KR20210103010A - Selective isolation of cells from microfluidic chips - Google Patents

Abstract

The present invention provides a method for selectively separating cells from a microfluidic chip. With the method according to the invention, after co-culturing one or more types of cells, only desired cells can be selectively isolated and used for biological analysis. The method comprises the following steps of: i) preparing a microfluidic chip; ii) injecting a hydrogel containing cells into each of the first and second hydrogel channels; iii) co-culturing the cells; iv) injecting a medium and injecting a hydrogel degrading enzyme; v) incubating the microfluidic chip; and vi) extracting the cells.

Description

Method for selective isolation of cells from microfluidic chips

The present invention relates to a method for selectively separating cells from a microfluidic chip.

In the case of cell culture technology using microfluidic chips, it is showing excellent results in academia and industry. As technology advances, more complex cell culture methods have been developed, and a technology of culturing several types of cells in one array instead of one type of cell has been widely used in recent years. Since various types of cells are cultured in the microfluidic chip, the method of analyzing the characteristics of the cells cultured in the microfluidic chip was performed based on imaging or cell culture medium. However, for clear and detailed characterization of cells, analysis of the cells itself is essential, and genomic analysis must be performed by extracting cells. In the previous simple cell culture model, the process of extracting cells was not complicated, but in the recently developed complex cell culture model, a method for isolating cells individually without mixing different types of cells is required.

In the case of a mixture of various cells, a method for separating them is flow cytometry (fluorescence-activated cell sorting, FACS). However, in the case of FACS, antibodies and FACS analyzers are required to distinguish different cells. However, FACS equipment is expensive. In addition, the microfluidic chip is not suitable for using the FACS equipment because a small amount of cells are cultured. Therefore, it is required to develop a method for extracting cells by sequentially separating cells grown in a common space.

In the case of the conventional method, the method of extracting cells from the microfluidic chip again was very limited. For example, there are 1) a method of extracting a cell lysate by adding a buffer that dissolves the entire cell, and 2) a method of separating the bound microfluidic chip again and extracting cells from it. In the case of method 1), since the space in the microfluidic chip has a porous structure such as collagen, the cell lysate is delivered through the porous structure, and when different types of cells are cultured, the cell lysate is mixed with each other. . Therefore, when co-culturing various cells on one microfluidic chip, there is a disadvantage that the cells cannot be distinguished. In the case of method 2), there is a high probability that the separation fails in the process of separating the microfluidic chip again, and the efficiency is very low due to the loss of cells during the separation process.

Accordingly, the present inventors completed the present invention by continuing research on a technology for selectively separating cells from a microfluidic chip.

US 9,121,847

An object of the present invention is to provide a method for selectively separating cells from a microfluidic chip.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a method for selectively separating cells from a microfluidic chip comprising:

i) a first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and preparing a microfluidic chip in which a second hydrogel degrading enzyme channel is arranged in a series order;

ii) injecting a hydrogel containing cells into each of the first and second hydrogel channels;

iii) co-culturing the cells by incubating the cells and the hydrogel-infused microfluidic chip;

iv) injecting a medium of 5° C. or less into the medium channel, and injecting a hydrogel degrading enzyme having a degrading enzyme activation temperature into all of the hydrogel degrading enzyme channels;

v) incubating the microfluidic chip injected with medium and hydrogel degrading enzyme at a hydrogel degrading enzyme activation temperature; and

vi) selectively extracting cells from the first and second hydrogel channels.

In step i), the microfluidic chip

the first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and the second hydrogel lyase channel is respectively connected to the reservoir, and the water level of the reservoir of the first medium channel is the water level of the reservoir of the first and second hydrogel lyase channel so that a water pressure is generated. It can be higher than the height (FIG. 1).

