阅读说明：本技术 一种细胞切向粘附力的自动化测量方法 (Automatic measurement method for cell tangential adhesion ) 是由 孙明竹 赵新 李璐 姚亚彤 于 2020-05-07 设计创作，主要内容包括：本发明公开一种细胞切向粘附力的自动化测量方法,包括以下步骤：将细胞测量区和洗针区设置于同一个培养皿中,微管在洗针区润洗后移动至细胞测量区；微管自动移动至细胞上方,并使气液交界面移动至微管前端；在微管下降的过程中进行自动化接触检测,待微管接触细胞表面时,微管自动停止下降；减小微管内平衡压,记录细胞与微管间刚形成密封时和细胞刚被吸进微管时的平衡压压强值,计算细胞切向粘附力。本发明将细胞测量区和洗针区设置于同一个培养皿,免去卸下微管进行润洗的步骤；通过检测微管下降过程中的运动情况判断微管是否与细胞接触,剔除人为因素的影响；通过平衡压测量吸附力,计算细胞切向粘附力,实现了细胞切向粘附力的自动化测量。(The invention discloses an automatic measurement method of cell tangential adhesion, which comprises the following steps: arranging the cell measuring area and the needle washing area in the same culture dish, and moving the microtube to the cell measuring area after the microtube is wetted in the needle washing area; the micro-tube automatically moves to the upper part of the cell and the gas-liquid interface moves to the front end of the micro-tube; carrying out automatic contact detection in the process of descending the microtube, and automatically stopping descending the microtube when the microtube contacts the cell surface; reducing the equilibrium pressure in the microtube, recording the equilibrium pressure intensity values when a seal is just formed between the cell and the microtube and when the cell is just sucked into the microtube, and calculating the tangential adhesion of the cell. According to the invention, the cell measuring area and the needle washing area are arranged on the same culture dish, so that the step of removing the microtube for rinsing is omitted; whether the microtubules are in contact with the cells or not is judged by detecting the movement condition of the microtubules in the descending process, and the influence of human factors is eliminated; the adsorption force is measured through the equilibrium pressure, the cell tangential adhesion force is calculated, and the automatic measurement of the cell tangential adhesion force is realized.)

1. An automated method for measuring cell tangential adhesion, comprising: the method comprises the following steps:

a. arranging the cell measuring area and the needle washing area in the same culture dish, and moving the microtube to the cell measuring area after the microtube is fully wetted in the needle washing area;

b. the micro-tube moves to the upper part of the cell and the gas-liquid interface moves to the front end of the micro-tube;

c. carrying out automatic contact detection in the process of descending the microtube, and stopping descending the microtube when the microtube contacts the cell surface;

d. reducing the equilibrium pressure in the microtube, recording the pressure value of the equilibrium pressure between the cell and the microtube when a seal is formed and the equilibrium pressure when the cell is sucked into the microtube, and calculating the tangential adhesion of the cell.

2. The method for automated measurement of cell tangential adhesion of claim 1, wherein: in the step a, the cell measuring area and the needle washing area are placed in the following modes: inoculating cells to be detected on a culture dish; dropping a proper amount of pancreatin on the half piece of cover glass; sticking the culture dish and the cover glass on another larger culture dish cover by distilled water in parallel respectively to ensure that no air bubbles exist between the culture dish and between the culture dish and the cover glass; and the movement direction of the microtube in the cell measuring area and the needle washing area is vertical to the microtube.

3. The method for automated measurement of cell tangential adhesion of claim 1, wherein: in step a, the measuring microtubes are continuously sucked, rinsed and rinsed in the needle washing area by using an electric injector.

4. The method for automated measurement of cell tangential adhesion of claim 1, wherein: in the step c, the movement of the microtubules and the cells is detected by using a movement history image method, and specifically, whether the microtubules contact the cells is judged by using whether the pixel average value of the moving part reaches a threshold value.

5. The method for automated measurement of cell tangential adhesion of claim 1, wherein: in step d, the value of the cell tangential adhesion is obtained by the following formula:

wherein r represents the inner radius of the microtube, α represents the angle between the x-axis and the microtube, and P_ICDenotes the equilibrium pressure immediately after the formation of the seal, P' denotes the equilibrium pressure immediately after the cell is taken into the microtube, σ denotes the surface tension coefficient of the culture solution, β_Cβ' shows the contact angles of the gas-liquid interface with the tube wall, R, respectively, immediately after the seal is formed and immediately after the cells are taken into the microtubes_CAnd R' respectively represent the inner wall radius of the micro-tube at two moments.

