A method for cell holding and releasing detection based on microtubule air pressure change

Abstract

The application discloses a cell suction and release detection method based on microtube air pressure change, relates to the technical field of cell operation, and comprises the following steps: S1: suction and release of cells are carried out through a microtube, and a cell suction model is established; S2: in-situ pressure of the microtube before and after suction and release of cells is modeled based on the Clapeyron equation; and S3: based on the modeling result, the suction and release of cells are detected through microtube air pressure change detection; the application obtains the air pressure change trend of the microtube in the suction and release process through air pressure modeling of the cells in the suction and release process, and realizes the detection of the suction and release operation of the cells based on the air pressure change; experimental results show that the cell suction and release detection has a high success rate, and the development ability of the cells after the operation is not affected, and the application has wide applicability; the application is free of dependence on microscopic vision for the cell suction and release detection, and significantly promotes the popularization and application of the cell operation technology in the above-mentioned occasions.

Description

Technical Field

[0001] This invention relates to the field of cell manipulation technology, and in particular to a method for detecting cell adsorption and release based on microtubule pressure changes. Background Technology

[0002] Microtubule-based cell holding and release is a crucial step in cell manipulation. Currently, cell holding and release are primarily detected through microscopic visual feedback by measuring the relative position of the microtubule opening and the cell. This reliance on microscopic vision makes traditional holding and release detection methods unsuitable for cell manipulation scenarios lacking microscopic vision. For example, in fully enclosed cell culture equipment, space constraints make it difficult to install a microscope, yet numerous cell transport operations during culture still require cell holding and release detection. Furthermore, in the manipulation of fluorescently stained cells, exposure to light fields can lead to photobleaching, making it inconvenient to provide microscopic visual feedback for these cell manipulations. To facilitate cell manipulation in these situations without visual feedback, it is essential to research cell holding and release methods that do not rely on visual feedback.

[0003] The changes in intratubular pressure caused by cell blockage and cell removal can be used to detect cell adsorption and release. However, since the microtubes need to manipulate cells in the culture medium, the flow of liquid inside the tube can interfere with the changes in intratubular pressure. Therefore, this invention provides a method for detecting cell adsorption and release based on microtube pressure changes by modeling the changes in microtube pressure caused by cell adsorption and release in the liquid environment. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention discloses a method for detecting cell adsorption and release based on microtubule pressure changes, comprising the following steps:

[0005] S1: Establish a cell holding model by holding and releasing cells through microtubules;

[0006] S2: Modeling of intratubular pressure before and after cell adsorption and release in microtubules based on the Clapeyron equation;

[0007] S3: Based on the above modeling results, the adsorption and release of cells are detected by detecting changes in microtubule air pressure.

[0008] Furthermore, in step S1, the inner diameter of the microtube is smaller than the cell diameter, and it generates a sufficiently large fluid force to cause cell movement.

[0009] Furthermore, in step S1, the model occurs during cell adsorption, and the maximum microtubule inner diameter capable of adsorbing solid and liquid cells is obtained based on the relationship between adsorption pressure and the length of the microtubule into which the cell is adsorbed.

[0010] Furthermore, in step S2, the adsorption and release of the cell involves four processes, namely: the cell is adsorbed and moves toward the tube opening, the cell is adsorbed and stuck at the tube opening, the cell is released and detaches from the tube opening, and the cell is released and detaches from the tube opening.

[0011] Furthermore, in step S2, the pressure change trend during the holding and releasing processes is determined based on the established model and through calculation.

[0012] Furthermore, in step S2, the deformation of the gas pipeline caused by temperature and pressure changes of the gas inside the pipe is ignored, and the relationship between the gas pressure and volume inside the pipe satisfies the Clapeyron equation.

[0013] Furthermore, in step S3, the cell adsorption and release procedure is as follows:

[0014] Step 1: Apply negative pressure to the microtube. When the change in air pressure slope is observed to exceed the threshold, the cell has been successfully adsorbed by the microtube opening.

[0015] Step 2: After completing the cell manipulation, positive pressure is applied to the microtube. When the change in the pressure slope exceeds a specific threshold, the cell is successfully released from the microtube opening.

[0016] Further, zebrafish egg experiments were conducted through steps S1, S2, and S3, and the success rate was statistically analyzed.