The flow of fluid in the microfluidic chip is caused by a waterhead pressure. At this time, the head difference is generated by the difference in volume of the fluid contained in the reservoir of the first medium channel and the hydrogel degrading enzyme channel. In other words, since the bottom area of the reservoir is the same, the height of the water surface of the reservoir containing the bulky solution is higher.

the first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; And that the second hydrogel degrading enzyme channel is arranged in a series order, one side of the first medium channel is adjacent to one side of the first hydrogel channel, and the other side of the first hydrogel channel is the first hydrogel channel. One side of the gel-degrading enzyme channel is adjacent, the other side of the first hydrogel-degrading enzyme channel is adjacent to one side of the second hydrogel channel, and the other side of the second hydrogel channel is second hydrogel degradation. It means adjacent to one side of the enzyme channel (FIG. 1).

In the ii) step of injecting the hydrogel including cells into each of the first hydrogel channel and the second hydrogel channel, the types of the cells may be the same or different from each other.

The cell may also be a disease (eg, cancer) cell.

Since the hydrogel of the hydrogel channel has a porous structure, material exchange between each channel is possible.

The exchange of substances includes mutual transfer and/or exchange of substances.

The hydrogel is, collagen (Collagen), 　 alginate (Alginate), fibrin (Fibrin), hyaluronic acid (Hyaluronic Acid), agarose (Agarose), PHEMA (Polyhydroxyethylmethacrylate), 　 PVA (Polyvinyl alcohol), 　 PEG (Poly (ethylene glycol) )), PEO(Poly(ethylene oxide)),　PEGDA(Polyethylene (glycol) diacrylate), Gelatin, Matrigel,　PLLA(poly(L-lactic acid)),　Carboxymethylcellulose, It may be selected from the group consisting of dextran, chitosan, and mixtures thereof. It is not necessarily limited thereto, and any hydrogel available in the art may be used.

The hydrogel-degrading enzyme injected into the hydrogel-degrading enzyme may be any hydrogel-degrading enzyme known in the art. For example, there are collagenase (collagenase), MMP, hyaluronidase (hyaluronidase), plasmin (Plasmin) and the like.

In the step of iv) injecting a medium of 5° C. or less into the first medium channel, and injecting a hydrogel degrading enzyme at 30-40° C. into all the enzyme channels, in the medium channel, the medium channel and the first hydrogel A flow occurs in the first medium channel in the direction of the first hydrogel degrading enzyme channel by the waterhead pressure between the degrading enzyme channels, and the hydrogel degrading enzyme in the first hydrogel degrading enzyme channel in the direction of the first hydrogel channel. By preventing diffusion (diffusion), the concentration of the hydrogel degrading enzyme reaching the first hydrogel channel may be lower than the concentration of the hydrogel degrading enzyme reaching the second hydrogel channel.

The step v) incubating the microfluidic chip injected with medium and the hydrogel degrading enzyme at the optimal temperature for culturing the hydrogel enzyme is a step of controlling the hydrogel degrading enzyme reaction of the first hydrogel channel and the second hydrogel channel As a result, the concentration of the hydrogel-degrading enzyme reaching the second hydrogel channel is higher than that of the first hydrogel channel, and the temperature of the second hydrogel channel is higher than that of the first hydrogel channel, so that the second hydrogel channel is higher than the first hydrogel channel. The hydrogel degradation reaction can occur rapidly in the gel channel.

In step iv), the medium injected into the medium channel may be a medium of 5° C. or less, preferably a medium of 0-4° C. or less. And the hydrogel-degrading enzyme activation temperature injected into the hydrogel-degrading enzyme channel may be preferably 30-40 ℃.

In step v), the hydrogel degrading enzyme activation temperature may be 30 to 40 °C. Preferably, it may be 37°C.

In the vi) step of selectively extracting cells from the first and second hydrogel channels, the hydrogel degrading enzyme reaction occurs faster in the second hydrogel channel than in the first hydrogel channel, so that the hydrogel of the second hydrogel channel By first dissolving, cells can be selectively separated from the second hydrogel first.

In the step of preparing the microfluidic chip of step i), the microfluidic chip may include: the first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and a microfluidic chip disposed by further adding a third hydrogel channel and a second medium channel to the second hydrogel degrading enzyme channel in a series order.

the first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and a second hydrogel lyase channel; A third hydrogel channel and a second medium channel are respectively connected to the reservoir. And the water level of the reservoir of the first medium channel and the second medium channel to generate a water pressure difference (water pressure) may be higher than the water level of the lower chamber of the first, second and third hydrogel degrading enzyme channels ( 2).

