CN112852628A - Method for constructing muscle model based on micro-fluidic chip - Google Patents
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Abstract
The invention provides a method for establishing a muscle model based on a microfluidic chip. The micro-fluidic chip mainly comprises a cell suspension inlet, a cell culture chamber, cell fixing anchor points, an air communication port and a liquid storage tank, wherein a small column-shaped communication part is arranged at the junction of the liquid storage tank and the cell culture chamber and can be used for material transmission. The muscle model building method comprises the following steps: (1) two-dimensional culture of muscle cells; (2) modifying the chip; (3) inoculating and culturing cells in the chip; (4) and (5) establishing a muscle model. The model can be used for observing the investigation of the activity of cells in a chip and the characterization of functional change and for evaluating the toxicity of drugs.
Description
Technical Field
The invention belongs to the technical field of research on applying a microfluidic technology to tissue bionics to detect cell biology, and particularly relates to a construction method of a muscle model based on a microfluidic chip.
Background
Muscle cells are the most abundant cell type in the human body. The main function of muscles is to produce strength. However, when muscles are damaged or diseased, normal function is impaired. There are many musculoskeletal diseases that tissue engineers are working to better understand and seek new and improved treatments. Many studies of muscle are performed in two-dimensional cell culture, but in this case many physiological conditions are lacking, and thus it is not possible to switch well between two-dimensional culture and work in vivo. In vitro models have therefore been extensively explored in recent years. In vitro models have the same advantages of high throughput analysis as two-dimensional culture models, but also have the additional advantage of mimicking in vivo physicochemical cues.
In recent years, significant progress has been made in the field of microfluidics. Highly sophisticated microfabrication techniques pave the way for the development of complex in vitro models that can integrate and measure the real-time response of multiple cell types interacting in a single system. The muscle technology on a chip has improved greatly and has become a platform for drug screening of many muscle diseases, such as muscular dystrophy, tendinopathy, fibromyalgia, mitochondrial myopathy, and myasthenia gravis. The establishment of microfluidic muscle chip models allows researchers to better understand disease pathology and to provide the strength of high throughput screening therapies for muscle diseases.
Disclosure of Invention
The invention aims to provide a micro-fluidic chip and a method for establishing a three-dimensional muscle model based on the micro-fluidic chip, and the method is applied to drug evaluation.
A micro-fluidic chip is formed by irreversibly sealing an upper layer chip material and a lower layer chip material, wherein the upper layer chip material is a light-permeable and air-permeable PDMS polymer with a cavity and a channel, and the cavity and the channel are arranged on the upper layer chip material and cover patterns on the surface of a substrate; the lower chip material is a glass slide.
The upper chip of the microfluidic chip mainly comprises a cell suspension inlet 1, a cell culture chamber 2, cell fixing anchor points 3, an air communicating port 4 and a liquid storage tank 5, wherein a communicating part 6 in a small column shape is arranged at the junction of the liquid storage tank 5 and the cell culture chamber 2, and the upper chip can be used for applying substance transmission. The structure is as described in fig. 1 and 2.
The upper chip material is irreversibly sealed on the lower material by plasma treatment for 30-60s, and the cell culture chamber of the upper chip covers the area of the substrate with the structure.
The height of all channels is 300- & gt 400 um.
A method for establishing a muscle model based on a microfluidic chip comprises the following steps:
(1) two-dimensional culture of skeletal muscle in mice
Mouse skeletal muscle myoblasts (C2C12) were cultured in DMEM medium (4.5g/L D-glucose, 110mg/L sodium pyruvate) supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin-streptomycin at 37 ℃ under 5% carbon dioxide. When the cell density reaches 70-80%, 0.2% (v/v) trypsin is used for digestion for standby.
(2) Chip modification
Infiltrating a chip cell suspension inlet (1) and a cell culture chamber (2) with 4% (w/v) Pluronic F-127 for 1h, washing with distilled water for 3-5 times, and drying in an oven at 80 ℃ for later use;
(3) inoculation and culture of cells in chip
Collagen cell suspension: C2C12 cells were distributed in collagen at a cell density (I collagen gel (2.4mg/ml) and 10% Matrigel). Precooling the chip, injecting the cell collagen mixture into the chip from the cell suspension inlet, standing for 30min at 37 ℃, and adding 0.5ml of basal medium (H-DMEM, 10% fetal calf serum, 1% penicillin-streptomycin) into the liquid storage tanks at both sides.