Technical Field

The invention belongs to the technical field of cell-level measurement, and particularly relates to an automatic measurement method for tangential cell adhesion.

Background

The cell adhesion is closely related to the physiological processes of cell growth, differentiation and the like, and the measurement of the cell adhesion has important significance in the medical fields of researching the mechanical properties of cells, and researching the tumor formation process, even the canceration of cells and the like. At present, methods for measuring cell adhesion are diversified, for example, techniques such as a microtube holding method, an optical tweezers method, a magnetic tweezers method, an environmental scanning electron microscope, and an atomic force microscope. Among them, the microtubule holding method is widely used because of its low equipment requirement, little damage to cells, and high integration with other instruments.

The traditional microtubule holding method is used for measuring the tangential adhesion of cells, in order to reduce the friction between microtubules and cells, the microtubules are required to be detached and washed in pancreatin when measuring one cell, and the measuring efficiency is greatly reduced. Meanwhile, in the measurement process, the relative height of the tip of the microtube and the cell at the holding position is judged by the experience of an operator, and an objective standard is lacked, so that the requirement on the operator is high, and the measurement result has great uncertainty due to the influence of subjective factors. In addition, in the conventional measurement process, a gas-liquid interface is formed between the gas in the pneumatic injector and the liquid in the culture dish due to capillary force, and the liquid at the gas-liquid interface is subjected to the capillary force, so that the measurement result is greatly influenced. Therefore, it is necessary to design a fast method for automatically measuring the tangential adhesion of cells, which can automatically detect the descending height of the microneedle and eliminate the influence of capillary force.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides an automatic measuring method for the cell tangential adhesion, which can be used for automatically detecting the contact between a microtube and a cell and eliminating the influence of capillary force on a measuring result without the processes of dismounting and rinsing the microtube, and can be used for quickly and accurately measuring the cell tangential adhesion.

The technical scheme adopted by the invention is as follows: an automated method for measuring cell tangential adhesion, comprising the steps of:

a. arranging the cell measuring area and the needle washing area in the same culture dish, continuously sucking and fully rinsing the measuring micro-tube in the needle washing area by using an electric injector, and then moving the micro-tube to the cell measuring area;

the cell measuring area and the needle washing area are placed in the following modes: inoculating cells to be detected on a culture dish; dropping a proper amount of pancreatin on the half piece of cover glass; sticking the culture dish and the cover glass on another larger culture dish cover by distilled water in parallel respectively to ensure that no air bubbles exist between the culture dish and between the culture dish and the cover glass; and the movement direction of the microtube in the cell measuring area and the needle washing area is vertical to the microtube.

b. The micro-tube moves to the upper part of the cell and the gas-liquid interface moves to the front end of the micro-tube;

adjusting the measuring microtube to be 10-20 μm higher than the cell in the z direction, and moving the microtube to the upper part of the cell in the x-y plane; and controlling the electric injector to continuously apply positive pressure to move the gas-liquid interface to the front end of the micro-tube so as to balance the capillary force.

c. Carrying out automatic contact detection in the process of descending the microtube, and stopping descending the microtube when the microtube contacts the cell surface;

the method comprises the steps of detecting the movement of the microtubules and the cells in the descending process of the microtubules, and judging whether the microtubules contact the cells or not by acquiring a region of interest (ROI), detecting the movement of the microtubules and the cells in the region by using a movement History Image (ROI) method, and judging whether the pixel average value of the moving part in the region of interest reaches a proper threshold value or not. In motion detection, only the part where motion occurs appears as a white pixel. When the microtubules contact the cell surface, the cell will have motion that is difficult to distinguish by the naked eye, and the number of white pixels in the ROI will increase.

