LU505383B1 - Microfluidic chip for screening migration of mesenchymal stem cell (msc) to tumor, and preparation method and use method thereof - Google Patents
Microfluidic chip for screening migration of mesenchymal stem cell (msc) to tumor, and preparation method and use method thereof Download PDFInfo
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- LU505383B1 LU505383B1 LU505383A LU505383A LU505383B1 LU 505383 B1 LU505383 B1 LU 505383B1 LU 505383 A LU505383 A LU 505383A LU 505383 A LU505383 A LU 505383A LU 505383 B1 LU505383 B1 LU 505383B1
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Abstract
The present disclosure discloses a microfluidic chip for screening a migration of a mesenchymal stem cell (MSC) to a tumor, and a preparation method and use method thereof. The microfluidic chip includes: a chip body with microstructural channels provided on a surface of the chip body, and a substrate tightly attached to the chip body, where the microstructural channels on the chip body each include a central cell cultivation channel and a plurality of independent cell migration units symmetrically arranged at two sides of the central cell cultivation channel; the plurality of independent cell migration units each include an inner matrigel perfusion channel and an outer migrated cell cultivation channel; and an interception structure is provided between the central cell cultivation channel and a matrigel perfusion channel of each cell migration unit, and between a matrigel perfusion channel and a migrated cell cultivation channel of a same cell migration unit.
Description
DESCRIPTION LU505383
MICROFLUIDIC CHIP FOR SCREENING MIGRATION OF MESENCHYMAL STEM
CELL (MSC) TO TUMOR, AND PREPARATION METHOD AND USE METHOD
THEREOF
The present disclosure belongs to the field of biotechnologies, and relates to a monitoring device for tumor chemotaxis of a mesenchymal stem cell (MSC). In particular, the present disclosure relates to a microfluidic chip for screening a migration of an MSC to a tumor, and a preparation method and a use method thereof.
Malignant tumors are a worldwide problem seriously threating the health of all mankind currently, and according to data reported by the American Cancer Society, there were nearly 19.3 million of newly-diagnosed tumor patients and 10 million of tumor deaths globally in 2020. Chemotherapeutic drugs such as platinum have been widely used in tumor treatments, but are easy to cause systemic toxicity and drug resistance.
Therefore, it is of great significance to establish an accurate targeted drug delivery platform to allow an accurate tumor treatment.
MSCs are a class of cells with a self-renewing function and a multi-directional differentiation potential. MSCs have characteristics such as widely-available tissue sources, easy proliferation, and low immunogenicity, and studies have proved that MSCs have unique migration chemotaxis for malignant tumor tissues, indicating that MSCs are a desired carrier for targeted drug delivery. However, although MSCs derived from different tissues have similar morphologies and immunophenotypes, MSCs are different from each other in biological behaviors such as proliferation, cytokine secretion, and cell migration activity. Therefore, it is particularly important to compare and find MSCs with the strongest tumor migration chemotaxis.
Models for evaluating cell migration properties are divided into in vivo models and (n,505383 vitro models. Compared with the in vivo models, the in vitro cell migration models have a low cost and a simple operation, can avoid the ethical problems involved in animal experiments, and can also avoid the phenomenon of inconsistent experimental results caused by species-specific differences between humans and experimental animals. The traditional in vitro models mainly rely on a scratch experiment and a transwell experiment.
The scratch experiment has poor experimental repeatability and is easy to damage cells, and the transwell experiment involves complicated operations and is not suitable for real-time dynamic monitoring.
The microfluidic chip technology has developed rapidly in the field of cell manipulation and analysis due to its advantages such as flexible structural design and large-scale integration. A microfluidic chip involves a micro-scale tube size, a small cell consumption, and a small reagent consumption, can well simulate a microenvironment in vivo, does not damage cells, and is suitable for dynamic observation. However, most of the current microfluidic chips for monitoring cell migration are single-channel chips with shortcomings such as low throughput, poor integration level, and poor experimental repeatability. In addition, the research and analysis on monitoring and screening of tumor cell migration chemotaxis of a plurality of MSCs on a same microfluidic chip is still in a blank stage.
In view of the shortcomings of the prior art, the present disclosure provides a microfluidic chip for screening a migration of an MSC to a tumor, and a preparation method and use thereof. The microfluidic chip allows the synchronous co-cultivation of various types of MSCs with a tumor cell, involves simple operations, reduces the consumption of actual samples, and can simulate the chemotaxis of human MSCs for tumor cells and allow synchronous comparison of differences, which can provide a theoretical basis for screening of an efficient drug carrier for targeted tumor therapy.
