CN111826283A - Micro electric field device and method for observing cell migration and directional arrangement in real time - Google Patents

Abstract

The invention aims to overcome the defect that the existing device can not observe the cell state in real time, provides an electric stimulation cell culture device which can directly observe the cell state in real time under an electron microscope based on an integrated electrode of a 3D printing technology, and provides a micro electric field device which is characterized by comprising the following components: the cell culture device comprises a power supply, a 3D printing component, a cell inoculation part and a microscope, wherein the 3D printing component comprises an electrode, a conductive liquid container and a salt bridge, the cell inoculation part comprises a glass slide, a cover glass and a culture dish, the cell inoculation part is arranged at the lower part of the microscope, the 3D printing component is connected with the cell inoculation part, the power supply is connected with the 3D printing component, and the cell culture device is characterized by comprising the following steps of: s1: performing aseptic treatment; s2: culturing cells; s3: and (4) performing electric stimulation and observation.

Description

Micro electric field device and method for observing cell migration and directional arrangement in real time

Technical Field

The invention relates to the field of medical instruments, in particular to a micro-electric field device and a method for observing cell migration and directional arrangement in real time.

Background

The growth environment of cells is a very complex microenvironment, and cells are usually stimulated by micro-current in biological tissues except for chemical molecular signals, biological factor signals, mechanical stimulation and the like in the environment. Electric fields naturally occurring in a living body, in addition to electric signals generated by nerve signal conduction, also include micro electric fields existing at wound sites, which are not only important for the growth and differentiation of cells, but also essential conditions for the cells to exert their functions. The normal life activity of the organism can not be separated from the electric signal, and as a regulation and control means, the electric field can be estimated to influence the cell migration according to the phenomenon that the electric field appears at the wound part.

Compared with the traditional processing technology, the biological 3D printing technology has remarkable advantages on the manufacturing of complex organizational structures and the spatial distribution of different components.

It has been proved that many cells in human body rely on normal conduction of electric charge to exert their physiological functions, for example, the loss of a large number of normal functional cardiomyocytes can lead to abnormal conduction of cardiomyocytes, and finally, myocardial infarction is often caused. The electrical stimulation also has very important significance on the tissue cells such as osteoblasts, nerve cells and the like which are affected by the in-vivo micro-current stimulation.

In the 19 th century, the german scientist Emil Du Bois-Reymond described the relationship between electricity and nerve excitation, muscle contraction and wound, and he even measured the voltage generated at the wound of his finger; wilhelm Roux attempts to apply an electric field to the egg of an animal and observe its response, but at that time he has not explored this in depth, nor has he sought an electric field generated by the cell or organism itself; according to the study on hydranth, Albert Mathews finds that the electric field naturally generated in the organism can influence the growth of the hydranth; laud, along with other scientists, have conducted more intensive research and have found that during the regeneration of a steel bulb, the hydrozoa appears at one end near the anode if an electric field is applied, whereas normally the hydrozoa appears at the top of a steel bulb; chun et al developed a conductive fabric in which cardiomyocytes on a conductive membrane were interconnected under an applied electrical stimulus to form a population of cells, the cells growing in an ordered direction within the material. After the culture, the cell connexin 43 can be clearly observed in the cell group, and all the myocardial cells can synchronously beat; zhuang et al used capacitive coupling stimulation to load the mouse osteoblasts with 20mV/cm electrical stimulation, which indicated that the electrical stimulation promoted osteoblast differentiation and increased osteoblast DNA synthesis.

However, most of the existing electrical stimulation cell culture devices are complex in structure, difficult in later-stage signal detection, collection and calculation, and difficult to realize direct real-time observation of the cell state under an electron microscope, so that the study of the electrical stimulation device capable of observing the cell state in real time has very important significance.

Disclosure of Invention

Based on the above problems, the present invention aims to overcome the defect that the existing device cannot observe the cell state in real time, and provides an electrical stimulation cell culture device which can directly observe the cell state in real time under an electron microscope based on the integrated electrode of the 3D printing technology.

According to a first aspect of the present invention, there is provided a micro electric field device characterized by comprising: the cell inoculating device comprises a power supply, a 3D printing component, a cell inoculating part and a microscope, wherein the 3D printing component comprises an electrode, a conductive liquid container and a salt bridge, the cell inoculating part comprises a glass slide, a cover glass and a culture dish, the cell inoculating part is arranged at the lower part of the microscope, the 3D printing component is connected with the cell inoculating part, and the power supply is connected with the 3D printing component.

Further, the power supply is an adjustable direct current power supply of 0-60V.

Further, the 3D printing component is printed and integrally formed.

