KR20110068165A - Method of preparing cell attachment using micro-fluidics channel and plasma - Google Patents

Abstract

PURPOSE: A method for fixing cells using a mcirofluidic channel and plasma is provided to simplify process and to change cell fixation site into hydrophilic property. CONSTITUTION: A method for fixing cells comprises: a step of laminating a structure having a microfluidic channel on a first substrate; a step of generating plasma in the microfluidic channel; and a step of seeding cells on the first sustrate and washing. The width of the microfluidic channel is 10-400 um. The depth of the microfluidic channel is 20-50 um. The first substrate is polydimethylsiloxane(PDMS), polymethylmethacrylate(PMMA), polycyclic olefins, polyimides, polyurethans, or polyester.

Description

Method of preparing cell attachment using Micro-fluidics channel and Plasma

 The present invention relates to a cell fixation method using a microfluidic channel and a plasma. More specifically, the present invention relates to a microfluidic channel and a plasma, in which a plasma is applied to a microfluidic channel to hydrophilize a pattern portion of a first substrate to fix cells therein. It relates to a cell fixation method used.

The ultimate purpose of the cell-related research in vivo (in vitro all the action of the cells occurs in vivo) (in in vitro ). Looking at the in vivo environment from macro-sized organs to micro-sized cells, it can be seen that all the basic functions are performed in molecular units in vivo. Thus, for in vitro cell research, cell regulation in the microenvironment is essential.

Most of the cells constituting 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 directly attaching a cell to a substrate, such as a protein chip or a DNA chip, is also important in the technology of immobilizing the cell on the substrate.

Such cell immobilization technology has been used in the fields of basic biology, biosensor, drug test, tissue engineering, etc., and researches for effectively fixing cells continue. In particular, as the research on the technology to be used industrially for a long time has been progressed, the cell fixation technology has been developed mainly in the technique of self-assembled monolayers (SAMs) using a soft lithography method, such as ECM protein or BSA By coating the surface locally with antibodies, chemicals, etc., the cells could be effectively immobilized on the surface in the desired position. However, the SAMs method has limitations in terms of industrial production due to complicated processes.

In more detail, it is known that there are a number of methods of immobilizing cells on a substrate, and may be classified into immobilization using biomaterials and immobilization using physical and chemical properties of the substrate itself. As a method using biomaterials, a peptide or a protein is first immobilized on a substrate, and then a cell is immobilized using a cell receptor possessed by 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 the method using the physical and chemical properties of the substrate as a method using the hydrophobic properties of the substrate, a method using the electrical properties, a method using the surface structure [Curtis AS, Wilkinson CD. Reactions of cells to topography. J Biomater SciPolym Ed 1998; 9 (12): 1313 -.29], 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 fixation method has a complicated process and a long time-consuming problem, and furthermore, it is difficult to commercialize it immediately due to low productivity.

The present invention aims to provide a cell fixation method which is more economical and highly commercialized since the process can be simplified and the cells can be fixed in a short time.

One aspect of the present invention comprises the steps of: laminating a structure in which a microfluidic channel is formed on a first substrate and generating plasma into the microfluidic channel; And a fixing step of washing and seeding cells on the first substrate from which the structure is separated.

The cell fixation method according to the present invention simplifies the process than the conventional SAMs technology, and changes the cell fixation site to hydrophilicity in a short time using plasma, thereby shortening the cell adhesion time by the hydrophilic functional groups on the surface. Furthermore, the fabrication of the structure using the MEMS process increased the economics and the possibility of commercialization.

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

The cell fixation method according to the present invention includes a plasma generation step and a cell fixation step.

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

plasma  Development stage

The plasma generation step is a step of generating a 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 the second substrate and the microfluidic channel 10 formed on the electrode layer.

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

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

The second substrate 20 may use a silicon wafer, glass, plastics, etc., but is not particularly limited thereto.

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 may 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) and the like can be used. .

The electrode layer 30 may be formed by a 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 the conductive polymer, but is not limited thereto.

In order 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 known steps such as photolithography, etching, thin film deposition, thermal oxidation and cleaning.