In order to achieve the above objective, the present disclosure provides a microfluidic chip for screening a migration of an MSC to a tumor, including: a chip body with microstructural channels provided on a surface of the chip body, and a substrate tightly attached to the chip body, where the microstructural channels on the chip body include a central cell cultivation channel and a plurality of independent cell migration units symmetrically arranged at two sides of the central cell cultivation channel;
the plurality of independent cell migration units each include an inner matrigel perfusign,505383 channel and an outer migrated cell cultivation channel, a sample inlet and a sample outlet are formed at two ends of each of the central cell cultivation channel, the matrigel perfusion channel, and the migrated cell cultivation channel, respectively; the central cell cultivation channel is configured to cultivate a tumor cell; the matrigel perfusion channel is configured to fill a matrigel; a plurality of migrated cell cultivation channels are configured to cultivate MSCs derived from different tissues; an interception structure is provided between the central cell cultivation channel and a matrigel perfusion channel of each cell migration unit, and between a matrigel perfusion channel and a migrated cell cultivation channel of a same cell migration unit; the interception structure includes a plurality of columnar structures arranged at a specified interval, and a signal transmission and interaction between cells and a cell migration are conducted through the interception structure between the central cell cultivation channel and a matrigel perfusion channel of each cell migration unit, and between a matrigel perfusion channel and a migrated cell cultivation channel of a same cell migration unit.
Further, in the microfluidic chip for screening the migration of the MSC to the tumor provided by the present disclosure, in each of the plurality of cell migration units, two ends of each of the matrigel perfusion channel and the migrated cell cultivation channel both are bent outwards.
Further, in the microfluidic chip for screening the migration of the MSC to the tumor provided by the present disclosure, a material of the chip body is selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), and polycarbonate (PC); and a material of the substrate is a glass or a silicon wafer.
Further, in the microfluidic chip for screening the migration of the MSC to the tumor provided by the present disclosure, the chip body is tightly attached to the substrate through a plasma bonding process.
Further, in the microfluidic chip for screening the migration of the MSC to the tumor provided by the present disclosure, the central cell cultivation channel and the migrated cell cultivation channel each are coated with a medium matrix before being used for cultivating a tumor cell and an MSC, and the medium matrix is one or more selected from the group consisting of gelatin, chitosan, silk fibroin (SF), rat tail collagen, fibrin glue (FG), and matrigel.
Further, in the microfluidic chip for screening the migration of the MSC to the tum@{,505383 provided by the present disclosure, the central cell cultivation channel and each matrigel perfusion channel and migrated cell cultivation channel of the chip body are located in a same plane, and have a same width and height of 1,000 um and 100 um, respectively; the matrigel perfusion channel has a length of 1.0 cm, and the migrated cell cultivation channel has a length of 0.8 cm; a length of the central cell cultivation channel depends on a number of cell migration units; the interception structure includes five hexagonal prism structures arranged at equal intervals; and each hexagonal prism structure has a side length of 100 um, and an interval between two adjacent hexagonal prism structures is 50 um.
The present disclosure also provides a preparation method of the microfluidic chip for screening the migration of the MSC to the tumor, including the following steps: step 1: designing and drawing a pattern of the microstructural channels on the chip body with computer-aided software; step 2: preparing the chip body with the microstructural channels by a micromachining technology, and forming a sample inlet and a sample outlet for each of the central cell cultivation channel and each matrigel perfusion channel and migrated cell cultivation channel by a manual puncher; and step 3: cleaning the chip body and the substrate with absolute ethanol, autoclaving the chip body and the substrate, and finally plasma-bonding the chip body and the substrate to obtain the microfluidic chip.
