KR101138536B1 - Cell fixation method using microfluidic channel and plasma - Google Patents

Abstract

The present invention relates to a microfluidic channel that fixes cells by hydrophilizing a pattern portion of a first substrate by applying a plasma inside the microfluidic channel and a cell fixation method using plasma. The cell fixation method according to the present invention has simplified the process than the conventional SAMs technology, and the cell fixation site was rapidly changed to hydrophilicity by using plasma, thereby shortening the cell attachment time by the hydrophilic functional group on the surface. Furthermore, as the structure was fabricated using the MEMS process, the economics and the possibility of commercialization were increased.

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Description

Method of preparing cell attachment using micro-fluidics channel and plasma

 The present invention relates to a method for fixing a cell using a microfluidic channel and a plasma, and more specifically, to apply a plasma inside the microfluidic channel to hydrophilize the pattern portion of the first substrate to fix the microfluidic channel and the plasma. It relates to the method of fixing the cells used.

The ultimate purpose of the cell-related research in vivo (in In vivo , all the actions of the cells occurring in vitro ( in in vitro ). Looking at the in vivo environment from the macro-sized organ to the micro-sized cell, it can be seen that all the basic actions in vivo are performed at the molecular level. Therefore, in the case of cell research in vitro, cell regulation in a microenvironment can be said to be essential.

Most of the cells that make up the human body are epithelial cells. The basic culture mechanism of epithelial cells consists of fixation/attaching/adhering and growth/proliferation. In particular, a cell chip based on attaching cells directly to a substrate, such as a protein chip or a DNA chip, is also important for immobilizing cells on the substrate.

These cell immobilization technologies are used in fields such as basic biology, biosensors, drug testing, and tissue engineering, and research is being conducted to effectively fix cells. In particular, as research on technology to be used industrially for a long time has been conducted, cell immobilization technology has been mainly developed based on the technique of self-assembled monolayers (SAMs) using a soft lithography method, and this method is ECM protein or BSA. , By coating the surface with antibodies, chemicals, etc. locally, it was possible to effectively immobilize the cells at the desired location on the surface. However, the SAMs method has a limitation in that it is less efficient in terms of industrial production due to complicated processes.

In more detail, it is known that there are various methods for immobilizing cells on a substrate, which can be largely divided into immobilization by a method using biomaterials and immobilization by using physical and chemical properties of the substrate itself. As a method using biomaterials, there is a method of first immobilizing peptides or proteins on a substrate, and then immobilizing cells using the cell receptors of these biomaterials [Mann BK, Tsai AT, Scott-Burden T, West JL. Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition. Biomaterials 1999;20(23-.24):2281-6].

And as a method using the physical and chemical properties of the substrate, a method using a hydrophobic property of a substrate, a method using an electrical property, a method using a surface structure [Curtis AS, Wilkinson CD. Reactions of cells to topography. J Biomater SciPolym Ed 1998;9(12):1313 -.29], Method of immobilizing cells using collagen [On-chip transfection of PC12 cells based on the rational understanding of the role of ECM molecules: efficient, non-viral transfection of PC12 cells using collagen IV, Neuroscience Letters 378 (2005) 40 -43.

However, such a cell fixing method has a problem that the process is complicated and takes a lot of time, and furthermore, it is difficult to commercialize immediately due to low productivity.

The present invention is to simplify the process, and to provide a method for fixing the cell, which is economical and has high potential for commercialization, because it can fix cells in a short time.

One aspect of the present invention is a step of generating a plasma into the microfluidic channel after laminating a structure having a microfluidic channel formed on a first substrate; It relates to a cell fixing method comprising a fixing step of washing the cells after seeding the cells on the separated first substrate.

The cell fixation method according to the present invention has simplified the process compared to the conventional SAMs technology, and the cell fixation site was rapidly changed to hydrophilicity by using plasma, thereby shortening the cell attachment time due to the hydrophilic functional groups on the surface. Furthermore, as the structure was fabricated using the MEMS process, the economics and the possibility of commercialization were increased.