The positions of the medium channel and the enzyme channel may be interchanged.

In another embodiment, the present invention provides a method for selectively separating cells from a microfluidic chip comprising the steps of:

i) a first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and a second hydrogel lyase channel; a third hydrogel channel; and preparing a microfluidic chip in which a second medium channel is arranged in a series order;

ii) injecting a hydrogel containing cells into each of the first hydrogel channel, the second hydrogel channel, and the third hydrogel channel;

iii) co-culturing the cells by incubating the cells and the hydrogel-infused microfluidic chip;

iv) injecting a medium of 5° C. or less into the medium channel, and injecting a hydrogel degrading enzyme at 30-40° C. into all the enzyme channels;

v) incubating the microfluidic chip into which the medium and the hydrogel degrading enzyme are injected, at the optimum temperature for culturing the hydrogel enzyme; and

vi) selectively extracting cells from the first, second and third hydrogel channels.

In the vi) step of selectively extracting cells from the first, second and third hydrogel channels, cells may be selectively extracted first from the second hydrogel channel.

A detailed description of each step is the same as above, and thus will be omitted.

The present invention relates to a technique for sequentially dissolving a hydrogel matrix in a microfluidic collection.

In the present invention, the term "microfluidic chip" refers to a platform that processes a sample by controlling the flow of fluid in a micrometer-diameter micro tube.

In the present invention, "convection" refers to convection that occurs when a fluid flow is forcibly generated by an external force (water head difference).

In the present invention, "diffusion" refers to a phenomenon in which molecules move and spread by themselves.

A representative example of a hydrogel is an extracellular matrix. Since the porous hydrogel is capable of mass transfer, when different waterhead pressures exist on both sides, flow occurs through the porous hydrogel matrix from a high head to a low location. The flow rate at this time occurs until the head difference becomes 0, and it depends on the flow resistance of the porous hydrogel substrate and the size of the head difference (convection).

In addition, the hydrogel degrading enzyme is present in a dissolved state in the buffer solution. At this time, in order to dissolve the hydrogel substrate smoothly, it must be able to satisfy specific temperature and specific concentration conditions. The hydrogel degrading enzyme moves from a high concentration to a low concentration by diffusion, diffuses through the porous hydrogel matrix, and stops when the concentrations on both sides become the same (diffusion). However, in the case of mass transfer, since the mass transfer rate due to the convection phenomenon caused by the head difference is faster than the diffusion rate, if the convection and diffusion directions are reversed, mass transfer due to diffusion can be prevented to some extent. Therefore, using this technique, the concentration of the enzyme in the hydrogel substrate of a specific channel in the microfluidic chip can be adjusted to be maintained lower than the concentration required for the enzyme reaction (FIG. 2).

As mentioned above, hydrogel degrading enzymes work properly only when a certain temperature is met, and this activation temperature is in the range of 30 to 40 ° C. Therefore, when the temperature of the hydrogel-degrading enzyme located in contact with the hydrogel is low, the enzyme reaction rate is slow.

Heat transfer in a space filled with fluid occurs through two pathways: convective heat transfer, in which heat is transferred by mixing fluids as the fluid moves, and conduction heat transfer, in which heat is transferred using a fluid as a medium. Using the law on the rate of enzyme reaction and the law of heat transfer, heat can be applied quickly or slowly only to a specific channel region, so that the temperature of the enzyme reaction can be controlled. can do it

The present invention designed an experiment so that the concentration of the hydrogel degrading enzyme in the porous hydrogel substrate of a specific channel region is maintained lower than the concentration required for the reaction by using mechanical engineering knowledge, and succeeded in controlling the reaction rate of the hydrogel degrading enzyme did. Through this, we succeeded in selectively extracting cells from a microfluidic channel containing a plurality of porous hydrogel channels (FIG. 3). In an embodiment of the present invention, the enzymatic reaction is designed to occur first in the second hydrogel channel, and the design can be changed so that the enzymatic reaction occurs first in the first and/or third hydrogel using the aforementioned principle. have.