The present disclosure also provides a use method of the microfluidic chip for screening the migration of the MSC to the tumor, including the following steps: step 1: pretreatment of the microfluidic chips: taking three microfluidic chips as an experimental microfluidic chip, a normal cell control microfluidic chip, and a blank control microfluidic chip; coating of a medium matrix: for each microfluidic chip, filling a medium matrix solution into the central cell cultivation channel and each migrated cell cultivation channel, and incubating the microfluidic chip in a 37°C incubator for 1 h, such that surfaces of the central cell cultivation channel and each migrated cell cultivation channel each are coated with the medium matrix; and restoration of hydrophobicity: for each microfluidic chip, rinsing the central cell cultivation channel and each migrated cell cultivation channel with sterile water to remove the excess medium matrix solution, and placing the microfluidic chip in an oven at 80°C for 1 h to 2 h, such that the microfluidic chip is dried and hydrophobicity of the microfluidic chip is restored;
step 2: fluorescent labeling of cells: taking a tumor cell line with an excellent growth,505383 status, a normal cell line derived from the same organ as the tumor cell line, and MSCs derived from different tissues, and labeling each of the tumor cell line, the normal cell line derived from the same organ as the tumor cell line, and the MSCs with a live cell tracer, where a live cell tracer to label the tumor cell line and the normal cell line derived from the same organ as the tumor cell line has a different color from a live cell tracer to label the MSCs; step 3: inoculation and cultivation of the cells: for each microfluidic chip, injecting a matrigel into each matrigel perfusion channel, and incubating the microfluidic chip in a 37°C incubator for 30 min to promote curing of the matrigel; adding a medium to each of the tumor cell line and the normal cell line derived from the same organ as the tumor cell line that each are labeled with a live cell tracer to prepare cell suspensions, and injecting the cell suspensions into central cell cultivation channels of the experimental microfluidic chip and the normal cell control microfluidic chip, respectively; injecting a medium into a central cell cultivation channel of the blank control microfluidic chip; adding a medium to each of the MSCs labeled with a live cell tracer to prepare cell suspensions, and injecting the cell suspensions into migrated cell cultivation channels of different cell migration units of each microfluidic chip, respectively, where the three microfluidic chips are inoculated identically; and incubating the three microfluidic chips in an incubator; and step 4: observation and monitoring of cell migration: after the MSCs are attached to a wall, taking the microfluidic chips out, and observing and recording migration conditions of different MSCs in respective matrigel perfusion channels under an inverted fluorescence microscope, which is denoted as 0 h; after the MSCs are further cultivated for n h, taking the microfluidic chips out, and observing and recording migration conditions of different MSCs in respective matrigel perfusion channels under an inverted fluorescence microscope, which is denoted as n h; and measuring and comparing migration areas and maximum migration distances of chemotactic migrations of different
MSCs to the tumor cell line, the normal cell line derived from the same organ as the tumor cell line, and the medium within n h.
Further, in the use method of the microfluidic chip for screening the migration of th&,505383
MSC to the tumor provided by the present disclosure, before the medium matrix is coated, the microfluidic chips each are sterilized; and a method for the sterilization is as follows: rinsing a central cell cultivation channel and each matrigel perfusion channel and migrated cell cultivation channel of each microfluidic chip with 75% (volume fraction) alcohol, sterilizing the microfluidic chip under ultraviolet (UV) irradiation overnight, and after the 75% alcohol is completely volatilized, coating the medium matrix.
Further, in the use method of the microfluidic chip for screening the migration of the
MSC to the tumor provided by the present disclosure, in step 3, the cell suspensions of the tumor cell line and the normal cell line derived from the same organ as the tumor cell line injected into the central cell cultivation channels each have a cell density of 2 x 108/mL; and the cell suspensions of the MSCs injected into the migrated cell cultivation channels each have a cell density of 1 x 108/mL.
The present disclosure has the following beneficial effects: According to specific tumor chemotaxis of MSCs and based on the microfluidic chip technology, the present disclosure provides a microfluidic chip for screening a migration of an MSC to a tumor, and a preparation method and use method thereof. The microfluidic chip allows the synchronous co-cultivation of various types of MSCs with a tumor cell, and can allow the simultaneous comparison of chemotaxis among different types of MSCs. Specifically: 1. The microfluidic chip of the present disclosure includes a plurality of channels with a micro-scale size, involves very small cell and reagent consumption, and can reduce the cost and cell damage. 2. The microfluidic chip of the present disclosure includes a central cell cultivation channel configured to cultivate a tumor cell and a plurality of independent cell migration units with a high integration level that are symmetrically arranged at two sides of the central cell cultivation channel, which can simultaneously allow the observation and monitoring of migration characteristics of MSCs from various tissues. Compared with the scratch experiment and the transwell experiment, the microfluidic chip of the present disclosure can reduce a cost and improve the experimental efficiency and experimental repeatability. 3. Each cell migration unit of the microfluidic chip of the present disclosure includes a matrigel perfusion channel, which can allow observation of migration abilities of different MSCs at a three-dimensional (3D) level. 4. The microfluidic chip of the present disclosure allows the synchronous co-cultivation of various types of MSCs with a tumor cell, involves simple operations,
reduces the consumption of actual samples, and can simulate the chemotaxis of hUman,595383
MSCs for tumor cells and allow synchronous comparison of differences, which can provide a theoretical basis for screening of an efficient drug carrier for targeted tumor therapy.