The cell fixation method according to the present invention relates to a cell fixation method using a microfluidic channel and plasma to fix cells after hydrophilizing the pattern portion of the first substrate by applying plasma inside the microfluidic channel.

Cell fixing method according to the present invention includes a plasma generation step and a cell fixing step.

Hereinafter, each step will be described in detail with reference to the drawings.

plasma  Stage of occurrence

The plasma generating step is a step of generating plasma by injecting (air) oxygen into the microfluidic channel after laminating the structure on which the first substrate and the microfluidic channel are formed.

1 is an example of structures 100 usable in the present invention. Referring to FIG. 1, the structure 100 includes a second substrate 20, an electrode layer 30 and a microfluidic channel 10.

The structure 100 may include an electrode layer formed on a second substrate and a microfluidic channel 10 formed on the electrode layer.

The microfluidic channel 10 represents a concave to recessed portion having a predetermined width W and depth H. A fluid such as oxygen or air may flow along the microfluidic channel.

There is no limitation on the shape of the microfluidic channel 10, and as an example, there may be a straight line shape as shown in FIG. 2 a, a cross shape as b, and a recessed cylindrical shape as c.

The second substrate 20 may be made of silicon wafer, glass or plastic, and is not particularly limited.

The electrode layer 30 is required for plasma generation. Therefore, the electrode layer 30 is electrically connected to the plasma generator 40.

The electrode layer 30 can be used without limitation as long as it is conductive. For example, gold (Au), silver (Ag), copper (Cu), aluminum (Al), nickel (Ni), molybdenum (Mo), tungsten (W), indium tin oxide (ITO), indium zinc oxide ( IZO), polythiophene, polyaniline, polyacetylene, polypyrrole, polyphenylene vinylene, PEDOT (polyethylenedioxythiophene)/PSS (polystyrenesulfonate) can be used. .

The electrode layer 30 may be formed by a vapor deposition method such as chemical vapor deposition in the case of the metal material, and may be formed using a spin coating method in the case of a conductive polymer, but is not limited thereto.

To increase the surface adhesion of the electrode layer 30, Cr, Ni, Ti, Zr, Pt, etc. may be first deposited on the second substrate 20, and then the electrode layer 30 may be formed.

The method of forming the microfluidic channel 10 on the electrode layer 30 may be performed by steps such as known photolithography, etching, thin film deposition, thermal oxidation, and cleaning.

In addition, it can also be produced by soft lithography (soft lithography) technology, as a typical example, by using an elastic mold such as PDMS (polydimethylsiloxane) can be modified and patterned surface.

In one embodiment, a method of forming a microfluidic channel on the electrode layer includes coating a photoresist composition on the electrode layer; Selectively exposing using a mask; And etching.

The photoresist composition is a photosensitive material, and both a positive photoresist or a negative photoresist can be used. As an embodiment, SU-8 (a type of epoch negative photoresist) having good resolution and biocompatibility may be used.

When the negative photoresist composition is coated on the electrode layer, a microfluidic channel can be formed by selectively exposing the desired microfluidic channel using a patterned mask and etching the unexposed portion.

At this time, the photoresist composition is applied on the electrode layer 30 by a conventional polymer coating method, for example, spin coating, to form a photoresist layer 11.

Referring to FIG. 1, the microfluidic channel 10 is a portion selectively removed from the applied photoresist layer 11.

2 shows the step of generating plasma by injecting oxygen into the microfluidic channel 10 after laminating the structure 100 and the first substrate 200.

Referring to FIG. 2, the first substrate 200 may be divided into a pattern portion 210 facing the microfluidic channel 10 and covering the microfluidic channel 10 and an area 211 in contact with the photoresist layer 11. have. When laminating the first substrate 200 and the structure 100, the pattern portion 210 is a partial region of the first substrate corresponding to and covering the microfluidic channel 10 (engraved portion). The shape of the pattern portion 210 is determined by the shape of the microfluidic channel 10. The pattern portion 210 is a portion where the surface properties are modified by plasma generation to fix the cells.

