzle 11 was used as described above. That is, the shape of the ground electrode eventually becomes a pin-to-pin electrode type. This pin-to-pin electrode type is an optimal electrode type that can be attached to the substrate as the liquid column itself, in particular, in the conical liquid column mode without splitting the droplet into droplets, that is, using an intact jet. . The nozzle 11 and the ground pin 13, which is a ground electrode, are connected to a power supply 21 capable of supplying a voltage of 15 kV or more.

The precision movement stage unit 3 is a configuration for patterning a predetermined material according to the command of the control unit 4, and the precision movement stage unit 3 is a linear motor (not shown) and a stage (not shown) of two axes xy. Consists of. The stage is positioned so as to be movable between the nozzle 11 and the ground pin 13, the linear motor and the stage can be moved up to 100 mm, and the repetitive movement error occurring during the movement is 5 m or less.

The control unit 4 is a device capable of moving the precision moving stage unit 3 to a desired position and controlling the speed. Generally, the CPU of the pc may correspond to this.

Substrates for patterning biopolymers

In the present invention, a glass cover slip or the like, which is a transparent substrate, may be used as the patterning substrate 5 for easy observation. On the other hand, in order to obtain a substrate in a hydrophilic state with little impurities, it is preferable that the substrate is washed with a slightly heated Piranha solution in which hydrogen peroxide and sulfuric acid are mixed at a predetermined ratio. This method makes the glass surface very clean and hydrophilic, facilitating surface treatment with biomaterials such as collagen.

FIG. 5 illustrates a patterning process for a substrate for patterning. In general, the surface of the substrate is required to have a high contrast characteristic of cell-friendly and cell-incompatible properties for specific fixation of cells.

Patterning solution

As a cell-friendly material to be patterned by an electro-hydraulic spray method, sodium chloride may be added to a mixed solution such as collagen and gelatin, or collagen and glycerin, and the background of the substrate except for the patterned region may be used as a background (background). Agar media are coated as cell-incompatible materials used as). Sodium chloride in the material is a material that controls the conductivity of the liquid. The electro-hydraulic spray method uses the conductivity of the liquid to make the monopolar charged liquid and the material is sprayed by the electric field formed by the potential difference, so the conductivity of the liquid is very important.

Collagen is most commonly used as a protein constituting the body of all living organisms, and is also cell-friendly, easily recognized by cell receptors, and thus has the property of improving cell adhesion.

As described above, since the substrate is already coated by agar medium having cell-incompatible properties, it is possible to control cells by providing a strong contrast effect when using collagen, which is a cell-friendly material, as a patterning solution.

On the other hand, the electro-hydraulic printing mode described above depends on the viscosity of the solution, the electrical conductivity of the solution, the flow rate of the solution and the applied voltage. Thus, the collagen solution must be modified to meet high viscosity requirements, since higher viscosities allow electro-hydraulic spraying in the cone-jet mode with smaller diameter stable jets. In order to satisfy this high viscosity, a predetermined amount of gelatin, glycerin, and the like may be added, such as a collagen 30 mg / ml solution containing 10% of gelatin 1 mg / ml. In addition, a predetermined amount of sodium chloride may be added, which serves to allow the patterning solution to obtain a predetermined electrical conductivity.

Patterning method by electro-hydraulic spray method

The method of implementing the patterning of a cell adhesion biomaterial such as collagen on a substrate using the electro-hydraulic spraying device described above is as follows.

First, the mixture containing the metal to be patterned is sprayed into the nozzle 11 uniformly using the syringe pump 12, and then a voltage is applied to the nozzle 11 so that the spray form becomes a conical mode. The conditions under which a stable conical mode can be formed should be identified.

Thereafter, the precision moving stage 3 holding the substrate 5 is moved in accordance with the pattern shape previously input to the controller. Eventually, the intact jet sprayed from the conical mode is attached to the substrate 5 positioned between the electrodes to form a desired cell attachment biomaterial pattern, and then, the cell culture process described below is performed to culture the desired cells on the pattern. To be possible.

Cell culture

Various cells such as Hela (ATCC CCL-2) cells may be cultured simultaneously on a substrate on which a cell-adhering biomaterial such as collagen is patterned. For example, in Hela (ATCC CCL-2) cell culture, cells were treated with 10% Fetal Bovine Serum (FBS) and 1% antibiotics under basic conditions (37 ° C, 5% CO ₂ ). (antibiotics) can be incubated using Dulbecco's Modified Eagles Media (DMEM: Cambrex, USA).

On the other hand, since the agar plate is pre-coated as a cell-incompatible material used as a background on the substrate except for the patterned area, the cells cannot be cultured in this area, resulting in a strong contrast effect. To provide cellular control.

EXAMPLE

In this embodiment, glass cover slips (Microscope cover slips; Marienfeld, Germany), which are transparent substrates, were used as the substrate 5 for patterning. Meanwhile, in order to obtain a substrate in which the impurities are low and hydrophilic, the Piranha solution mixed with hydrogen peroxide and sulfuric acid at a ratio of 1 to 3 is heated at a temperature of about 70 ° C for 20 minutes. It was. In addition, the substrate surface was coated with 0.15% agar solution (BD biosciences, USA) sterilized by autoclave and dried at room temperature for 3 hours to obtain the surface conditions necessary for specific fixation of cells. After that, it was sterilized for 2 hours or more with ultraviolet light.

As a result of the treatment, in the characteristics of the substrate coated with agar, the agar was uniformly coated with a thickness of about 2 μm on a cover glass whose surface properties were changed, and the surface roughness of the coated agar was 1500Å ± 500 It was.