(4) Establishment of muscle model
After the C2C12 cells are completely paved on the bottom surface of the cell culture chamber through precipitation, the cells are in a bundle shape, after one day, the liquid storage tank is replaced by a differentiation medium (H-DMEM, 2% horse serum, 1% penicillin-streptomycin) for induced differentiation, and the cells are continuously cultured for 3-7 days.
The invention utilizes the microfluidic technology to construct a three-dimensional muscle tissue model, and can be used for muscle tissue research and drug evaluation. In particular, additional equipment can be added to evaluate the influence of electrical stimulation or mechanical stimulation on muscle maturation in the process of muscle formation and establish a muscle model of a disease source for characteristic drug screening.
The invention has the beneficial effects that:
the invention utilizes the micro-fluidic chip technology to culture muscle cells by providing a three-dimensional matrix, and establishes a three-dimensional muscle tissue model. The micro-fluidic chip mainly comprises a cell suspension inlet, a cell culture chamber, cell fixing anchor points, an air communication port and a liquid storage tank, wherein a small column-shaped communication part is arranged at the junction of the liquid storage tank and the cell culture chamber and can be used for material transmission. The collagen and the Matrigel are improved to be used as a three-dimensional culture medium to form a three-dimensional muscle bundle to construct a muscle model, and the three-dimensional muscle bundle can be used for observing the activity investigation of cells in a chip and the characterization of functional change and for evaluating the toxicity of medicaments.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a microfluidic chip according to the present invention
Wherein: 1 represents a cell suspension inlet, 2 represents a cell culture chamber, 3 represents a cell fixing anchor point, 4 represents an air communicating port, 5 represents a liquid storage tank, and 6 represents a communicating part with a small column shape at the junction of the liquid storage tank and the cell culture chamber.
FIG. 2 is a diagram of a microfluidic chip according to the present invention
FIG. 3 growth of the microfluidic chip of example 1 in which C2C12 cells were seeded on the chip
FIG. 4C 2C12 cell death and survival staining and muscle expression protein identification in the microfluidic chip of example 2;
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto
Example 1
Inoculation and growth of muscle cells on microfluidic chip
The microfluidic chip mainly comprises a cell suspension inlet, a cell culture chamber, cell fixing anchor points, an air communication port and a liquid storage tank, wherein a small column-shaped communication part is arranged at the junction of the liquid storage tank and the cell culture chamber and can be used for material transmission, as shown in figure 1. The heights of all channels of the microfluidic chip are 300-400 um.
The microfluidic chip is formed by irreversibly sealing an upper layer chip material and a lower layer chip material, wherein the upper layer chip material is a light-permeable and air-permeable PDMS polymer with a cavity and a channel, and the cavity and the channel are arranged on the upper layer chip material and cover the surface pattern of the substrate; the lower chip material is a glass slide. The upper chip material is irreversibly sealed on the lower material by plasma treatment for 30-60s, and the cell culture chamber of the upper chip covers the area of the substrate with the structure.
The method for establishing the muscle model of the microfluidic chip is characterized by comprising the following steps of:
(1) two-dimensional culture of skeletal muscle in mice
Mouse skeletal muscle myoblasts (C2C12) were supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin in DMEM medium (4.5g/L D-glucose, 110mg/L sodium pyruvate). Culturing at 37 deg.C under 5% carbon dioxide. When the growth rate reaches 70-80%, 0.2% trypsin is used for digestion and standby.
(2) Chip modification
Soaking the chip glue filling channel for 1h by using 4% (w/v) Pluronic F-127, washing the chip glue filling channel for 3-5 times by using distilled water, and drying the chip glue filling channel in an oven at 80 ℃ for later use.
(3) Inoculation and culture of cells in chip
Collagen cell suspension: C2C12 cells were distributed in collagen at a cell density of 1x10^7cells/ml (I collagen gel (2.4mg/ml) and 10% Matrigel). Precooling the chip, injecting the cell collagen mixture into the chip from the cell suspension inlet, standing for 30min at 37 ℃, and adding 0.5ml of basal medium (H-DMEM, 10% fetal calf serum, 1% penicillin-streptomycin) into the liquid storage tanks at both sides.
(4) And (3) characterization: the cells seeded on the chip were subjected to bright field characterization as shown in FIG. 3.