FIG. 1 is a schematic structural diagram of the microfluidic chip of the present disclosure;
FIG. 2 is an enlarged view of a zone Ain FIG. 1;
FIG. 3 shows test results of biocompatibility of the microfluidic chip of the present disclosure; and
FIG. 4 shows observation and comparison results of chemotaxis of MSCs derived from different tissues for a cervical cancer cell on the microfluidic chip.
In order to make a person skilled in the art well understand the technical solutions of the present disclosure, the technical solutions of the present disclosure are further described below with reference to specific embodiments.
Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; and unless otherwise specified, the reagents, methods, and devices used are conventional reagents, methods, and devices in the prior art.
The present disclosure provides a microfluidic chip for screening a migration of an
MSC to a tumor, including a chip body with microstructural channels provided on a surface of the chip body, and a substrate tightly attached to the chip body, where the microstructural channels are formed between the chip body and the substrate.
As shown in FIG. 1, the microstructural channels on the chip body include central cell cultivation channel 1 and four independent cell migration units 2 symmetrically arranged at two sides of the central cell cultivation channel 1; and the plurality of independent cell migration units 2 each include inner matrigel perfusion channel 21 and outer migrated cell cultivation channel 22. The "inner" and "outer" refer to directions relatively close to and away from the central cell cultivation channel 1, respectively.
A plurality of cell migration units 2 may be arranged symmetrically at two sides of th&,505383 central cell cultivation channel 1.
A sample inlet and a sample outlet along a thickness direction of the chip body are formed at two ends of each of the central cell cultivation channel 1, the matrigel perfusion channel 21, and the migrated cell cultivation channel 22, respectively;
The central cell cultivation channel 1 is configured to cultivate a tumor cell; the matrigel perfusion channel 21 is configured to fill a matrigel; and a plurality of migrated cell cultivation channels 22 are configured to cultivate MSCs derived from different tissues.
Interception structure 3 is provided between the central cell cultivation channel 1 and matrigel perfusion channel 21 of each cell migration unit 2, and between matrigel perfusion channel 21 and migrated cell cultivation channel 22 of a same cell migration unit 2; the interception structure 3 includes a plurality of columnar structures 31 arranged at a specified interval; and a signal transmission and interaction between cells and a cell migration are conducted through the interception structure 3 between the central cell cultivation channel 1 and matrigel perfusion channel 21 of each cell migration unit 2, and between matrigel perfusion channel 21 and migrated cell cultivation channel 22 of a same cell migration unit 2.
In a preferred embodiment, two ends of each of the matrigel perfusion channel 21 and the migrated cell cultivation channel 22 in each cell migration unit 2 both are bent outwards, and a bending structure may increase a distance between a sample inlet and a sample outlet of each channel, which is convenient for processing of a sample inlet and a sample outlet of each channel on the one hand, and is also convenient for inoculation of cells or perfusion of a matrigel in each channel on the other hand.
A material of the chip body may be selected from the group consisting of PDMS,
PMMA, and PC; and a material of the substrate may be a glass or a silicon wafer. The chip body is tightly attached to the substrate through a plasma bonding process.
The central cell cultivation channel 1 and the migrated cell cultivation channel 22 each are coated with a medium matrix before being used for cultivating a tumor cell and an MSC, and the medium matrix is one or more selected from the group consisting of gelatin, chitosan, SF, rat tail collagen, FG, and matrigel.
The central cell cultivation channel 1 and each matrigel perfusion channel 21 and,505383 migrated cell cultivation channel 22 of the chip body are located in a same plane, and have a same width and height of 1,000 um and 100 um, respectively; the matrigel perfusion channel 21 has a length of 1.0 cm, and the migrated cell cultivation channel 22 has a length of 0.8 cm; a length of the central cell cultivation channel 1 depends on a number of cell migration units 2. In this embodiment, two cell migration units 2 are arranged in a length direction of the central cell cultivation channel 1, and the central cell cultivation channel 1 has a length of 2.6 cm. Preferably, the interception structure 3 includes five hexagonal prism structures arranged at equal intervals; and each hexagonal prism structure has a side length of 100 um, and an interval between two adjacent hexagonal prism structures is 50 um.
The present disclosure also provides a preparation method of the microfluidic chip for screening the migration of the MSC to the tumor, including the following steps: step 1: a pattern of the microstructural channels on the chip body is designed and drawn with computer-aided software (CAD); step 2: the chip body with the microstructural channels is prepared by a micromachining technology such as lithography, soft-lithography, molding, or the like, and a sample inlet and a sample outlet for each of the central cell cultivation channel 1 and each matrigel perfusion channel 21 and migrated cell cultivation channel 22 are formed by a manual puncher; and step 3: the chip body and the substrate are cleaned with absolute ethanol, autoclaved, and finally plasma-bonded to obtain the microfluidic chip.