[0017] The beneficial effects of this invention compared with the prior art are as follows: (1) This invention obtains the trend of microtubular pressure change during the above process by modeling the cell pressure during the holding and releasing process, and uses this trend to realize cell holding and releasing operation detection based on pressure change. Experimental results show that this method can complete cell holding and releasing detection with a success rate of 90%, and the cell development ability after the operation is not affected; (2) This invention is implemented on a cell operating system composed of a conventional holding needle and a pneumatic injector containing a pressure sensor, and is applicable to holding and releasing detection in typical cell operations including cell injection, enucleation, and plucking, and has wide applicability; (3) In the method of this invention, cell holding and releasing detection is freed from the dependence on microscopic vision, so it can be applied to occasions where microscopic vision cannot be provided, such as fully enclosed cell workstations and all-black-box staining cell operating systems, which will significantly promote the application of cell manipulation technology in the above occasions. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the overall process of the present invention.

[0019] Figure 2This is a schematic diagram of the cell holding model of the present invention (where a is a liquid cell holding model; b is a solid cell holding model).

[0020] Figure 3 This is a schematic diagram of the cell adsorption and release process of the present invention (where a is cell aspiration (before adsorption); b is cell adsorption; c is cell release (before detachment from microtubules); d is cell adsorption (after detachment from microtubules)).

[0021] Figure 4 These are microscopic images and corresponding pressure change curves of zebrafish egg adsorption and release processes in an embodiment of the present invention.

[0022] Figure 5 These are microscopic images of cell culture as described in this invention (where ac is a microscopic image of cell culture after the holding and releasing operation; df is a microscopic view of cell culture without the holding and releasing operation). Detailed Implementation

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0024] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0025] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0026] Example: Figure 1As shown, a method for detecting cell adsorption and release based on microtubule pressure changes includes the following steps:

[0027] S1: Establish a cell holding model. The cell operating system consists of a conventional holding needle and a pneumatic injector containing a pressure sensor. The cell operating system determines the maximum microtubule inner diameter that can hold cells by the relationship between holding pressure and the length of the cell drawn into the microtubule.

[0028] S2: Based on the Clapeyron equation, model the intratubular pressure before and after microtubule cell adsorption and release to determine the pressure change trend during adsorption and release.

[0029] S3: Based on the above modeling results, the adsorption and release of cells are detected by detecting changes in microtubule air pressure.

[0030] In step S1 of this embodiment: the inner diameter of the microtube is smaller than the diameter of the cell, thereby preventing the cell from entering the microtube under the action of the holding pressure, and the inner diameter of the microtube can generate sufficient fluid force to make the cell move. Therefore, a cell holding model is established, and the maximum inner diameter of the microtube that can hold solid and liquid cells is obtained according to the relationship between the holding pressure and the length of the cell being drawn into the microtube.

[0031] For cells that exhibit fluid properties, such as Figure 2 (a) The suction pressure P required to aspirate cells into a micropipette A It can be calculated as follows:

[0032]

[0033] Where T C Cortical tension, R, can be obtained through microtubule aspiration. C and R P These are the outer radius of the microtubule and the inner radius of the microtubule, respectively. According to the continuous droplet model in equilibrium, when L... A Equal to R P At that time, P A The value reaches its peak. After that moment, according to (1), since R C As the diameter of the microtubule R decreases, the required suction pressure will decrease. According to (1), the inner diameter R of the microtubule that can block the cell... Pmax The maximum value is:

[0034]

[0035] Where P Amax It is the maximum suction pressure provided by the pump. When L A Equal to R P At that time, obtain R Pmax The key parameter is R CThe value of . Assuming the cell volume remains constant during adsorption, then:

[0036] V0 = V O +V In =4πR0 3 / 3 (3)

[0037] Where V0 is the volume of the cell before aspiration, R0 is the radius of the cell before aspiration, and V O and V In These represent the cell volume outside the microtubule and the cell volume inside the microtubule, respectively. Observations revealed that the cells inside the microtubule are roughly hemispherical. For example... Figure 2 As shown in (a), the cellular portion outside the micropipette also approximates a sphere cut by the plane of the micropipette opening. Therefore, V O and V In It can be calculated as follows:

[0038]

[0039] R C The value of can be obtained through (3), (4), and (5). Then, according to (2), we know that R Pmax The value of .

[0040] For cells that exhibit solid properties, such as Figure 3 (b) According to the continuous solid model under equilibrium conditions, P A and L A The relationship between them remains linear throughout the microtubule aspiration process. This linear relationship is defined as follows:

[0041]

[0042] E is the Young's modulus of the cell, and Φ is a term that is weakly dependent on the ratio of the wall thickness to the microtubule radius. A typical value for Φ is 2.1.