According to the method of the present invention, only desired cells can be selectively isolated by co-culturing a plurality of cells on a microfluidic chip.

1 schematically shows a three-dimensional microfluidic chip according to an embodiment of the present invention.
2 and 3 schematically show a method of separating cells using a microfluidic chip according to an embodiment of the present invention.
4 shows a three-dimensional microfluidic chip manufactured according to an embodiment of the present invention.
5 is a photograph showing the result of separating cells from a microfluidic chip according to an embodiment and a comparative example of the present invention.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Example]

Example 1. Three-dimensional microfluidic chip fabrication

Microfluidic chips are designed to co-culture two or more types of cells. The microfluidic has four units, each of which has two medium channels (a, g in Fig. 2), three hydrogel channels (b, d and f in Fig. 2), and two hydrogelase channels (Fig. 2 c, e) (FIGS. 2 and 4). Each medium channel has two reservoirs for supplying medium, separated by a hydrogel channel. The first media channel, the first hydrogel channel, the first hydrogel lyase channel, the second hydrogel channel, the second hydrogel lyase channel, the third hydrogel channel and the second medium channel were arranged in a series order. .

The microfluidic chip was fabricated using a previously reported method (Y. Shin et al. , "Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels," Nat. Protoc. , vol. 7, no. 7, pp. 1247-1259, 2012.). Briefly, SU-8 photoresists with a thickness of 180-200 μm were first patterned on a silicon wafer using photolithography techniques. A polydimethylsiloxane mixture (PDMS, SYLGARD 184 Silicone Elastomer Kit, Dow Corning) was prepared by mixing a curing agent and a PDMS prepolymer in a weight ratio of 1:10. The PDMS mixture was poured onto the patterned wafer. The PDMS mixture was then degassed in a vacuum chamber for 15 minutes to remove air bubbles. The PDMS mixture was baked in a drying oven at 80° C. for 4 hours. The cured PDMS mold was separated, trimmed and punched with a biopsy punch and blunt needle. The PDMS mold was autoclaved at 120° C. for 30 minutes. The sterilized PDMS mold (top) and micro coverglass (bottom) were bonded after oxygen plasma treatment (FEMTO science). The bonded microchip was dried in an oven at 80° C. for 24 hours to recover hydrophobicity.

Example 2. Cell co-culture

2-1. 2D cell culture

Normal human astrocytes (Astrocyte, Cell systems) are derived from serum, CultureBoost ?? (Cell systems) and Astrocytes Growth Medium BulletKit (AGM, Lonza) were cultured in Complete Classic Medium and preserved according to the manufacturer's standard protocol.

Cancer cells from xenograft mice were isolated from donors by the same method as previously reported (HW Lee et al. , “Patient-derived xenografts from non-small cell lung cancer brain metastases are valuable translational platforms for the development of personalized targeted therapy,” Clin. Cancer Res. , vol. 21, no. 5, pp. 1172-1182, 2015.). The isolated tumor cells were treated with 2% B27 supplement (50X, Gibco), 1% N2 supplement (100X, Gibco), 1% L-glutamine (Gibco), 200ng/ml epidermal growth factor (EGF, R&D systems), 200 ng/ml of basic fibroblast growth factor (bFGF, R&D systems), 30ng/ml of recombinant human neuregulin-1 beta1/heregulin-beta1 (rhNRG-1-b1/HRG-b1(EGF domain), R&D systems), 200ng Neurobasal Media (NBA, Gibco) containing /ml of rhIGF-1 (R&D systems) was cultured in a cell culture flask for suspension culture. Tumor cell spheres were grown to a diameter of <600um, and cancer spheres were collected into culture medium and washed with phosphate buffered saline (PBS, IX, Gibco). The collected cell pellet was dissociated with Accutase (Gibco) to make single cells and neutralized with PBS.