A biocompatibility test of the microfluidic chip for screening the migration of the MSC to the tumor provided in the present disclosure: The biocompatibility of each of three microfluidic chips with each of a cervical cancer cell (SiHa cell), a normal cervical epithelial cell (ECT1/E6E7 cell), and an adipose tissue-derived MSC (AT-MSC) is tested.
The three microfluidic chips each are sterilized, coated with a medium matrix, and subjected to hydrophobicity restoration. The SiHa cell and the ECT1/E6E7 cell each are then labeled with a red-fluorescence live cell tracer (CellTracker™ Red CMTPX), and the
MSC is labeled with a green-fluorescence live cell tracer (CellTracker™ Green CMFDA).
The SiHa cell and the ECT1/E6E7 cell that each are labeled with CellTracker™ Red,505383
CMTPX each are collected through trypsin digestion and then centrifugation, and then a medium (DMEM medium, the same below) is added to prepare cell suspensions each with a density of 2 x 108/mL; and the prepared cell suspensions of the SiHa cell and the
ECT1/E6E7 cell each are pipetted by a pipette and added to central cell cultivation channels of two microfluidic chips, where an addition speed is controlled such that a cell suspension is slowly injected into the central cell cultivation channel. The AT-MSC labeled with CellTracker™ Green CMFDA is collected through trypsin digestion and then centrifugation, and a medium is added to prepare a cell suspension with a density of 1 x 108/mL; and the prepared cell suspension of the AT-MSC is pipetted by a pipette and added to a migrated cell cultivation channel of another microfluidic chip, where an addition speed is controlled such that the cell suspension is slowly injected into the migrated cell cultivation channel. The three microfluidic chips are placed in Petri dishes, and the Petri dishes each are filled with phosphate buffered saline (PBS) to surround the chips and then incubated in a 37°C incubator. After cells are attached to a wall, at 0 h, 24 h, and 48 h of incubation, the chips are taken out and observed under an inverted fluorescence microscope to record growth conditions of the SiHa cell, ECT1/E6E7 cell, and AT-MSC, and results show that the SiHa cell, ECT1/E6E7 cell, and AT-MSC grow well on the microfluidic chips. The results in FIG. 3 show that the microfluidic chips have excellent biocompatibility. In other embodiments, the detection of biocompatibility of the microfluidic chip may also be conducted by a live cell/dead cell in situ double-staining method such as DAPI/PI and Calcein-AM/PI.
The present disclosure also provides a use method of the microfluidic chip for screening the migration of the MSC to the tumor, including the following steps:
Step 1: Pretreatment of microfluidic chips:
Three microfluidic chips are taken as an experimental microfluidic chip, a normal cell control microfluidic chip, and a blank control microfluidic chip.
Sterilization: A central cell cultivation channel and each matrigel perfusion channel and migrated cell cultivation channel of each microfluidic chip are rinsed with 75% (volume fraction) alcohol, then the microfluidic chip is sterilized under UV irradiation overnight, and after the 75% alcohol is completely volatilized, the medium matrix is coated.
Coating of a medium matrix: For each microfluidic chip, a diluted rat tail collagen,595383 type | solution is filled into the central cell cultivation channel and each migrated cell cultivation channel, and the microfluidic chip is incubated in a 37°C and 5 viv% CO2 incubator for 1 h, such that surfaces of the central cell cultivation channel and each migrated cell cultivation channel each are coated with rat tail collagen type | to promote the cell attachment.
Restoration of hydrophobicity: For each microfluidic chip, the central cell cultivation channel and each migrated cell cultivation channel are rinsed with sterile water to remove the excess rat tail collagen type | solution, and the microfluidic chip is placed in an oven at 80°C for 1 h to 2 h, such that the microfluidic chip is dried and hydrophobicity of PDMS is restored.
