阅读说明：本技术 基于三维环境中对细胞运动追踪的方法及微流控芯片 (Method for tracking cell movement based on three-dimensional environment and microfluidic chip ) 是由 傅雄飞 何彩云 白阳 储攀 朱静雯 于 2020-11-30 设计创作，主要内容包括：本申请涉及微流控芯片应用技术领域,提供了一种基于三维环境中对细胞运动追踪的方法,包括以下步骤：制作具有微型通道的载体；微型通道为三维通道,微型通道的高度在一定范围内；用制作好的载体对细胞进行运动追踪。一种微流控芯片,包括微型通道,微型通道的高度范围为40-55μm,宽度范围为0.6-2.5mm,长度范围为1-2cm；微型通道具有进样口和出样口,进样口和出样口的孔径范围均为0.8-1.5mm。本申请通过设计具有微型通道的载体,且微型通道的高度在一定范围内,不仅能够模拟细胞在三维立体空间中运动,同时还能够满足显微镜的焦距范围,使得用显微镜观察细胞运动时不会出现失焦现象,使得采集到的数据更具真实性。(The application relates to the technical field of microfluidic chip application, and provides a method for tracking cell movement based on a three-dimensional environment, which comprises the following steps: manufacturing a carrier with a micro channel; the micro channel is a three-dimensional channel, and the height of the micro channel is within a certain range; the cell was followed by movement using the prepared vector. A micro-fluidic chip comprises a micro-channel, wherein the height range of the micro-channel is 40-55 μm, the width range is 0.6-2.5mm, and the length range is 1-2 cm; the micro channel is provided with a sample inlet and a sample outlet, and the aperture ranges of the sample inlet and the sample outlet are both 0.8-1.5 mm. This application has microchannel's carrier through the design, and microchannel's height is in certain extent, not only can simulate the cell and move in three-dimensional spatial space, can also satisfy microscopical focus range simultaneously for the phenomenon of losing focus can not appear when observing cell movement with the microscope, makes the data of gathering have more the authenticity.)

1. A method for tracking cell movement based on a three-dimensional environment is characterized by comprising the following steps:

manufacturing a carrier with a micro channel; wherein the microchannel is a three-dimensional channel, and the height of the microchannel is in a certain range;

the cell was followed by movement using the prepared vector.

2. The method for tracking cell movement in three-dimensional environment according to claim 1, wherein the carrier is fabricated by the following steps:

determining the three-dimensional size of the microchannel according to the characteristics of the microorganism escherichia coli;

and manufacturing the carrier according to the determined three-dimensional size.

3. The method for tracking the movement of cells in a three-dimensional environment according to claim 2, wherein the height of said microchannel is in the range of 40-55 μm.

4. The method for tracking the movement of cells in a three-dimensional environment according to claim 3, wherein the width of the microchannel is in the range of 0.6-2.5mm and the length is in the range of 1-2 cm;

the carrier is provided with a sample inlet and a sample outlet which are communicated with the micro channel, and the aperture ranges of the sample inlet and the sample outlet are both 0.8-1.5 mm.

5. The method of claim 2, wherein the microchannel has a rectangular, cylindrical or square shape.

6. The method for tracking movement of cells in a three-dimensional environment according to claim 2, wherein said carrier is made of polydimethylsiloxane material.

7. The method for tracking cell movement in a three-dimensional environment according to claim 6, wherein the carrier is a microfluidic chip.

8. The method for tracking cell movement based on three-dimensional environment according to any one of claims 2 to 7, wherein the method for tracking cell movement by using the carrier comprises the following steps:

diluting and injecting cultured escherichia coli into the micro channel;

sealing the sample inlet and the sample outlet of the micro channel;

the movement of E.coli was observed in real time with a microscope.

9. The method for tracking cell movement in a three-dimensional environment according to claim 8, wherein the sample inlet and the sample outlet are sealed by a sealing piece; wherein, the block tablet is prepared by mixing lanolin and vaseline according to the ratio of 1: 1.

