CN106811414A - One kind visualization Transwell chips and preparation method thereof - Google Patents

Abstract

One kind visualization Transwell chips and preparation method thereof, particular for the micro-fluidic chip with complex passages structure for being loaded with porous membrane.The micro-fluidic chip is mainly made up of the top layer chip by form, porous membrane, bottom chip, and the lower surface of the top layer chip designed with form by irreversible sealing-in using porous membrane, porous membrane connects the upper surface of bottom chip under being bonded by PDMS.The micro-fluidic chip has the function of Transwell cells and micro-fluidic chip simultaneously, and bottom chip can be directly observed by form, can be applied to the biological studies such as drug metabolism, cell invasion.

Description

A visualized Transwell chip and its preparation method

technical field

The invention relates to the field of microfluidic chip preparation, in particular to a visualized Transwell chip and a preparation method thereof.

Background technique

Transwell experimental technology, the traditional method is to put the Transwell chamber into the culture plate, the chamber is called the upper chamber, the culture plate is called the lower chamber, the upper chamber is filled with the upper layer culture solution, the lower chamber is filled with the lower layer culture solution, and the upper and lower layer culture solution is made of polycarbonate separated by ester membranes. We planted the cells in the upper chamber, and because the polycarbonate membrane is permeable, the components in the lower layer of the culture medium can affect the cells in the upper chamber, so that we can study the effects of the components in the lower layer of the culture medium on cell growth and movement. However, due to the limitation of the properties of the intermediate membrane material, the Transwell experiment cannot directly observe the phenomenon of the lower layer, and can only observe it through fluorescence, which limits its application.

Microfluidic chip laboratory, also known as chip laboratory or microfluidic chip, refers to the basic operation units involved in the fields of biology and chemistry, such as sample preparation, reaction, separation, detection, cell culture, sorting, lysis, etc. Integrated or basically integrated on a chip of a few square centimeters (or even smaller), a network of microchannels is formed, and a controllable fluid runs through the entire system to replace various functions of conventional chemical or biological laboratories. As a rapidly developing science and technology, microfluidic chip technology has demonstrated its unique advantages in the field of biomedicine, and because it matches the size of cells, the environment is similar to the physiological environment, and it can provide more in terms of time and space. It is an important platform for the new generation of biomimetic and cell research because of its precise manipulation and easy flexible design to realize the research of various cell functions. However, microfluidic chips are difficult to realize the functions of Transwell experiments, and are greatly limited in the research of drug metabolism and cell invasion.

At present, there is no method to directly observe the phenomenon under the Transwell chamber in real time. It is still in the blank stage to use the visualized Transwell chip for relevant research and analysis. If it can be realized, it will have great application prospects in biological research and pharmaceutical research and development.

Contents of the invention

The purpose of the present invention is to provide a visualized Transwell chip and its preparation method. The prepared microfluidic chip has both the functional advantages of the Transwell chamber and the microfluidic chip, can directly observe the underlying chip, and can be applied to drug metabolism and cell invasion and other biological research.

The invention provides a visualized Transwell chip. The microfluidic chip is mainly composed of a top chip with a window, a porous filter membrane and a bottom chip.

The top-layer chip is designed with a window, and the position of the window can be in the area of the porous filter membrane or outside the area of the porous filter membrane.

The underlying chip can be observed in real time through the window.

A method for preparing a visualized Transwell chip is carried out according to the following steps: the porous filter membrane is irreversibly sealed to the lower surface of the top chip, and the porous filter membrane is bonded to the upper surface of the bottom chip through PDMS.

The chip material is light-transmitting and air-permeable PDMS polymer, the ratio of PDMS monomer to initiator is 5:1, and the porous filter membrane material is polycarbonate membrane, and the pore size of the polycarbonate membrane is 0.01um-10um.

The lower surface of the top chip of the microfluidic chip is irreversibly sealed to the porous filter membrane, and the upper surface of the bottom chip is bonded to the porous filter membrane by PDMS.

The irreversible sealing method is UV activation for 1 hour, silanization treatment for 30 minutes, and oxygen plasma sealing.

The PDMS bonding method is to use a PDMS polymer with a monomer-to-initiator ratio of 20:1, throw it on a glass sheet with a thickness of 10um-50um, dip the upper surface of the chip into PDMS, and irreversibly seal the porous surface with the upper chip. The filter membrane is aligned and sealed, put into an 80-degree oven, and heated for 30 minutes.

When the window position is in the porous filter membrane, the window is a columnar structure, which is slightly smaller than the window area. The porous filter membrane at the window position is removed, and PDMS is used to bond the window column and the porous filter membrane.

When the position of the window is outside the area of the porous filter membrane, the channel structure reagent of the lower layer chip should be extended to the outside of the area of the porous filter membrane to coincide with the position of the window.

The Transwell chip based on microfluidic technology provided by the present invention can inoculate different cells on different layers of the chip according to the experimental design.

The microfluidic chip of the present invention has the functions of a Transwell chamber and a microfluidic chip at the same time, the underlying chip can be directly observed through the window, and can be applied to biological researches such as drug metabolism and cell invasion.

Description of drawings

Fig. 1 is a schematic diagram of the microfluidic chip a of the present invention, the left side is a physical diagram, and the right side is a schematic diagram of a cross-sectional structure;

Fig. 2 is a schematic diagram of the microfluidic chip b of the present invention, the left side is a physical diagram, and the right side is a schematic diagram of a cross-sectional structure;

1 top chip, 2 porous membrane, 3 bottom chip, 4 observation window, 5 observation area, 6 bottom chip channel, 7 top chip channel. ,

Figure 3 is the cell observation diagram of the microfluidic chip of the present invention under a microscope, (a) the bright field photo of the observation area, (b) the bright field photo of the cells in the bottom chip through the observation area.

detailed description

The following examples will further illustrate the present invention, but do not limit the present invention thereby.