Step 2: Fluorescent labeling of a cervical cancer cell (SiHa cell), a normal cervical epithelial cell (ECT1/E6E7 cell), and MSCs derived from different tissues:
Before fluorescent labeling, a green-fluorescence live cell tracer (CellTracker™
Green CMFDA) and a red-fluorescence live cell tracer (CellTracker™ Red CMTPX) each are prepared with dimethyl sulfoxide (DMSO) into a 10 mM stock solution, and then a medium is added to the stock solution to prepare a live cell tracer working solution with a final concentration of 5 uM. The SiHa cell, ECT1/E6E7 cell, and MSCs derived from different tissues in a logarithmic growth phase and an excellent growth status each are taken, culture supernatants are discarded, and then the cells each are washed twice with
PBS. 6 mL of a CellTracker™ Red CMTPX working solution is slowly added by a pipette to each of Petri dishes respectively with the SiHa cell and ECT1/E6E7 cell, and 6 mL of a
CellTracker™ Green CMFDA working solution is slowly added by a pipette to Petri dishes respectively with the MSCs derived from different tissues; and the Petri dishes are incubated in a 37°C and 5% CO» incubator for 1 h. After the incubation is completed, the Petri dishes are taken out and observed under an inverted fluorescence microscope to determine fluorescent labeling conditions, and results show that the SiHa cell,
ECT1/E6E7 cell, and MSCs derived from different tissues are successfully labeled by the fluorescent tracers. Then, the tracer working solutions are discarded, the cells are washed with PBS once, then a corresponding medium is added, and the cells are further cultivated in a 37°C and 5% CO: incubator.
Step 3: Inoculation and cultivation of cells: For each microfluidic chip, 8505383 matrigel/medium (in a volume ratio of 1:1) dilution pre-cooled on ice is slowly injected by a pipette through a sample inlet of each matrigel perfusion channel, and the microfluidic chip is placed in a petri dish and incubated in a 37°C and 5% CO» incubator for 30 min to promote curing of the matrigel.
The SiHa cell and ECT1/E6E7 cell that each are labeled with CellTracker™ Red
CMTPX each are collected through trypsin digestion and then centrifugation, and a medium is added to prepare cell suspensions each with a density of 2 x 10%/mL. Each microfluidic chip is taken out, and a suspension of the SiHa cell, a suspension of the
ECT1/E6E7 cell, and a cell-free DMEM are added by a pipette to central cell cultivation channels of the experimental microfluidic chip, the normal cell control microfluidic chip, and the blank control microfluidic chip, respectively, where an addition speed is controlled such that the suspension of the SiHa cell, the suspension of the ECT1/E6E7 cell, and the cell-free DMEM each slowly flow into a corresponding central cell cultivation channel. Each microfluidic chip is placed in a corresponding Petri dish, and the Petri dish is filled with PBS to surround the chip and then incubated in a 37°C incubator for 6 h to 8 h. After the SiHa cell and ECT1/E6E7 cell are attached to a wall, MSCs derived from different tissues that each are labeled with CellTracker™ Green CMFDA each are collected through trypsin digestion and then centrifugation, and a medium is added to prepare cell suspensions each with a density of 1 x 108/mL. Each microfluidic chip is taken out, and the suspensions of the MSCs derived from different tissues are added by a pipette to migrated cell cultivation channels of different cell migration units of each microfluidic chip, respectively, where an addition speed is controlled such that the cell suspensions of the MSCs each are slowly injected into a corresponding migrated cell cultivation channel. Each microfluidic chip is placed in a corresponding Petri dish, and the Petri dish is further incubated in a 37°C and 5% CO: incubator.
Step 4: Observation and monitoring of cell migration (observation and comparison of tumor chemotaxis of MSCs):
After the inoculation of MSCs is completed, each microfluidic chip is incubated in a,505383 37°C and 5% CO: incubator for 6 h to 8 h; and after each MSC is attached to a wall, each microfluidic chip is taken out and observed under an inverted fluorescence microscope, growth statuses of the SiHa cell, the ECT1/E6E7 cell, and the MSCs derived from different tissues are recorded, and migration behaviors of the MSCs derived from different tissues in each matrigel perfusion channel are recorded, which is denoted as 0 h. Each microfluidic chip is further incubated in a 37°C and 5% CO 2 incubator for 24 h; and then each microfluidic chip is taken out and observed under an inverted fluorescence microscope, growth statuses of the SiHa cell, the ECT1/E6E7 cell, the MSCs derived from different tissues are recorded, and migration behaviors of the
MSCs derived from different tissues in each matrigel perfusion channel are recorded, which is denoted as 24 h. Migration areas and maximum migration distances of chemotactic migrations of different MSCs to the SiHa cell, the ECT1/E6E7 cell, and the cell-free DMEM in matrigel perfusion channels within 24 h are measured and compared by Image-Pro Plus software.
In this embodiment, four types of tissue-derived MSCs are adopted, including
AT-MSCs, umbilical cord-derived MSCs (UC-MSCs), amniotic membrane-derived MSCs (AM-MSCs), and chorionic plate-derived MSCs (CP-MSCs). Results are shown in FIG. 4.