2-2. Co-culture on 3D microfluidic chips

A collagen solution was prepared by mixing rat tail collagen type 1 solution (BD), 10X PBS, distilled water (DW) and sodium hydroxide (NaOH, 0.1N). The final concentration and pH of the collagen gel mixture were adjusted to 2.0 mg/ml and pH 7.0-7.4, respectively. To embed cells in collagen, ^{20 μl of each cell culture solution having a density of 1.0 × 10 7} cells/ml (10 times higher than the final cell density) and 180 μl of a 2.22 mg/ml collagen solution were mixed well.

The collagen solution and collagen-cell mixture were injected into the hydrogel channel and incubated in a _{CO 2} incubator at 37° C. for 30 min at high humidity (~80%) to prevent drying during gelation.

2-3. Cell Extraction from Microfluidic Chips

After co-culturing the cells on the microfluidic chip, in order to isolate the tumor cells from the hydrogel channel, the collagen scaffold filled with the hydrogel channel was degraded by collagenase type D (Roche). The collagenase solution was prepared at 1.0 mg/ml in PBS. (b) 60ul of the collagenase solution preheated to 37°C is filled in the hydrogel-degrading enzyme channel, and 120ul of 4°C PBS is filled in the first and second medium channels to generate water pressure, so that the medium channel A continuous fluid flow was made in the direction of the hydrogel channel (Fig. 2B, Fig. 3A). This flow provides a lower temperature and lower enzyme concentration than conditions under which the enzymatic reaction can occur to the first hydrogel channel (and the third hydrogel channel) to prevent collagenase from degrading collagen. The microfluidic chip was incubated in a CO ₂ incubator at 37° C. for 30 minutes. Lysed collagen gel and cells in the second hydrogel channel were collected with a pipette in a conical tube (Fig. 2C, Fig. 3B). To extract the cell pellet, the mixture of collagen gel and cells was centrifuged at 1000 rpm for 3-5 minutes. After removing the supernatant except for the pellet, it was washed once at 4°C and centrifuged again. DNA and RNA were extracted from the washed cell pellet using TRIzol ^{™ (Fig. 2D).}

comparative example

Cells were isolated by injecting only collagenase at 37° C. into both the medium channel and the hydrogel degrading enzyme channel of the microfluidic chip prepared in Example 1 (control in FIG. 5). As a result, if you look at the control group of FIG. 5, cells are not properly separated from the hydrogel channel and the cells are mixed as compared to the situation before cell extraction. On the other hand, when the method of the present invention is used, as shown in the experimental group of FIG. 5 , it can be seen that the collagenase solution is already extracted together with the cells in the central channel and the peripheral channel, and only bubbles remain in the peripheral channel. That is, it can be seen that cell separation was well performed.

The present invention relates to a method for selectively dissolving the hydrogel and selectively separating cells embedded in the hydrogel. Because hydrogels are porous substrates that allow mass transfer, it is very difficult to process hydrogel-degrading enzymes only on specific parts of the hydrogel. In addition, as the hydrogel dissolution proceeds, the mass transfer rate also increases. Therefore, if the hydrogel degrading enzyme is treated without any special treatment, the dissolving agent is diffused in all the porous hydrogel substrates, so it is impossible to dissolve only a specific part of the hydrogel substrate because the cells included in the hydrogel substrate are mixed.

The present invention is a technology that overcomes the barriers of the prior art, and by controlling the flow and temperature of the fluid, the reaction of the hydrogel degrading enzyme occurs first only in a specific hydrogel channel, thereby selectively separating cells.

By using the cell isolation method of the present invention, a plurality of cells in a disease-related tissue can be simultaneously cultured to selectively isolate and analyze only desired cells.