Example 2
Differentiation and characterization of muscle cells
The structure of the microfluidic chip designed and manufactured by a laboratory is shown in figure 1. After the chip modification, a muscle model was established using the same cell inoculation and culture method as in example 1. Cell death and viability staining and cell immunofluorescence staining were performed 7 days after the differentiation medium was changed, and the detection protein was myosin heavy chain (MYCH). The method comprises the following steps: 4% paraformaldehyde for cell, washing with PBS buffer solution for three times, each time for 10-15 min; allowing 0.1% triton X-100 pore-forming agent to act for 20min, washing with PBS buffer solution for three times, each time for 10-15 min; sealing and cleaning goat for 1h, diluting primary antibody (mouse-anti-mouse MYHC) at a ratio of 1:500, incubating overnight, washing with PBS buffer solution for three times, each time for 10-15 min; a secondary antibody (Alexa Fluor 594 labeled goat anti-mouse IgG (H + L)) is diluted at a ratio of 1:1000, incubated for 1H at normal temperature in the dark, and washed with PBS buffer solution for three times, wherein each time lasts for 10-15 min; adding DAPI working solution diluted by 1:5000 after washing for incubation for l5 min; the expression of the corresponding protein was recorded by 2 PBS buffer washes and pictures taken under a fluorescence microscope, and the results are shown in FIG. 4.
Claims (7)
1. A microfluidic chip, characterized in that: the microfluidic chip is formed by irreversibly sealing an upper layer chip material and a lower layer chip material, wherein the upper layer chip material is a light-permeable and air-permeable PDMS polymer with a cavity and a channel, and the cavity and the channel are arranged on the upper layer chip material and cover the surface pattern of the substrate; the lower chip material is a glass slide.
2. The microfluidic chip of claim 1, wherein: the upper chip of the microfluidic chip mainly comprises a cell suspension inlet (1), a cell culture chamber (2), cell fixing anchor points (3), an air communicating port (4) and a liquid storage tank (5), wherein a communicating part (6) in a small column shape is arranged at the junction of the liquid storage tank (5) and the cell culture chamber (2), and the upper chip can be used for applying substance transmission.
3. The microfluidic chip according to claim 1, wherein: the upper chip material is irreversibly sealed on the lower material by plasma treatment for 30-60s, and the cell culture chamber of the upper chip covers the area of the substrate with the structure.
4. The microfluidic chip according to claim 2, wherein: the height of all channels is 300- & gt 400 um.
5. A method for establishing a muscle model based on the microfluidic chip of any one of claims 1 to 4, comprising the following steps:
(1) two-dimensional culture of skeletal muscle in mice
DMEM medium (4.5g/L D-glucose, 110mg/L sodium pyruvate) was supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin.
Mouse skeletal muscle myoblasts (C2C12) were cultured in the above medium at 37 ℃ under 5% carbon dioxide conditions; when the cell density reaches 70-80%, digesting with 0.2% (v/v) trypsin for later use;
(2) chip modification
Infiltrating a chip cell suspension inlet (1) and a cell culture chamber (2) with 4% (w/v) Pluronic F-127 for 1h, washing with distilled water for 3-5 times, and drying in an oven at 80 ℃ for later use;
(3) inoculation and culture of cells in chip
Collagen cell suspension: C2C12 cells were distributed in collagen at a cell density (I collagen gel (2.4mg/ml) and 10% Matrigel); precooling the chip, injecting a cell collagen mixture into the chip from a cell suspension inlet, standing for 30min at 37 ℃, and adding 0.5ml of a basic culture medium (H-DMEM, 10% fetal calf serum and 1% penicillin-streptomycin) into liquid storage tanks at two sides;
(4) establishment of muscle model
After the C2C12 cells are completely paved on the bottom surface of the cell culture chamber through precipitation, the cells are in a bundle shape, after one day, the liquid storage tank is replaced by a differentiation medium (H-DMEM, 2% horse serum, 1% penicillin-streptomycin) for induced differentiation, and the cells are continuously cultured for 3-7 days.
6. Use of a microfluidic chip based muscle model according to claim 5, wherein: the model can be used for observing the investigation of the activity of cells in a chip and the characterization of functional change and for evaluating the toxicity of drugs.
7. Use according to claim 6, characterized in that: the model is used for evaluating the influence of electric stimulation or mechanical stimulation on muscle maturation in the process of muscle formation and establishing a muscle model of a disease source to carry out characteristic drug screening.
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