In more detail, when the first substrate 200 and the structure 100 having the microfluidic channel formed are opposed to each other and then injected with oxygen into the microfluidic channel, the region where oxygen contacts the photoresist layer 11 ( 211), but only in the pattern portion 210, when plasma is applied, only the pattern portion 210 of the first substrate 200 in which oxygen is present can be modified with hydrophilicity.

In general, it is known that when a surface of a hydrophobic material is plasma treated, the surface thereof is oxidized to change the surface properties to hydrophilicity.

The microfluidic channel may have a width (W) of 10 to 400 μm, preferably 10 to 100 μm, and if less than 10 μm, the area of the pattern portion 210 corresponding to the microfluidic channel is narrowed. As a result, the cells cannot be easily fixed to the pattern portion.

The microfluidic channel may have a depth (H) of 20 to 50 μm, preferably 20 to 30 μm, and if it is less than 20 μm, it is difficult to fix the cells to the pattern portion of the first substrate corresponding to the microfluidic channel.

The structure 100 may include a flow path portion 12 having a width of less than 10 μm to transfer oxygen to the microfluidic channel. The separation between the microfluidic channel and the flow path is based on the channel width (W). That is, if the width is less than 10 μm, it can be divided into a flow path portion and a microfluidic channel if more than 10 μm. The channel portion 12 has a small channel width and cannot fix cells to the pattern portion of the first substrate.

Referring to the structure 100 of FIG. 1C, when a straight channel is formed as a flow path portion 12 and a microfluidic channel 10 is formed as a recessed cylinder, the cell fixing pattern is independent islands on the first substrate. (island) structure.

The present invention relates to a method for fixing cells to a first substrate in a desired position or shape. To this end, in the present invention, a desired cell anchoring pattern is formed in an intaglio (microfluidic channel) on a structure facing and contacting the first substrate, and a plasma is generated thereon to locally fix the cell by hydrophilizing the first substrate facing the structure. As a result, the process was simplified compared to the existing SAMs technology, and the cell fixation site was rapidly changed to hydrophilicity using plasma, thereby shortening the cell adhesion time due to the hydrophilic functional groups on the surface. Furthermore, as the structure was fabricated using the MEMS process, the economics and the possibility of commercialization were increased.

Cell fixation step

The cell fixing step is a step of washing the cells after seeding the cells on the first substrate 200 from which the structure 100 is separated.

3 is a schematic diagram showing implantation and washing of cells on the first substrate 200. The implantation is preferably performed within about 3 hours, more preferably about 30 minutes to 3 hours.

In the fixing step, when the cells are seeded on the first substrate and then washed, the cells are fixed only on the pattern part 210.

4 is a graph measuring cells attached to the surface every 2 hours, 4 hours, 6 hours, 8 hours, 24 hours after seeding 2X10 ⁵ cell/ml AGS cells (adenocarcinoma cell human stomach cancer cells) on the surface of 1 cm X 1 cm. . The data shown in blue represents the number of cells attached to the culture dish, and the red color changes the PDMS surface from hydrophobic to hydrophilic by generating oxygen plasma for 2 minutes and 30 seconds using an oxygen plasma generator. It is the number of cells measured by seeding after seeding (PDMS subjected to plasma treatment). The green data is the number of cells measured after seeding cells on the PDMS hydrophobic surface without plasma treatment.