Claims (12)

i) a first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and preparing a microfluidic chip in which a second hydrogel degrading enzyme channel is arranged in a series order;
ii) injecting a hydrogel containing cells into each of the first and second hydrogel channels;
iii) co-culturing the cells by incubating the cells and the hydrogel-infused microfluidic chip;
iv) injecting a medium of 5° C. or less into the medium channel, and injecting a hydrogel degrading enzyme having a degrading enzyme activation temperature into all of the hydrogel degrading enzyme channels;
v) incubating the microfluidic chip injected with medium and hydrogel degrading enzyme at a hydrogel degrading enzyme activation temperature; and
vi) selectively extracting cells from the first and second hydrogel channels;
A method for selectively separating cells from a microfluidic chip comprising a.
According to claim 1,
In step i), the microfluidic chip
the first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and the second hydrogel lyase channel is respectively connected to the reservoir, and the water level of the reservoir of the first medium channel is the water level of the reservoir of the first and second hydrogel lyase channel so that a waterhead pressure occurs. higher than the height, the method.
According to claim 1,
In step ii),
The cell types are the same or different from each other, the method.
According to claim 1,
The hydrogel is made of a porous structure so that material exchange between each channel is possible, the method.
According to claim 1,
The hydrogel is, collagen (Collagen), alginate (Alginate), fibrin (Fibrin), hyaluronic acid (Hyaluronic Acid), agarose (Agarose), PHEMA (Polyhydroxyethylmethacrylate), PVA (Polyvinyl alcohol), PEG (Poly (ethylene glycol) )), PEO(Poly(ethylene oxide)), PEGDA(Polyethylene (glycol) diacrylate), Gelatin, Matrigel, PLLA(poly(L-lactic acid)), Carboxymethylcellulose, Dextran (Dextran), chitosan (Chitosan) and a method selected from the group consisting of mixtures thereof.
According to claim 1,
In step iv),
In the medium channel, a flow occurs in the direction of the first hydrogel degrading enzyme channel in the first medium channel by the water pressure difference between the medium channel and the first hydrogel degrading enzyme channel. By preventing the diffusion (diffusion) of the hydrogel degrading enzyme from the channel in the direction of the first hydrogel channel, the concentration of the hydrogel degrading enzyme reaching the first hydrogel channel is the hydrogel degrading enzyme reaching the second hydrogel channel is lower than the concentration of the method.
According to claim 1,
Step v) is,
In the step of regulating the hydrogel degrading enzyme reaction of the first hydrogel channel and the second hydrogel channel,
The concentration of the hydrogel-degrading enzyme reaching the second hydrogel channel is higher than that of the first hydrogel channel, and the temperature of the second hydrogel channel is higher than that of the first hydrogel channel, so that the hydrogel degradation reaction occurs faster.
According to claim 1,
In step v), the culture temperature is 30 to 40 ℃, the method.
According to claim 1,
In step vi),
The method of first separating cells from the second hydrogel by dissolving the hydrogel of the second hydrogel channel first because the hydrogel degrading enzyme reaction occurs faster in the second hydrogel channel than the first hydrogel channel.
According to claim 1,
In the step of preparing the microfluidic chip of step i), the microfluidic chip
the first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and a microfluidic chip disposed by further adding a third hydrogel channel and a second medium channel to the second hydrogel degrading enzyme channel in a series order.
i) a first medium channel; a first hydrogel channel; a first hydrogel degrading enzyme channel; a second hydrogel channel; and a second hydrogel lyase channel; a third hydrogel channel; and preparing a microfluidic chip in which a second medium channel is arranged in a series order;
ii) injecting a hydrogel containing cells into each of the first hydrogel channel, the second hydrogel channel, and the third hydrogel channel;
iii) co-culturing the cells by incubating the cells and the hydrogel-infused microfluidic chip;
iv) injecting a medium of 5° C. or less into the medium channel, and injecting a hydrogel degrading enzyme at 30-40° C. into all the enzyme channels;
v) incubating the microfluidic chip into which the medium and the hydrogel degrading enzyme are injected at the optimum temperature for culturing the hydrogel enzyme; and
vi) selectively extracting cells from the first, second and third hydrogel channels;
A method for selectively separating cells from a microfluidic chip comprising a.
12. The method of claim 11,
In step vi), the method of extracting cells from the second hydrogel channel first.

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