10. The microfluidic chip is characterized by comprising a micro-channel, wherein the height range of the micro-channel is 40-55 mu m, the width range of the micro-channel is 0.6-2.5mm, and the length range of the micro-channel is 1-2 cm; the micro-fluidic chip is also provided with a sample inlet and a sample outlet which are communicated with the micro-channel, and the aperture ranges of the sample inlet and the sample outlet are both 0.8-1.5 mm.

Technical Field

The application belongs to the technical field of micro-fluidic chip application, and particularly relates to a method for tracking cell movement in a three-dimensional environment and a micro-fluidic chip.

Background

Among the model organisms of biological science research, the research on the movement of microbial bacteria and eukaryotic cells has certain scientific significance. The movement of bacteria tends to be coupled with their growth, and in the study of the resource allocation and metabolic flux of bacterial growth, the movement of bacteria tends to occupy most of the energy and resource allocation. In recent years, in the research of bacteria-mediated tumor therapy, the realization of the fixed-point targeting of bacteria to a tumor region by utilizing the chemotactic movement of bacteria becomes a new idea.

In the past, most of movement research on bacteria and cells is focused on a uniform environment scene, and the movement of the cells is tracked in a two-dimensional environment. Usually, cultured microbial cells are diluted by a culture medium according to a certain proportion, and a small amount of diluted sample is placed on a glass sheet to form a single liquid layer so as not to be scorched under the observation of a microscopic environment. However, the real environment of the cell is a three-dimensional environment and is complex and variable. In scientific research, in order to simulate the complex environment where microorganisms are located more truly, research observation of cell motion tracking in a three-dimensional environment is necessary.

In the study of tracking cell movement in a three-dimensional environment, most of the conventional methods use a cell culture plate or a bacterial culture plate, spread a small amount of culture solution containing cells on the culture plate, and then perform microscopic observation. Because the focus of the microscope has a certain range, the sample obtained by the conventional sample preparation method often has a defocusing phenomenon when being observed under the microscope, so that ideal data cannot be acquired.

Disclosure of Invention

The embodiment of the application aims to provide a method for tracking cell motion based on a three-dimensional environment, so as to solve the technical problem that observation under a microscope is out of focus due to the fact that a cell culture plate or a bacterial culture plate is used for tracking the cell three-dimensional environment in the prior art.

In order to achieve the purpose, the technical scheme adopted by the application is as follows: a method for tracking cell movement based on a three-dimensional environment is provided, which comprises the following steps:

manufacturing a carrier with a micro channel; wherein the microchannel is a three-dimensional channel, and the height of the microchannel is in a certain range;

the cell was followed by movement using the prepared vector.

In one embodiment, the manufacturing process of the carrier comprises the following steps:

determining the three-dimensional size of the microchannel according to the characteristics of the microorganism escherichia coli;

and manufacturing the carrier according to the determined three-dimensional size.

In one embodiment, the height of the microchannel is in the range of 40-55 μm.

In one embodiment, the microchannel has a width in the range of 0.6 to 2.5mm and a length in the range of 1 to 2 cm;

the carrier is provided with a sample inlet and a sample outlet which are communicated with the micro channel, and the aperture ranges of the sample inlet and the sample outlet are both 0.8-1.5 mm.

In one embodiment, the microchannel is rectangular, cylindrical, or square.

In one embodiment, the carrier is made of a polydimethylsiloxane material.

In one embodiment, the carrier is a microfluidic chip.

In one embodiment, a method for cell movement tracking using a carrier comprises the steps of:

diluting and injecting cultured escherichia coli into the micro channel;

sealing the sample inlet and the sample outlet of the micro channel;

the movement of E.coli was observed in real time with a microscope.

In one embodiment, the sample inlet and outlet are sealed by a sealing agent; wherein, the block tablet is prepared by mixing lanolin and vaseline according to the ratio of 1: 1.