Referring to FIG. 4, it can be seen that as time passes, the number of cells attached to the surface of cells increases in all three different surfaces. It can be seen that a large number of cells were attached in the fastest time in the plasma-treated PDMS, and about 50 times more cells were attached in 2 hours than PDMS without surface treatment. In addition, it can be seen that about 1.5 times more cells are attached than the culture vessel that is mainly used for existing cell culture. After 6 hours, the amount of cells attached to the culture vessel and plasma-treated PDMS can be seen to be similar, because there is no space for cells to adhere to the surface. On the other hand, PDMS shows that after 24 hours, a smaller number of cells were attached to the culture vessel and plasma-treated PDMS for 2 hours, and the hydrophobic property of the PDMS surface was not suitable for cell adhesion. In addition, it can be seen that the PDMS on the hydrophilic surface can attach cells within a shorter time than the culture vessel.

5 is AGS cell morphology confirmed by SEM after 24 hours cell culture on (a) plasma treated PDMS surface and (b) (plasma untreated) PDMS surface. AGS cells are epithelial cells, which are attached to the surface and cultured. The shape of the polyhedron is formed when the cytoplasm of the cell is firmly attached to the surface. Referring to FIG. 5A, the AGS cell shows the shape of a polyhedron on the hydrophilic surface through plasma treatment, but as shown in FIG. 5B, the hydrophobic surface shows the shape of a round circle.

4 and 5, it can be confirmed that the plasma-treated PDMS is more suitable for cell culture.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples should not be interpreted as limiting the protection scope of the present invention.

Example  One

Cr and Cu are sequentially deposited on a slide glass using a thermal evaporator. Cr deposited 600 Pa, Cu deposited 1200 Pa. On the deposited Cu surface, a negative photoresist SU-8 (Micro-Chem, SU-8 2025) was coated with a spin coater at 2500 rpm for 3 seconds, and then subjected to a baking process at 95° C. for 10 minutes. After baking, the desired pattern is exposed through a chrome mask using a UV mask aligner by applying 180 mJ/cm 2 energy. After exposure, a baking process is performed at 95°C for 5 minutes using a hot plate. After baking, the desired pattern can be obtained by selectively removing unexposed areas by immersing it in a PGMEA (Micro-Chem, SU-8 developer) solution.

The structure in which the microfluidic channel is formed is connected to the generator of the plasma generator (Femto-Sicence, CUTE B). The connected microfluidic channels are adhered to the PDMS. After placing the PDMS bonded to the microfluidic channel on the ground plate, plasma is generated while injecting O2 (99.9%) at 1000 cc/min. Plasma was continued for 40 minutes at a frequency of 40 KHz, and the plasma applied voltage was set to 100 W.

After the plasma treatment was completed, the structure was separated, and the cells were seeded on the PDMS surface for 30 minutes to 3 hours, and then the unattached cells were washed and removed.

6 is a schematic view showing the entire process of Example 1.

7 is an example of a structure in which the microfluidic channel prepared in Example 1 is formed.

8 is a photograph showing a cell pattern fixed to the first substrate (PDMS) according to the shape of the microfluidic channel prepared in Example 1. FIG. 8A is a cell of FIG. 1, and FIG. 8B is a cell fixed to the first substrate (PDMS) using the structure of FIG. 1B. Referring to a, b of FIG. 8, it can be confirmed that the cells are fixed in a straight line shape or a cross shape like the microfluidic shape of the structure on the PDMS.

Example  2

In Example 2, the structure was the same as in Example 1 except that the structure was formed in the form of a in FIG. 1 and the channel width (W) was 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, and 30 μm. It was carried out.

9 is a photograph of a pattern in which cells are fixed on the PDMS according to Example 2. Referring to FIG. 9, it can be seen that as the width of the microfluidic channel decreases, the surface of the PDMS exposed from the plasma generator decreases, so that the immobilized cells decrease. In addition, when the channel width (W) is 30㎛, it can be confirmed that the cells were fixed in a line.

Example  3

In Example 3, the structure is in the form of c in FIG. 1, the diameter of the microfluidic channel (island structure) is 200 μm, the depth is constant to 30 μm, and the width (W) of the straight channel is 50 μm, 30 μm, 10 It was carried out in the same manner as in Example 1, except that three structures each having a different µm was used.