d. Controlling an injector to reduce the equilibrium pressure in the microtube, enabling the cells to be completely sucked from the bottom surface of the culture dish and enter the microtube, recording the pressure intensity values of the equilibrium pressure when the cells and the microtube just form a seal and the equilibrium pressure when the cells are just sucked into the microtube, measuring the adsorption force of the microtube on the cells by using the equilibrium pressure to replace the pressure of the injector, and calculating the tangential adhesion force of the cells;

the value of the cell tangential adhesion was obtained by the following formula:

wherein r represents the inner radius of the microtube, α represents the angle between the x-axis and the microtube, and P_ICDenotes the equilibrium pressure immediately after the formation of the seal, P' denotes the equilibrium pressure immediately after the cell is taken into the microtube, σ denotes the surface tension coefficient of the culture solution, β_Cβ' shows the contact angles of the gas-liquid interface with the tube wall, R, respectively, immediately after the seal is formed and immediately after the cells are taken into the microtubes_CAnd R' respectively represent the inner wall radius of the micro-tube at two moments.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the invention, the cell measuring area and the needle washing area are arranged in the same culture dish, so that the step of removing the microtube for rinsing is omitted, and the measuring efficiency is improved;

2. the invention judges whether the microtubule is in contact with the cells or not by detecting the movement condition of the microtubule in the descending process, determines the optimal height for measurement, eliminates the influence of human factors, and improves the measurement speed and accuracy;

3. the invention measures the adsorption force through the equilibrium pressure, calculates the cell tangential adhesion force, realizes the automatic measurement of the cell tangential adhesion force, and has high measurement speed and high accuracy of the measurement result.

Drawings

FIG. 1 is a flow chart of the present invention;

FIG. 2 is a schematic view of the needle wash and measurement area placement of the present invention;

FIG. 3 is an image of automated microtube motion detection of the present invention;

FIG. 4 is a schematic view of the force analysis of the present invention upon formation of a seal between a cell and a microtubule;

FIG. 5 is a schematic view of the force analysis of the cells of the present invention upon being drawn into the microtubules;

FIG. 6 is an image of the present invention with cells being aspirated off the bottom surface of the culture dish.

Detailed Description

In order to make the technical solutions of the present invention better understood, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The embodiment of the invention discloses an automatic measuring method of cell tangential adhesion, which comprises the following steps as shown in figure 1:

(1) obtaining mouse osteoblasts;

the mouse osteoblasts used in this example were supplied from the relevant partner, and after the cells were taken, they were placed at 37 ℃ in 5% CO₂And culturing in an incubator with 100% humidity. The culture medium was changed every 2-3 days, and the complete medium consisted of 89% DMEM (high sugar), 10% FBS and 1% diabody, which was added to the petri dish after being preheated for 10 minutes in a 37 ℃ water bath. And (3) carrying out passage when the cell is cultured to reach the density of more than 80 percent: sucking the cell culture medium to be abandoned; adding 2-3ml of sterile PBS into the culture dish, slightly shaking the culture dish to soak and wash the culture dish, and then sucking and discarding the culture dish; adding 1ml of pancreatin, slightly shaking the culture dish to immerse the pancreatin at all parts of the bottom of the dish, covering the dish, putting the dish into an incubator for digestion, observing cells under a microscope after 3 minutes, and adding 3ml of preheated complete culture medium to stop digestion if most cells are not attached to the wall any more; blowing and beating the cells on the bottom surface of the culture dish by using a pipette to ensure that the cells completely fall off; collecting cell suspension, centrifuging at 1000rpm for 3 min, discarding supernatant, adding 2ml complete culture medium, blowing the dividing plate, adding 9-10ml complete culture medium, shaking the culture dish lightly to distribute cells uniformly, and culturing the cells in an incubator. Experiments were performed after 24 hours of culture.

(2) Placement of the wash needle with the measurement area, as shown in FIG. 2;

in this example, mouse osteoblasts were seeded on a 3cm dish, pancreatin was dropped on a half clean cover glass, and the cover glass and dish were stuck to a 6cm dish cover with distilled water and placed on a stage together. Before the experiment, the electric injector continuously pumps and rinses the microtube so as to reduce the influence of the friction force between the inner wall of the microtube and cells. After the micro-operation arm and the object stage are fully rinsed, the micro-operation arm and the object stage are controlled to enable the micro-tube to rapidly move to the area to be measured.