The method for tracking the cell movement based on the three-dimensional environment has the advantages that: according to the method for tracking the cell movement in the three-dimensional environment, the micro channel is arranged in the carrier, the micro channel is the three-dimensional channel, the height of the micro channel is within a certain range, the micro channel is closer to a complex three-dimensional environment where an organism lives, and a three-dimensional space can be provided for the movement of the cell, so that the movement tracking research of the cell can be more authentic, and the method is favorable for the growth research of the cell and the research of a bacteria fixed-point targeting tumor area. Simultaneously, because the motion space of microchannel can certain altitude range, for the three-dimensional space that cell culture plate more can provide the cell motion, its third dimension is stronger, the motion of cell can not receive the constraint, the motion of cell more tends to the authenticity, simultaneously when observing through the microscope, as long as the focus is adjusted rationally, the microchannel of certain altitude range is deuterogamied, the phenomenon of losing focus just can not appear when observing, thereby make the data of gathering have more the authenticity, improved research value and research significance.

The application also provides a micro-fluidic chip which comprises a micro-channel, wherein the height range of the micro-channel is 40-55 mu m, the width range is 0.6-2.5mm, and the length range is 1-2 cm; the micro-fluidic chip is also provided with a sample inlet and a sample outlet which are communicated with the micro-channel, and the aperture ranges of the sample inlet and the sample outlet are both 0.8-1.5 mm.

The application provides a micro-fluidic chip's beneficial effect lies in: the micro-fluidic chip provided by the embodiment of the application not only can simulate the movement of cells in a three-dimensional space by setting the length, the width and the height of the micro-channel in a certain range respectively, but also can meet the focal range of a microscope, so that the phenomenon of defocusing can not occur when the cells are observed by the microscope, the observation visual field of the microscope is large enough, and the collected data is more authentic. In addition, the collapse phenomenon of the microfluidic chip can be prevented.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.

Fig. 1 is a schematic flowchart of a method for tracking cell movement in a three-dimensional environment according to an embodiment of the present disclosure;

FIG. 2 is a schematic diagram of a process for making the vector of FIG. 1;

FIG. 3 is a schematic diagram of the process for tracking cell movement using the vector of FIG. 1;

fig. 4 is a schematic view of a microchannel of a microfluidic chip provided in an embodiment of the present application;

FIG. 5 is a graph showing the distribution of E.coli observed under a 10-fold microscope;

FIG. 6 is a diagram showing the locus of E.coli movement under a 10-fold microscope.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application clearer, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element.

It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, as used herein, refer to an orientation or positional relationship indicated in the drawings that is solely for the purpose of facilitating the description and simplifying the description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be considered as limiting the present application.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present application, "a plurality" means two or more unless specifically limited otherwise.

The method for tracking cell movement in a three-dimensional environment provided in the embodiments of the present application will now be described. The method for tracking the cell movement based on the three-dimensional environment can simulate the tracking of the single cell movement in the three-dimensional environment, can simulate and reflect the complex environment of an organism more truly, and contributes to the research of cells and the realization of a technology of targeting a tumor region at a fixed point by bacteria. It is understood that in other embodiments of the present application, the method can also be used for observing the movement of eukaryotic cells, such as immune cells, and is not limited herein.

Referring to fig. 1, in particular, a method for tracking cell movement based on a three-dimensional environment includes the following steps:

s10: making a carrier with microchannels 10; the carrier is any carrier having the microchannel 10, and the material of the carrier should be a material that is favorable for the survival of cells and does not cause pollution or damage to the cells. The microchannel 10 is a three-dimensional channel, and the height of the microchannel 10 is within a certain range, that is, the height of the microchannel 10 can be limited according to actual requirements.

S20: the cell was followed by movement using the prepared vector.

Specifically, the movement of the cells in the carrier is tracked by a microscope.