FIG. 10 is an image obtained by SEM of a structure in which the diameter of the microfluidic channel (island structure) prepared in Example 3 is 200 μm and the width of the straight channel is 50 μm.

FIG. 11 shows the structure of Example 3 after the structure and PDMS are exposed to plasma, and the cells are immobilized and confocal microscope through live and dead staining to confirm the existence of the immobilized cells. ).

11A is a case where the width of a straight channel is 50 μm, b is 30 μm, and c is 10 μm. Referring to FIG. 11, since cells stained in green from viable staining are viable cells and cells stained in red are dead cells, it can be confirmed that all the cells immobilized in Example 3 survive.

In the case of a, b of FIG. 11, it is confirmed that the cells are fixed in the straight channel between the round island structures, but in the case of FIG. 11 c, almost no cells are fixed in the straight channel. That is, the microfluidic channel having a width of approximately 10 μm or more can fix cells on the first substrate, but the microfluidic channel formed with a width of less than 10 μm can only function as a flow path for supplying oxygen.

1 is an example of structures 100 usable in the present invention.

2 shows the step of generating plasma by injecting oxygen into the microfluidic channel 10 after laminating the structure 100 and the first substrate 200.

3 is a schematic diagram showing implantation and washing of cells on the first substrate 200.

FIG. 4 measures cells attached to the surface every 2 hours, 4 hours, 6 hours, 8 hours, 24 hours after seeding 2X10 ⁵ cell/ml AGS cells (adenocarcinoma cell human stomach cancer cells) on the surface of 1 cm X 1 cm. It is a graph.

5 is AGS cell morphology confirmed by SEM after 24 hours cell culture on (a) plasma-treated PDMS surface and (b) (non-plasma-treated) PDMS surface.

6 is a schematic view showing the entire process of Example 1.

7 is an example of a structure in which the microfluidic channel prepared in Example 1 is formed.

8 is a photograph showing the cell pattern fixed to the first substrate (PDMS) according to the shape of the microfluidic channel prepared in Example 1

9 is a photograph of a pattern in which cells are fixed on the PDMS according to Example 2.

FIG. 10 is an image obtained by SEM of a structure in which the diameter of the microfluidic channel (island structure) prepared in Example 3 is 200 μm and the width of the straight channel is 50 μm.

11 is an image obtained by immobilizing cells after exposing the structure and PDMS prepared in Example 3 to plasma.

[Description of the main parts of the drawing]

10: microfluidic channel 20: second substrate
30: electrode layer 40: plasma generator

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100: structure 200: first substrate

Claims (8)

Generating a plasma in the microfluidic channel after laminating a structure on which a microfluidic channel is formed on a first substrate, thereby hydrophilically modifying the pattern portion of the first substrate facing and covering the microfluidic channel; And And a fixing step of separating the first substrate and the structure, washing the cells after seeding the cells on the first substrate, wherein the fixing step includes cells only on the pattern portion of the first substrate. Cell fixing method characterized in that the fixed to form a pattern. The method of claim 1, wherein the microfluidic channel has a width (W) of 10 to 400 μm. The method of claim 1, wherein the microfluidic channel has a depth (H) of 20-50 μm. delete delete The method of claim 1, wherein the first substrate is polydimethylsiloxane (polydimethylsiloxane, PDMS), polymethyl methacrylate (polymethylmethacrylate, PMMA), polyacrylates (polyacrylates), polycarbonates (polycarbonates), polysilicon olefins ( Cell fixation method characterized in that it is selected from the group consisting of polycyclic olefins, polyimides, polyurethanes, and polyesters. The method of claim 1, wherein the structure comprises an electrode layer formed on a second substrate and a microfluidic channel formed on the electrode layer. The method of claim 1, wherein the structure further comprises a layer formed of a material selected from the group of Cr, Ni, Ti, Zr and Pt between the second substrate and the electrode layer.

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