It can be seen from comparison of results of the experimental microfluidic chip with results of the normal cell control microfluidic chip and the blank control microfluidic chip that each MSC has a chemotaxis ability for the SiHa cell. It can be seen from comparison of results of cell migration units of the experimental microfluidic chip that CP-MSC has the strongest chemotaxis ability for the SiHa cell, and thus can be used as a potential drug carrier for subsequent research on targeted cervical cancer therapy. In this embodiment, the microfluidic chip can be observed at any time point during cultivation, for example, the microfluidic chip can be observed at 24 h of cultivation.
The above are only preferred implementations of the present disclosure, and the protection scope of the present disclosure is not limited thereto. All technical solutions based on the idea of the present disclosure should fall within the protection scope of the present disclosure. It should be noted that several modifications and improvements made by those of ordinary skill in the art without departing from the principle of the present disclosure should fall within the protection scope of the present disclosure.
Claims (10)
1. A microfluidic chip for screening a migration of a mesenchymal stem cell (MSC) to a tumor, comprising: a chip body with microstructural channels provided on a surface of the chip body, and a substrate tightly attached to the chip body, wherein the microstructural channels on the chip body comprise a central cell cultivation channel and a plurality of independent cell migration units symmetrically arranged at two sides of the central cell cultivation channel; the plurality of independent cell migration units each comprise an inner matrigel perfusion channel and an outer migrated cell cultivation channel; a sample inlet and a sample outlet are formed at two ends of each of the central cell cultivation channel, the matrigel perfusion channel, and the migrated cell cultivation channel, respectively; the central cell cultivation channel is configured to cultivate a tumor cell; the matrigel perfusion channel is configured to fill a matrigel; a plurality of migrated cell cultivation channels are configured to cultivate MSCs derived from different tissues; an interception structure is provided between the central cell cultivation channel and a matrigel perfusion channel of each cell migration unit, and between a matrigel perfusion channel and a migrated cell cultivation channel of a same cell migration unit; the interception structure comprises a plurality of columnar structures arranged at a specified interval; and a signal transmission and interaction between cells and a cell migration are conducted through the interception structure between the central cell cultivation channel and a matrigel perfusion channel of each cell migration unit, and between a matrigel perfusion channel and a migrated cell cultivation channel of a same cell migration unit.
2. The microfluidic chip for screening the migration of the MSC to the tumor according to claim 1, wherein in each of the plurality of cell migration units, two ends of each of the matrigel perfusion channel and the migrated cell cultivation channel both are bent outwards.
3. The microfluidic chip for screening the migration of the MSC to the tum@(,505383 according to claim 1, wherein a material of the chip body is selected from the group consisting of polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), and polycarbonate (PC); and a material of the substrate is a glass or a silicon wafer.
4. The microfluidic chip for screening the migration of the MSC to the tumor according to claim 1, wherein the chip body is tightly attached to the substrate through a plasma bonding process.
5. The microfluidic chip for screening the migration of the MSC to the tumor according to claim 1, wherein the central cell cultivation channel and the migrated cell cultivation channel each are coated with a medium matrix before being used for cultivating a tumor cell and an MSC, and the medium matrix is one or more selected from the group consisting of gelatin, chitosan, silk fibroin (SF), rat tail collagen, fibrin glue (FG), and matrigel.
6. The microfluidic chip for screening the migration of the MSC to the tumor according to claim 1, wherein the central cell cultivation channel and each matrigel perfusion channel and migrated cell cultivation channel of the chip body are located in a same plane, and have a same width and height of 1,000 um and 100 um, respectively; the matrigel perfusion channel has a length of 1.0 cm, and the migrated cell cultivation channel has a length of 0.8 cm; the interception structure comprises five hexagonal prism structures arranged at equal intervals; and each hexagonal prism structure has a side length of 100 um, and an interval between two adjacent hexagonal prism structures is 50 um.
7. A preparation method of the microfluidic chip for screening the migration of the MSC to the tumor according to any one of claims 1 to 6, wherein the preparation method comprises the following steps: step 1: designing and drawing a pattern of the microstructural channels on the chip body with computer-aided software; step 2: preparing the chip body with the microstructural channels by a micromachining technology, and forming a sample inlet and a sample outlet for each of the central cell cultivation channel and each matrigel perfusion channel and migrated ce@ll,505383 cultivation channel by a manual puncher; and step 3: cleaning the chip body and the substrate with absolute ethanol, autoclaving the chip body and the substrate, and finally plasma-bonding the chip body and the substrate to obtain the microfluidic chip.