In the method for tracking cell movement based on the three-dimensional environment in the embodiment, the carrier is provided with the micro channel 10, the micro channel 10 is a three-dimensional channel, and the height of the micro channel 10 is within a certain range, so that the micro channel 10 is closer to a complex three-dimensional environment where an organism lives, and a three-dimensional space can be provided for the movement of the cell, so that the movement tracking research of the cell can be more realistic, and the method is favorable for the growth research of the cell and the research of a fixed-point target tumor area of bacteria. Simultaneously, because the motion space of microchannel 10 can certain altitude range, for the three-dimensional space that the cell culture plate more can provide the cell motion, its third dimension is stronger, the motion of cell can not receive the constraint, the motion of cell more tends to the authenticity, simultaneously when observing through the microscope, as long as the focus is adjusted rationally, the microchannel 10 of certain altitude range is deuterogamied, the phenomenon of losing focus just can not appear when observing, thereby make the data of gathering have more the authenticity, improved research value and research significance.

In a specific embodiment, referring to fig. 2, the manufacturing process of the carrier includes the following steps:

s11: determining the three-dimensional size of the microchannel 10 according to the characteristics of the microorganism escherichia coli;

the microorganism escherichia coli is a member of the enterobacteriaceae family, and is often widely used in scientific research as a model organism of bacteria. In addition, escherichia coli has flagella around the body, the flagella distribution of the flagella is beneficial to movement, and the escherichia coli is convenient to obtain and small in size, so that the escherichia coli is a good choice for tracking and analyzing cell movement. It is understood that in other embodiments of the present application, according to the specific research direction, when the research on the movement of other types of cells or bacteria is required, the escherichia coli can be replaced by other bacteria, such as marine bacteria, salmonella, etc., which are not limited herein.

S12: and manufacturing the carrier according to the determined three-dimensional size.

The three-dimensional size of each microchannel 10 is determined, and then the number of microchannels 10 provided on each carrier is determined, for example, one microchannel 10 is provided on each carrier, or two or more microchannels 10 are provided on each carrier; finally, the approximate size of the carrier is determined according to the size and number of the micro-channels 10.

In a specific embodiment, the escherichia coli is a rod-shaped bacterium, the width of the escherichia coli obtained through a large number of experiments is within the range of 0.2-0.5 μm, and the length of the escherichia coli is within the range of 1.3-2 μm, so that when the size of the escherichia coli is not limited by the environment, the moving range of the escherichia coli is at least 10 times of the size of the escherichia coli, the movement of the escherichia coli is not limited, and the movement simulation of cells in a three-dimensional environment is more real. In addition, when the microscope is used for observation, in order to enlarge the observation field of view, the magnification of the microscope is generally 10 times or less, for example, when the magnification of the microscope is 10 times, the height H range of the selected micro-channel 10 is 40-55 μm, and the movement range of escherichia coli can be in the gathering range of the microscope, so that the phenomenon of defocusing of microscope observation can not occur, and further, the microscope can acquire accurate number, which is beneficial to accurate research of cell trend movement and research of a bacteria fixed-point targeting tumor technology.

In a specific practical experiment, the height H of the microchannel 10 may be 40 μm, 45 μm, 50 μm or 55 μm. It is understood that in other embodiments of the present application, for example, when the microscope has a magnification of 4 times or 6 times, the range of the height H of the microchannel 10 may also be changed as appropriate according to the focusing requirement of the microscope, and is not limited herein.

In a specific embodiment, in order to clearly photograph the movement trace of the escherichia coli, it is necessary to ensure that the photographing field of the microscope is large enough, and thus the length and width of the microchannel 10 are designed as long as possible, but if the length of the microchannel 10 is too long, the structural strength of the microfluidic chip is reduced, and the microchannel 10 is easily collapsed. By combining the two factors, multiple experiments prove that when the width W of the micro channel 10 ranges from 0.6mm to 2.5mm and the length L of the micro channel 10 ranges from 1cm to 2cm, the micro channel 10 basically does not collapse, and the observation field of view is sufficiently large, clear and accurate data are acquired when the microscope is used for observing the movement track of escherichia coli.