In the cell adhesion printing solution used in the electrohydraulic patterning method, about 2.5% and 10% of sodium chloride was added to 1.0 mg / ml collagen solution (Sigma Chemical Inc., USA) of type I extracted from calf skin. Used. All solutions were stored at 4 ° C. and dried quickly after printing. The thickness of the patterned collagen solution was about 4000 mm ± 1000 mm.

The printing solution was printed in various types by electrohydraulic patterning equipment. The printing process was performed in a clean space, and the patterned collagen solution was photographed after drying for 1 hour.

Figure 6 shows a photograph of the collagen solution patterned by various modes of electro-hydraulic spraying method according to the present invention, A is dripping mode, B is multi-dripping mode, C It can be seen that is the contact mode (cone-jet mode), and D is a patterned state by the contact mode (contact mode) and that various patterns can be produced. Each of these modes is generated according to the applied voltage, when the spray flow rate of the syringe pump is 2.5 μl / min, the dropping mode is about ˜5.5 kV, and the multi dropping mode is about 5.5 kV to 8.5 kV, The contact mode was found at about 6.5 kV to 7.25 kV, and the contact mode was at about 6.5 kV to 9.5 kV.

The diameter of the droplets in the pattern was distributed over 50 ~ 500㎛ and typically corresponds to 200 ~ 300㎛, the width of the straight line of the pattern corresponded to 180 ± 20 ㎛ in contact mode, 500 ± 40 ㎛ in contact mode .

On the other hand, Figure 7 shows a photograph taken after the dyeing treatment with the Coomassie Blue solution, a kind of protein dye to confirm that the component of the pattern patterned by the electro-hydraulic spray method according to the present invention.

Each patterned 22 × 22 mm 2 cover slip was placed in a dish for 100 mm tissue culture and injected into each dish with 2 ml of cell culture from 40,000 to 50,000 cells / ml. . Cell culture medium was replaced at one to two day intervals, and cell growth was observed daily.

FIG. 8 is a photograph of HeLa cells cultured on patterned collagen, (A) a photograph of culture on untreated glass surface, (B) showing a sharp contrast between agar coated surface and collagen coated surface (C) shows a photograph of HeLa cells cultured on linearly patterned collagen, and (D) shows enlarged photographs of cells grown on the collagen-patterned surface. That is, HeLa cells did not grow in the area coated with the agar plate as described in detail above, it was confirmed that the collagen was attached to the surface cultured in the patterned area.

In addition, as shown in FIG. 9, the cells were cultured by thinning the thickness of the pattern during electro-hydraulic spraying of the collagen solution. (A) is a photograph showing HeLa cells cultured on a pattern of about 200μm thickness, (B) is an enlarged photograph.

On the other hand, it was confirmed through the above example that a single cell can be finely patterned by the electro-hydraulic patterning method according to the present invention. In addition to using such micropatterned and tissue cultured cell chips biologically, the electro-hydraulic patterning method allows the cultivation of more cell populations on spatially limited substrates or chips. By using high-throughput screening, it can be seen that the vast amount of reagents and experiments required for the cultivation of multi-cells in multiwells and the experiment of antibodies and antigens can be significantly reduced.

The present invention is not limited to the above-described embodiments, but can be variously modified and changed by those skilled in the art, which should be regarded as included in the spirit and scope of the present invention as defined in the appended claims. something to do.

As described above, in the present invention, unlike an inkjet printing method using an inkjet printer using a commercially known photolithography technique and a commercially available inkjet printer, the present invention utilizes a newly developed electro-hydraulic printing technique such as collagen. Adhesion of cells was controlled by patterning the cell-adhesive biomaterial into a desired form.

That is, by intensively developing the culture of single cells and cell populations using more sophisticated and simple cell patterning method than the existing method, it can make a great contribution to the study of cell and tissue regeneration of internal organs of the human body.

In addition, in the use of high-throughput screening in the development of new drugs, a vast amount of reagents and experiment costs for culturing multi-cells on multiwells and testing antibodies and antigens can be obtained through the present invention. The patterning technique of single cells and populations of cells creates effects that can be significantly reduced.

As a result, it is possible to manufacture new drugs with various new ingredients by shortening the development time and cost reduction related to new drug development, and moreover, it is comfortable to meet the trends of future society that values quality of life. It can help you live your life.

Claims (9)

delete delete In the method for patterning collagen (collagen) as a cell-fixing biomaterial on a substrate, Providing a nozzle in which the solution containing the cell-fixing biomaterial is sprayed at a constant hydraulic pressure, and a pin-shaped ground electrode installed at a distance separated by a position corresponding to the nozzle; Positioning the substrate between the nozzle and the ground electrode; Converting the spray form of the solution by applying a voltage between the nozzle and the ground electrode when the solution is sprayed from the nozzle; And Attaching the solution to the substrate while moving the substrate in a predetermined pattern shape while the solution is sprayed in the converted spray form. The method of claim 3, The substrate is a patterning method characterized in that the surface of the substrate is coated with an agar medium (agarose) solution, dried at room temperature and sterilized with ultraviolet rays. delete delete In the patterning device for patterning collagen (collagen) which is a cell-fixing biomaterial on a substrate, A nozzle for spraying the solution containing the cell-fixing biomaterial at a constant hydraulic pressure; A pin-shaped ground electrode installed at a distance spaced from the nozzle corresponding position; A stage positioned between the nozzle and the ground electrode to move the substrate while holding the substrate; And And a power supply device for converting the spray form of the solution by applying a voltage between the nozzle and the ground electrode, and attaching the solution to the substrate according to a predetermined pattern shape in the converted spray form. The method of claim 7, wherein The substrate is a patterning device, characterized in that the surface is coated with agar solution (agarose) solution. A cell chip formed by culturing predetermined cells on said patterned cell-fixing biomaterial in a patterned substrate by the patterning method according to claim 3 or 4.

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