According to a second aspect of the present invention, there is provided a method for observing cell migration and orientation alignment in real time, comprising the steps of:

s1: sterile processing, wiping the micro-electric field device of claim 1 with medical alcohol,

soaking the 3D printing component and the cell inoculation position in alcohol for 30min, irradiating for 2h by using an ultraviolet lamp for aseptic treatment, soaking the glass slide in medical alcohol, and taking out the glass slide on a super clean bench when in use;

s2: the cells were cultured, the slides were rinsed with sterile PBS to remove residual alcohol, and 20. mu.L of the solution containing

Dripping cell culture solution of cells into the center of the upper runner of the glass slide; placing the glass slide in a culture dish, and culturing for 3-4 hours at 37 ℃ under the condition of 5% carbon dioxide concentration until most cells are adhered and spread;

s3: and (3) performing electrical stimulation and observation, taking out the culture dish, adding a culture medium to the position of the inoculated cells on a super-clean bench, covering a cover glass, taking out and placing on a microscope, connecting all the components of the micro-electric field device, communicating a circuit, switching on a power supply, adjusting to required voltage, photographing and recording at certain intervals, and performing comparative analysis according to the positions of the cells on the picture.

Further, the cell comprises: fibroblasts and human umbilical vein endothelial cells.

The invention has the following beneficial effects:

(1) the device is simple to carry, low in cost and easy to clean and sterilize;

(2) the parameters of the electric signals used by the invention are adjustable;

(3) the electrode, the conductive liquid container and the salt bridge integrated structure manufactured by 3D printing is exquisite, and the space is saved;

(4) the micro electrostatic field device has novel structural design, and the cell shape and position can be observed in real time under a microscope after the cells are inoculated;

(5) the micro electrostatic field device can play a role in inducing the directional migration and directional arrangement of cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic view of a micro-electric field device of the present invention;

FIG. 2 is a 3D printed component layout of the present invention; the left drawing is an integrated electrode printing component, the left drawing is a conductive liquid container, the right drawing is a salt bridge, and the right drawing is a fragment carrying shielding structure;

FIG. 3 is a schematic diagram of an integrated electrode.

Description of specific symbols:

1-an electrode; 2-a power supply; 3-cell inoculation site; 4-microscope.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of the present invention.

Example 1:

the apparatus shown in figure 1 was used to load the fibroblasts with micro-electric field stimulation.

The micro-electric field device for stimulating cell migration and directional arrangement is wiped by medical alcohol, soaked for 30 minutes and irradiated by an ultraviolet lamp for 2 hours for aseptic treatment; the glass slides inoculated with the fibroblasts were assembled, soaked in 75% alcohol and removed on a clean bench for use. The slide was rinsed with sterile PBS to remove residual alcohol, and 20. mu.L of the cell culture medium containing fibroblasts was aspirated and dropped onto the center of the upper channel of the slide. The slides were placed in a petri dish and incubated at 25 ℃ for 3-4 hours in air until a majority of adherent spreading of the fibroblasts was observed. Taking out the culture dish, and adding a culture medium to the position of the inoculated cells on a super clean bench, wherein the components of the culture medium are as follows every 50 mL: SFM (39.5mL), fetal bovine serum (10mL), ECGS (1.5mg), double antibody (500. mu.L), coverslip, and place on the microscope, connect the device components, make the circuit through, power on, adjust the electric field strength to 155 mV/mm. And taking a picture and recording after electrifying for 3 hours, and comparing according to the positions of the cells on the picture to obtain the moving distance and the moving track of the cells.

Example 2:

the apparatus shown in figure 1 was used to load the fibroblasts with micro-electric field stimulation.

The micro-electric field device for stimulating cell migration and directional arrangement is wiped by medical alcohol, soaked for 30 minutes and irradiated by an ultraviolet lamp for 2 hours for aseptic treatment; the glass slides inoculated with the fibroblasts were assembled, soaked in 75% alcohol and removed on a clean bench for use. The slide was rinsed with sterile PBS to remove residual alcohol, and 20. mu.L of the cell culture medium containing fibroblasts was aspirated and dropped onto the center of the upper channel of the slide. The slides were placed in a petri dish and incubated at 37 ℃ for 3-4 hours at 5% carbon dioxide concentration until most adherent spreading of the fibroblasts was observed. Taking out the culture dish, and adding a culture medium to the position of the inoculated cells on a super clean bench, wherein the components of the culture medium are as follows every 50 mL: SFM (39.5mL), fetal bovine serum (10mL), ECGS (1.5mg), double antibody (500. mu.L), coverslip, and place on the microscope, connect the device components, make the circuit through, power on, adjust the electric field strength to 100 mV/mm. And taking a picture after electrifying for 5 hours, recording, and carrying out comparative analysis according to the positions of the cells on the picture to obtain the moving distance and the moving track of the cells.