In a specific practical experiment, the width W of the microchannel 10 may be 0.6mm, 1.0mm, 1.5mm, 2mm or 2.5mm, and the length L of the microchannel 10 may be 1cm, 1.3cm, 1.5cm or 2 cm. It is understood that in other embodiments of the present application, the length L of the microchannel 10 may be up to 3mm, 4mm, etc. when the size of the microscope stage allows, and is not limited herein.

Referring to fig. 4, the carrier has a sample inlet 20 and a sample outlet 30, the sample inlet 20 and the sample outlet 30 are respectively disposed at two ends of the microchannel 10 along the length direction, and the sample inlet 20 and the sample outlet 30 are respectively communicated with the microchannel 10 and used for injecting the diluted escherichia coli into the microchannel 10.

Specifically, the aperture of the sample inlet 20 and the sample outlet 30 is 0.8-1.5 mm. Generally, the aperture of the inlet 20 and outlet 30 is determined by the selected hole puncher, and if the aperture of the inlet 20 and outlet 30 is too large, air bubbles will enter the microchannel 10 easily, while if the aperture of the inlet 20 and outlet 30 is too small, the E.coli will not be injected into the microchannel 10 well. In addition, the aperture of the inlet 20 and the outlet 30 should be slightly smaller than the width and height of the microchannel 10, and the aperture of the inlet 20 and the outlet 30 is set to be 1mm in this application, and in other embodiments of this application, the aperture of the inlet 20 and the outlet 30 may also be 0.8mm, 1.2mm, or 1.5mm, etc., which is not limited herein.

In the specific embodiment, please refer to fig. 4, the micro channel 10 is rectangular, and has a simple structure and a convenient manufacturing. It should be understood that, in other embodiments of the present application, the microchannel 10 may be configured to have a cylindrical or square shape, or may even be any three-dimensional shape with other sizes adapted according to actual design conditions and specific requirements, and is not limited herein.

In a specific embodiment, the carrier is made of polydimethylsiloxane material, the polydimethylsiloxane has good biocompatibility and air permeability, so that the escherichia coli can conveniently absorb oxygen, the material is clean, and pollution or harm can not be caused to the escherichia coli, and after the escherichia coli solution is injected into the micro channel 10, the carrier can not influence or interfere the escherichia coli and the activity of the escherichia coli, so that the escherichia coli can be guaranteed to survive in the micro channel 10 and can move without hindrance.

In a specific embodiment, the carrier is a microfluidic chip, and the microfluidic chip technology integrates basic operation units of sample preparation, reaction, separation, detection and the like in biological, chemical and medical analysis processes on a chip with a micron scale, automatically completes the whole analysis process, and forms a network by the micro-channels 10 to control the whole system, thereby replacing a technology with various functions of a conventional biological or chemical laboratory. The carrier is arranged into the microfluidic chip, so that a micro environment can be provided, the usage amount of the escherichia coli sample is small, and the carrier is simple, stable, reliable and good in repeatability. It is understood that in other embodiments of the present application, the carrier may also be other simple structures made of polydimethylsiloxane material according to practical design conditions and specific requirements, and the structure may have all the microchannels 10 with a certain height, a certain width and a certain length, which is not limited herein.

The manufacturing method of the micro-fluidic chip is substantially the same as that of a common micro-fluidic chip, and comprises the following steps:

drawing a CAD drawing of the micro channel 10 according to the three-dimensional size; specifically, after the three-dimensional size of the microchannel 10 is selected according to the structure of escherichia coli, a drawing of the structure of the microchannel 10 is drawn through a CAD drawing.

Manufacturing a mask according to a CAD drawing; specifically, a CAD drawing is handed to a third party company, and a mask is manufactured by the third party company through the CAD drawing, specifically, a mask having a three-dimensional structure is manufactured by using a film.