8. A use method of the microfluidic chip for screening the migration of the MSC to the tumor according to any one of claims 1 to 6, wherein the use method comprises the following steps: step 1: pretreatment of microfluidic chips: taking three microfluidic chips as an experimental microfluidic chip, a normal cell control microfluidic chip, and a blank control microfluidic chip; coating of a medium matrix: for each microfluidic chip, filling a medium matrix solution into the central cell cultivation channel and each migrated cell cultivation channel, and incubating the microfluidic chip in a 37°C incubator for 1 h, such that surfaces of the central cell cultivation channel and each migrated cell cultivation channel each are coated with the medium matrix; and restoration of hydrophobicity: for each microfluidic chip, rinsing the central cell cultivation channel and each migrated cell cultivation channel with sterile water to remove the excess medium matrix solution, and placing the microfluidic chip in an oven at 80°C for 1 h to 2 h, such that the microfluidic chip is dried and hydrophobicity of the microfluidic chip is restored; step 2: fluorescent labeling of cells: taking a tumor cell line with an excellent growth status, a normal cell line derived from the same organ as the tumor cell line, and MSCs derived from different tissues, and labeling each of the tumor cell line, the normal cell line derived from the same organ as the tumor cell line, and the MSCs with a live cell tracer, wherein a live cell tracer to label the tumor cell line and the normal cell line derived from the same organ as the tumor cell line has a different color from a live cell tracer to label the MSCs; step 3: inoculation and cultivation of the cells: for each microfluidic chip, injecting a matrigel into each matrigel perfusion channel, and incubating the microfluidic chip in a 37°C incubator for 30 min to promote curing of the matrigel; adding a medium to each of the tumor cell line and the normal cell line derived from the same organ as the tumor cell line that each are labeled with a live cell tracer to prepare cell suspensions, and injecting the cell suspensions into central cell cultivation channels of the experimental microfluidic chip and the normal cell control microfluidig,505383 chip, respectively; injecting a medium into a central cell cultivation channel of the blank control microfluidic chip; adding a medium to each of the MSCs labeled with a live cell tracer to prepare cell suspensions, and injecting the cell suspensions into migrated cell cultivation channels of different cell migration units of each microfluidic chip, respectively; and incubating the three microfluidic chips in an incubator; and step 4: observation and monitoring of cell migration: after the MSCs are attached to a wall, taking the microfluidic chips out, and observing and recording migration conditions of different MSCs in respective matrigel perfusion channels, which is denoted as 0 h; after the MSCs are further cultivated for n h, taking the microfluidic chips out, and observing and recording migration conditions of different MSCs in respective matrigel perfusion channels, which is denoted as n h; and measuring and comparing migration areas and maximum migration distances of chemotactic migrations of different MSCs to the tumor cell line, the normal cell line derived from the same organ as the tumor cell line, and the medium within n h.
9. The use method of the microfluidic chip for screening the migration of the MSC to the tumor according to claim 8, wherein before the medium matrix is coated, the microfluidic chips each are sterilized; and a method for the sterilization is as follows: rinsing a central cell cultivation channel and each matrigel perfusion channel and migrated cell cultivation channel of each microfluidic chip with 75% alcohol, sterilizing the microfluidic chip under ultraviolet (UV) irradiation overnight, and after the 75% alcohol is completely volatilized, coating the medium matrix.
10. The use method of the microfluidic chip for screening the migration of the MSC to the tumor according to claim 8, wherein in step 3, the cell suspensions of the tumor cell line and the normal cell line derived from the same organ as the tumor cell line injected into the central cell cultivation channels each have a cell density of 2 x 10%/mL; and the cell suspensions of the MSCs injected into the migrated cell cultivation channels each have a cell density of 1 x 108/mL.
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| CN103627635B (en) * | 2013-11-18 | 2015-05-20 | 辽宁中医药大学 | Multifunctional micro-fluidic chip for cell migration and invasion assay |
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| CN106566863B (en) * | 2015-10-10 | 2020-11-20 | 中国科学院大连化学物理研究所 | Cell bidirectional invasion monitoring method based on micro-fluidic chip |
| CN107955783B (en) * | 2016-10-14 | 2021-11-30 | 中国科学院大连化学物理研究所 | Method for constructing in-vitro diabetic glomerulus model based on microfluidic chip |
| CN107955784B (en) * | 2016-10-14 | 2021-10-15 | 中国科学院大连化学物理研究所 | Three-dimensional cell sphere migration monitoring method based on micro-fluidic chip technology |
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