In addition, it may be prepared by soft lithography technology. For example, the surface may be modified and patterned using an elastomer mold such as polydimethylsiloxane (PDMS).

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

The photoresist composition may use both a positive photoresist and a negative photoresist as a photosensitive material. In one embodiment, SU-8 (a kind of epoch negative photoresist) with good resolution and biocompatibility may be used.

When the negative photoresist composition is coated on the electrode layer, the mask may be selectively exposed using a patterned mask of a desired microfluidic channel, and the unexposed portion may be etched to form the microfluidic channel.

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

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

2 illustrates a step of laminating the structure 100 and the first substrate 200 and injecting oxygen into the microfluidic channel 10 to generate plasma.

Referring to FIG. 2, the first substrate 200 may be divided into a pattern portion 210 facing and covering the microfluidic channel 10 and an area 211 in contact with the photoresist layer 11. have. When the first substrate 200 and the structure 100 are laminated, the pattern portion 210 is a partial region of the first substrate that corresponds to and covers the microfluidic channel 10 (the negative 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 surface properties are modified by plasma generation to fix cells.

In more detail, when the first substrate 200 and the structure 100 on which the microfluidic channel is formed are laminated to face each other, and oxygen is injected into the microfluidic channel, the oxygen is in contact with the photoresist layer 11 ( Since it is not present in 211 and is present only in the pattern portion 210, when the plasma is applied, only the pattern portion 210 of the first substrate 200 in which oxygen is present may be modified to have hydrophilicity.

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

The microfluidic channel may have a width (W) of 10 to 400 μm, preferably 10 to 100 μm, and if it is less than 10 μm, the area of the pattern portion 210 corresponding to the microfluidic channel may be 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. If the microfluidic channel is smaller than 20 µm, the microfluidic channel may hardly be fixed 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 for transferring oxygen to the microfluidic channel. The division of the microfluidic channel and the flow path part depends on the channel width (W). That is, if the width is less than 10 μm, the flow path may be divided into the microfluidic channel. The channel portion 12 has a small channel width, and thus cannot fix cells in the pattern portion of the first substrate corresponding thereto.

Referring to the structure 100 of FIG. 1c, when the straight channel is formed as the flow path part 12 and the microfluidic channel 10 is formed by the recessed cylinder, the island fixing pattern is formed on the first substrate. It can be formed into an island structure.

The present invention relates to a method for securing a cell to a first substrate in a desired position or shape. To this end, in the present invention, a desired cell fixation pattern is engraved (microfluidic channel) on the structure facing and contacting the first substrate, and plasma is generated therein to locally fix the first substrate facing the structure to fix the cells. As a result, the process is simplified compared to the existing SAMs technology, and the plasma fixation site is changed to hydrophilicity in a short time by using plasma, thereby shortening the cell adhesion time by hydrophilic functional groups on the surface. Furthermore, the fabrication of the structure using the MEMS process increased the economics and the possibility of commercialization.

Cell fixation step

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

3 is a schematic diagram illustrating implanting and washing cells on the first substrate 200. The implantation may be performed preferably within about 3 hours, more preferably over 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 portion 210.

Figure 4 is a graph measuring the cells attached to the surface every 2 hours 4 hours 6 hours 8 hours 24 hours after seeding 2X10 ⁵ cells / ml AGS cells (adenocarcinoma cell human stomach cancer cells) on the surface of 1cm X 1cm . The data shown in blue represents the number of cells attached to the culture dish, and the red one changes the PDMS surface from hydrophobic to hydrophilic by generating oxygen plasma for 2 minutes and 30 seconds using an oxygen plasma generator. The number of cells measured by seeding cells after seeding (plasma treated PDMS). The green data is the number of cells measured after seeding the cells on 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 the cells increases in all three different surfaces. Plasma-treated PDMS showed a large number of cells attached in the fastest time, and 50 times more cells attached in 2 hours than PDMS without surface treatment. In addition, it can be confirmed that about 1.5 times more cells are attached than the culture vessel mainly used for conventional cell culture. After 6 hours, the amount of cells attached to the culture vessel and the plasma-treated PDMS is similar, because there is no space for the cells to attach to the surface. In contrast, PDMS can be seen that less than 2 hours of culture vessel and plasma treated PDMS attached after 24 hours, the hydrophobic PDMS surface is not suitable for cell attachment. In addition, the hydrophilic surface of PDMS can confirm that the cells can be attached in a shorter time than the culture vessel.