Forming a micro-channel 10 structure on a silicon wafer through a mask in a photoetching mode; specifically, according to the size of a mask, corresponding silicon wafers are matched to be used as a film substrate, photoresist (SU-8) is thrown to the height of a required micro-channel 10 structure by a photoresist homogenizer, and the photoresist is exposed by utilizing the mask and ultraviolet lithography principles to form a silicon wafer mold with the micro-channel 10 structure.

Spreading a liquid polydimethylsiloxane material on a silicon wafer, and placing the silicon wafer in an oven at 80 ℃ to be dried until the liquid polydimethylsiloxane material is solidified;

after the polydimethylsiloxane block is solidified, taking the polydimethylsiloxane block down from the silicon wafer, and cutting the silicon wafer according to the size of the micro channel 10;

and bonding the polydimethylsiloxane structure on the clean glass slide through plasma bonding to form the microfluidic chip.

In one embodiment, referring to fig. 3, the method for tracking cell movement using a vector comprises the following steps:

s21: diluting the cultured escherichia coli and injecting the diluted escherichia coli into the micro channel 10;

specifically, a certain amount of escherichia coli can be cultured through the bacterial culture plate, and then the escherichia coli is used for diluting the culture medium, wherein the aim of dilution is to reduce the density of the escherichia coli, so that the movement between the escherichia coli cannot interfere with each other, the tendency movement of the escherichia coli is facilitated, the observation of the movement of the escherichia coli under a microscope is facilitated, and the research and development of a bacteria fixed-point targeting tumor area are facilitated.

In a more specific example, E.coli has a cell density of 10 after dilution in the culture medium⁴-10⁷That is, within this density range, the density of the escherichia coli can be ensured to be sufficiently small, the mutual interference of the movements of the escherichia coli can be prevented, and the observation by a microscope is facilitated.

S22: sealing the sample inlet 20 and the sample outlet 30 of the microchannel 10;

specifically, the injection port 20 and the outlet port 30 are sealed by a sealing tablet, so as to prevent the rapid evaporation of water from the culture medium in the microchannel 10, which leads to the death of E.coli due to drying, and the failure of cell movement tracking.

Specifically, the sealing agent is prepared by mixing lanolin and vaseline according to a ratio of 1:1, the sealing agent is liquid at a temperature of more than 50 ℃, and the sealing agent is solidified at a temperature of less than 50 ℃, so that the sample inlet 20 and the sample outlet 30 are sealed.

S23: the movement of E.coli was observed in real time with a microscope.

Specifically, before injecting the escherichia coli into the microchannel 10, the escherichia coli is labeled with fluorescence, for example, green fluorescence, then the vector is placed under a microscope for real-time observation, and the microscope is adjusted to be magnified by 10 times to photograph the movement locus of the escherichia coli. Referring to FIG. 5, for single cell tracking identification of fluorescent bacteria under 10-fold microscope, the green dots in the figure are fluorescent bacteria. FIG. 6 is a trace diagram of the movement of E.coli under a microscope, wherein each line represents the trace diagram of E.coli in 15 seconds, thereby achieving the purpose of research.

Referring to fig. 4, the present application further provides a microfluidic chip, including a micro channel 10, wherein the height of the micro channel 10 ranges from 40 to 55 μm, the width ranges from 0.6 to 2.5mm, and the length ranges from 1 to 2 cm; the micro-fluidic chip is also provided with a sample inlet 20 and a sample outlet 30 which are communicated with the micro-channel 10, and the aperture ranges of the sample inlet 20 and the sample outlet 30 are both 0.8-1.5 mm. The micro-fluidic chip provided by the embodiment of the application not only can simulate the movement of cells in a three-dimensional space by setting the length, the width and the height of the micro-channel 10 in a certain range respectively, but also can meet the focal range of a microscope, so that the defocusing phenomenon can not occur when the cells are observed by the microscope to move, the observation visual field of the microscope is large enough, and the collected data is more authentic. In addition, the collapse phenomenon of the microfluidic chip can be prevented.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.