Example 1

Visual Chip Fabrication

Since the top chip 1 and the bottom chip 3 are transparent PDMS chips, and the porous filter membrane 2 is opaque, the porous filter membrane 2 is punched at a predetermined position as the observation window 4, so that the porous filter membrane 2 can be observed Window 4 is used to observe the state of cells in channel 6 of the bottom chip. Put the porous filter membrane 2 on a glass plate for UV activation for 1 hour, then silanize it for 30 minutes, and then heat the porous filter membrane 2 and the top chip 1 together with the irreversible oxygen plasma for 30 minute. Use the PDMS polymer with a monomer-to-initiator ratio of 20:1, throw a 10um-50um thick film on a glass plate, treat the upper surface of the bottom chip 3 with oxygen plasma, dip the thin PDMS, and seal it with The porous filter membrane 2 of the top chip 1 is aligned and bonded, heated in an oven at 80 degrees, and cured completely in 30 minutes. After taking the sealed chip out of the oven, cut it into the required size, as shown in Figure 2.

Example 2

Visual Chip Fabrication

Since the top chip 1 and the bottom chip 3 are transparent PDMS chips, and the porous filter membrane 2 is opaque, in order to observe the cell state in the bottom chip channel 6, the porous filter membrane 2 is cut off at a predetermined position as the observation area 5 . After cutting the porous filter membrane 2 to a suitable size, put it on a glass sheet for UV activation for 1 hour, and then silanize it for 30 minutes, perform oxygen plasma sealing with the top chip 1, and place it in an oven at 80 degrees for 30 minutes. Use PDMS polymer with a ratio of monomer to initiator of 20:1, throw a 10um-50um thick film on a glass plate, treat the upper surface of the bottom chip 3 with oxygen plasma, dip the thin PDMS, and seal it with The porous filter membrane 2 of the top chip 1 is aligned and bonded, heated in an oven at 80 degrees, and cured completely in 30 minutes. After the sealed chip is taken out of the oven, it is cut into the required size. As shown in Figure 1.

Example 3

Visual chip application

Put the chip in a petri dish and sterilize it by ultraviolet light for 2 hours. Use a pipette gun to add cell culture medium from the inlets of the bottom chip channel 6 and the top chip channel 7, so that the culture medium fills the entire bottom chip channel 6 and top chip channel 7. Digest the HePG2 cells in the culture flask with trypsin, stop the digestion after the cells fall off the wall, suck the cell suspension in the culture flask into a centrifuge tube for centrifugation, suck the supernatant and throw it away, add Resuspend the cells with a new medium, draw the cell suspension with a pipette gun, add it from the inlet of channel 6 of the bottom chip, suck out the corresponding volume of medium from the outlet of the liquid, and replace it. After repeating three times, the bottom chip can be regarded as Channel 6 has been filled with cell suspension, and medium is added to the culture dish to reduce the volatilization of the medium in the chip. Place the dish in a 37°C incubator to keep still to allow the cells to attach. After 12 hours, the petri dish was taken out, and the state of the cells was observed through the observation area 5 under a microscope, and photographed, as shown in FIG. 3 .

Claims (9)

1. A visual Transwell chip, characterized in that: the microfluidic chip is mainly composed of a top chip with an observation window, a porous filter membrane, and a bottom chip. 2. A visual Transwell chip according to claim 1, characterized in that: the top chip is designed with an observation window, and the location of the observation window can be in the porous membrane area or outside the porous membrane area. 3. A visualized Transwell chip according to claim 1, characterized in that: the underlying chip can be observed in real time through the window. 4. according to the preparation method of a kind of visualized Transwell chip described in any claim in claim 1～3, it is characterized in that, carry out according to the following steps: porous filter membrane is irreversibly sealed with the lower surface of the top layer chip of window design, The porous filter membrane is attached to the upper surface of the underlying chip by PDMS bonding. 5. according to the preparation method of a kind of visualized Transwell chip described in claim 4, it is characterized in that described chip material is the PDMS polymer that can transmit light and air, the ratio of PDMS monomer and initiator is 5:1, porous filter membrane The material is polycarbonate membrane, and the pore size of the polycarbonate membrane is 0.01um-10um. 6. The method for preparing a visualized Transwell chip according to claim 4, wherein the irreversible sealing method is UV activation for 1 hour, silanization treatment for 30 minutes, and oxygen plasma sealing. 7. according to the preparation method of a kind of visual Transwell chip described in claim 4, it is characterized in that described PDMS bonding method is to use the PDMS polymer that monomer and initiator ratio are 20:1, throw 10um- 50um thick, after the upper surface of the chip is dipped in PDMS, align and seal with the porous filter membrane that has been irreversibly sealed with the upper chip, put it in an 80-degree oven, and heat for 30 minutes. 8. according to the preparation method of a kind of visualized Transwell chip as claimed in claim 4, it is characterized in that: when the window position is in the porous filter membrane area, the window is a columnar structure, and the porous filter membrane at the window position is slightly smaller than the window area , using PDMS to bond the window post to the porous filter membrane. 9. The preparation method of a visualized Transwell chip according to claim 4, characterized in that: when the window position is outside the porous filter membrane, the channel structure of the lower layer chip is designed to extend outside the porous filter membrane area to coincide with the window position.