FIG. 5 shows AGS cell morphology confirmed using SEM after 24-hour cell culture on (a) plasma-treated PDMS surface and (b) (non-plasma-treated) PDMS surface. AGS cells are epithelial cells that are attached to the surface and cultured. The general form is polyhedral on the surface. The polyhedron forms as the cytoplasm of cells adheres firmly to the surface. Referring to a of FIG. 5, the AGS cell shows the shape of a polyhedron at the hydrophilic surface through plasma treatment, but as shown in b of FIG. 5, the AGS cell shows a round shape at the hydrophobic surface.

4 and 5 it can be seen 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 construed as limiting the protection scope of the present invention.

Example  One

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

The structure in which the microfluidic channel is formed is connected to a generator of a plasma generator (Femto-Sicence, CUTE B). The connected microfluidic channel is adhered to PDMS. After the PDMS adhered to the microfluidic channel is placed on the ground plate, plasma is generated while injecting O 2 (99.9%) at 1000 cc / min. The plasma was maintained for 40 minutes at a frequency of 40 KHz, and the plasma applied voltage was 100W.

After the plasma treatment was completed, the structure was separated and the cells were seeded for 30 minutes to 3 hours on the PDMS surface, and the non-attached cells were washed out.

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

7 is an example of a structure in which a 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. 1A and FIG. 8B is a cell of the first substrate PDMS using the b-type structure of FIG. Referring to a and b of FIG. 8, it can be seen 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 that of Example 1 except that the structure was formed in form a of FIG. 1 and the channel width W was set to 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, or 30 μm. Was carried out.

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

Example  3

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

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

FIG. 11 is a view of immobilized cells after exposing the structure prepared in Example 3 and PDMS to plasma and confocal microscope through live and dead staining to confirm the survival of the immobilized cells. ) Is obtained by using

In FIG. 11A, the width of the straight channel is 50 μm, b is 30 μm, and c is 10 μm. Referring to FIG. 11, the cells stained with green in survival staining are viable cells, and the cells stained with red are dead cells, and thus, all of the cells immobilized in Example 3 are alive.

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

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

2 illustrates a step of laminating the structure 100 and the first substrate 200 and injecting oxygen into the microfluidic channel 10 to generate plasma.

3 is a schematic diagram illustrating implanting and washing cells on the first substrate 200.

Figure 4 measures the cells adhered to the surface every 2 hours 4 hours 6 hours 8 hours 24 hours after seeding 2X10 ⁵ cells / ml AGS cells (adenocarcinoma cell human stomach cancer cells) on the surface of 1cm X 1cm One graph.

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

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

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

8 is a photograph showing a cell pattern fixed on 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 PDMS according to Example 2. FIG.

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

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

[Description of main part of drawing]

10 microfluidic channel 20 second substrate

30 electrode layer 40 plasma generator

100 structure 200 first substrate

Claims (8)

Stacking a structure in which a microfluidic channel is formed on a first substrate and generating a plasma in the microfluidic channel; And a fixing step of seeding and washing the cells on the separated first substrate. 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 to 50 μm. The method of claim 1, wherein the plasma generating step comprises generating a plasma in the microfluidic channel to modify the pattern portion of the first substrate facing and covering the microfluidic channel with hydrophilicity. 5. The method of claim 4, wherein in the fixing step, when the cells are seeded on the first substrate and washed, the cells are fixed only on the pattern portion to form a pattern. The method of claim 1, wherein the first substrate is made of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyacrylates, polycarbonates, polysilicon ( Cell cyclic method, characterized in that selected from the group consisting of polycyclic olefins, polyimides (polyimides), polyurethane (polyurethans) and polyester (polyester). The method of claim 1, wherein the structure comprises an electrode layer formed on the 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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