Example 3:

the device shown in figure 1 is used for loading micro-electric field stimulation to human umbilical vein endothelial cells.

The micro-electric field device for stimulating cell migration and directional arrangement is wiped by medical alcohol, soaked for 30 minutes and irradiated by an ultraviolet lamp for 2 hours for aseptic treatment; the glass slide inoculated with the human umbilical vein endothelial cells is assembled and soaked in 75% alcohol, and is taken out on a super clean bench when in use. The slide was rinsed with sterile PBS to remove residual alcohol, and 20. mu.L of cell culture medium containing human umbilical vein endothelial cells was pipetted down onto the center of the upper channel of the slide. The slide glass is placed in a culture dish and is placed in air condition at 25 ℃ for 3-4 hours until most of human umbilical vein endothelial cells are observed to spread adherent to the wall. Taking out the culture dish, and adding a culture medium to the position of the inoculated cells on a super clean bench, wherein the components of the culture medium are as follows every 50 mL: SFM (39.5mL), fetal bovine serum (10mL), ECGS (1.5mg), double antibody (500. mu.L), coverslip, and place on the microscope, connect the device components, make the circuit through, power on, adjust the electric field strength to 270 mV/mm. And taking a picture and recording after electrifying for 3 hours, and carrying out comparative analysis according to the positions of the cells on the picture to obtain the moving distance and the moving track of the cells.

Example 4:

the device shown in figure 1 is used for loading micro-electric field stimulation to human umbilical vein endothelial cells.

The micro-electric field device for stimulating cell migration and directional arrangement is wiped by medical alcohol, soaked for 30 minutes and irradiated by an ultraviolet lamp for 2 hours for aseptic treatment; the glass slide inoculated with the human umbilical vein endothelial cells is assembled and soaked in 75% alcohol, and is taken out on a super clean bench when in use. The slide was rinsed with sterile PBS to remove residual alcohol, and 20. mu.L of cell culture medium containing human umbilical vein endothelial cells was pipetted down onto the center of the upper channel of the slide. The slide glass is placed in a culture dish and is cultured for 3-4 hours under the condition of 37 ℃ and 5% carbon dioxide concentration until most of human umbilical vein endothelial cells are observed to spread adherent. Taking out the culture dish, and adding a culture medium to the position of the inoculated cells on a super clean bench, wherein the components of the culture medium are as follows every 50 mL: SFM (39.5mL), fetal bovine serum (10mL), ECGS (1.5mg), double antibody (500. mu.L), coverslip, and place on the microscope, connect the device components, make the circuit through, power on, adjust the electric field strength to 100 mV/mm. After the power-on, the photograph was recorded every 1 hour, and the cell position on the photograph was compared. And after 3 hours, changing the direction of the power plant from left to right to left, photographing and recording every 1 hour after electrifying, and performing comparative analysis according to the positions of cells on the pictures to obtain the moving distance and the moving track of the power plant.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (5)

1. A micro-electric field device, comprising: the cell inoculating device comprises a power supply, a 3D printing component, a cell inoculating part and a microscope, wherein the 3D printing component comprises an electrode, a conductive liquid container and a salt bridge, the cell inoculating part comprises a glass slide, a cover glass and a culture dish, the cell inoculating part is arranged at the lower part of the microscope, the 3D printing component is connected with the cell inoculating part, and the power supply is connected with the 3D printing component.

2. The micro-electric field device of claim 1, wherein the power source is a regulated dc power source of 0-60V.

3. The micro-electric field device of claim 1, wherein the 3D printed member is printed as a single piece.

4. A method for observing cell migration and orientation in real time, comprising the steps of:

s1: sterile processing, wiping the micro-electric field device of claim 1 with medical alcohol, wherein the 3D printing component and the cell inoculation position are placed in order to be soaked in alcohol for 30min, then irradiated by an ultraviolet lamp for 2h for sterile processing, and the glass slide is soaked in medical alcohol and taken out on a super clean bench when in use;

s2: cell culture, washing the glass slide with sterile PBS to remove residual alcohol, sucking 20 μ L of cell culture solution containing cells, and dripping into the center of the upper flow channel of the glass slide; placing the glass slide in a culture dish, and culturing for 3-4 hours at 37 ℃ under the condition of 5% carbon dioxide concentration until most cells are adhered and spread;

s3: and (3) performing electrical stimulation and observation, taking out the culture dish, adding a culture medium to the position of the inoculated cells on a super-clean bench, covering a cover glass, taking out and placing on a microscope, connecting all the components of the micro-electric field device, communicating a circuit, switching on a power supply, adjusting to required voltage, photographing and recording at certain intervals, and performing comparative analysis according to the positions of the cells on the picture.

5. The method of claim 4, wherein the cells comprise: fibroblasts and human umbilical vein endothelial cells.

Images