(3) Automated microtube motion detection, as shown in FIG. 3;

in this embodiment, positive pressure is applied to move the gas-liquid interface (GLI) behind the front end of the microtube as the microtube is moved into position in the x-y plane. At this time, the height of the microtube is 10-20 μm different from that of the cell, and the movement detection of the microtube and the cell is started. The method comprises the steps of obtaining a region of interest (ROI), detecting the movement of the microtubules and the cells in the region by a movement History Image (Motion History Image), and judging whether the average value of pixels of the moving part in the region of interest reaches a proper threshold value to judge whether the microtubules contact the cells. Fig. 3(a) is a selected image of a region of interest (ROI), fig. 3(b) is an image of a part of microtubules and cells for motion detection, and fig. 3(c) is a motion of the microtubules and cells in the current frame ROI. In motion detection, only the part where motion occurs appears as a white pixel. When the microtubules contact the cell surface, the cell can generate movement which is difficult to distinguish by naked eyes, the number of white pixel points in the ROI can be increased, and through experiments, when the average value of images in the ROI exceeds 10, the microtubules are judged to be in contact with the cell, and the microtubules reach the target position.

(4) Measurement of cell tangential adhesion based on equilibrium pressure model, as shown in fig. 4, 5;

and applying positive pressure to balance capillary force, moving the GLI to the front end of the microtube, and controlling the electric injector to slowly reduce the balance pressure after the microtube reaches a target position so as to obtain the adsorption force of the microtube on the cell.

FIG. 4 is a schematic view of the force analysis just before the seal between the cell and the microtubules is formed due to the decrease in equilibrium pressure, which is as follows:

P_IC＝2σcosβ_C/R_C

wherein, β_CDenotes the angle of contact of GLI with the tube wall when the seal is formed, R_CIndicating the radius of the inner wall of the microtube at GLI when the seal is formed.

Fig. 5 is a schematic diagram of force analysis when a cell is just sucked into a micro-tube, and at this time, the equilibrium pressure is P ', since the cell seals the micro-tube, the liquid outside the micro-tube cannot enter the micro-tube, and a holding pressure Δ P is formed at the micro-tube opening, and P' is represented by the following formula:

P′＝2σcosβ′/R′-ΔP

where β 'and R' are the contact angle and inner wall radius at GLI as soon as the cell is taken up in the microtubule.

The holding pressure Δ P obtained by working out the above two formulae is:

ΔP＝(P_IC-P′)+2σ(cosβ′/R′-cosβ_C/R_C)

in this embodiment, the objective lens used in the operation has a large magnification, so that the length of the microtube in the field of view is limited, and the inner diameter of the microtube does not change much in a range of small length. Therefore, assuming that the inner diameter of the micro-tube is constant at all times, the capillary pressure is not changed, i.e. the second term of the above equation is zero. The holding pressure Δ P can be obtained by the following equation:

ΔP＝P_IC-P′

(5) calculating the tangential adhesion force of the cells;

the value of the cell tangential adhesion can be obtained according to the following formula:

P_τ＝πr²p/cosα

where p is the pressure on the cell, r represents the radius in the microtubule orifice and α represents the angle of the x-axis to the microtubule, 30 °.

The holding pressure Δ P is the pressure P acting on the cell, and Δ P is substituted to obtain the final measurement formula:

P_τ＝πr²(P_IC-P’)/cosα

FIG. 6 is an image of cells being aspirated from the bottom of a culture dish during an experiment; FIG. 6(a) is an image of the cell and the microtube just before the seal is formed in the experiment, and P corresponds to P in the image_IC21.329kPa, FIG. 6(b) is an image of a cell immediately after it was taken into a microtube, and P' in the image is 9.793kPa, and the inner radius of the microtube opening in both images is 7.935 μm, and the tangential adhesion of the cell in the image is calculated to be 2.635 × 10^-1Dyne. to reduce the effect of measuring multiple cells on the inner wall of the microtube, one microtube was replaced for every three to four cells measured in this example, the tangential adhesion of 5 cells was measured using this method and found to be 3.231. + -. 0.845 × 10^-1dyne, and literatureThe human osteoblast adhesion measured in (1) is consistent, and the operating speed of the method is about 4 times that of the existing method (the traditional method: 20-30min/cell, the method of the invention: 5-7 min/cell).

The present invention has been described in detail with reference to the embodiments, but the description is only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The scope of the invention is defined by the claims. The technical solutions of the present invention or those skilled in the art, based on the teaching of the technical solutions of the present invention, should be considered to be within the scope of the present invention, and all equivalent changes and modifications made within the scope of the present invention or equivalent technical solutions designed to achieve the above technical effects are also within the scope of the present invention.