[0043] like Figure 2 (b) When the entire cell is drawn into the microtubule, its volume can be expressed as:

[0044]

[0045] If the cell volume is considered constant during aspiration, then L A The value is:

[0046]

[0047] According to (7), R Pmax The value is calculated using (6) and (8):

[0048]

[0049] In step S2 of this embodiment, cell adsorption and release involve four processes: cell adsorption and movement towards the tube opening, cell adsorption and jamming at the tube opening, cell release before detachment from the tube opening, and cell release and detachment from the tube opening. The cell adsorption process model is as follows: Figure 3 (a) and Figure 3 As shown in (b), ignoring the temperature change of the gas during cell aspiration, the pressure P(t) and the volume V(t) within the channel can be expressed by the following formula:

[0050] P0V0=P(t)V(t) (10)

[0051] Where P0 and V0 are the initial pressure and volume of the gas channel when cell aspiration begins, respectively. The value of V(t) during cell holding can be determined by the velocity v at the gas-liquid interface (GLI). G (t) and the moving speed v of the air pump piston T (t) is obtained from the following formula:

[0052]

[0053] Where R T and R P These are the radius of the pump tube and the inner diameter of the micropipette, respectively. Therefore, the pressure can be expressed as:

[0054]

[0055] The rate of change of air pressure before blockage can be obtained:

[0056]

[0057] Where t B This is the moment when cells are held captive by the microtubule openings, leading to blockage. (via v) T and v G Determine the rate of pressure drop before blockage. Due to the buffering effect of the gas passage caused by the deformation of the plastic tube and the resistance between the liquid and the inner surface of the microtube, the movement of the GLI lags behind the movement of the pump piston, resulting in an increase of V in (11), a decrease of P(t) in (12), and a negative value of P′(t) in (13). According to fluid mechanics theory, as the flow velocity increases, the flow resistance increases, leading to a decrease in V. G The upper limit of v. P When approaching the upper limit, the microtubule radius R P R has a significant impact on the rate of change of P in (12). P The larger the value, the slower the rate of decrease of P before absorption.

[0058] After the cell is adsorbed, the GLI's migration velocity v GReduce to zero. For example... Figure 3 As shown in (b), the volume of gas channel V can be obtained from the following formula:

[0059]

[0060] V(t B t is the volume of the gas passage at the moment of blockage. S This is the moment the piston stops. According to (12) and (13), the pressure P and its rate of change are respectively:

[0061]

[0062] P(t B ) and V(t B (15) and (16) represent the pressure and volume of the gas in the channel when the cell is blocked. According to (15) and (16), P continues to decrease after the tube opening is blocked by the cell. The rate of decrease of V is determined by the piston's velocity after the blockage. T Decision. According to (11) and (14), the rate of increase of V after the pipe is blocked is greater than the rate of increase of V before the blockage. Therefore, according to (12), (13), (15) and (16), at the same v T Below, after the tube opening is blocked, the rate of pressure decrease increases. Therefore, the increase in the rate of pressure decrease during cell adsorption can be used to determine the cell adsorption status. As mentioned above, R P The larger the value, the smaller the rate of descent of P before suction. Simultaneously, the rate of increase of V is solely determined by the piston's velocity after blockage. T Decision. Therefore, R P The larger the value, the greater the difference in the rate of pressure drop before and after adhesion. This means that R... P It should be large enough to produce a detectable difference in the rate of pressure drop, thus determining whether the cell has been adsorbed.

[0063] Cell release process model, such as Figure 3 (c) and Figure 3 (d) Due to the adhesion between the cells and the inner surface of the micropipette, the blockage at the microtube opening will persist for a period of time, even after the piston has been pushed forward to generate a positive pressure P in the gas channel. Figure 3 As shown in (c), the volume of gas channel V can be obtained from the following formula:

[0064]

[0065] Where V0 is the initial volume of the gas channel before the piston begins to move forward. Before the cell leaves the microtubule, the pressure P and its change P′ satisfy:

[0066]

[0067] Where v T P0 is the forward movement speed of the piston, and P0 and V0 are the initial pressure and volume of the gas passage before it is pushed back, respectively. According to (18), the pressure in the gas passage increases before the cell is released.

[0068] When P increases sufficiently to overcome the adhesive forces, the cell is released. After this moment, the volume V of gas in the channel can be obtained according to the following formula:

[0069]

[0070] like Figure 3 (d), the corresponding pressure and its rate of change are obtained from the following formula:

[0071]

[0072] Where P(t) R ) and V(t R ) represents the initial pressure and volume of the channel immediately after cell release. According to (17), since the pressure has increased for a period of time before cell release, according to (21), the pressure at cell release is v T Large enough to produce a negative value P ′ This means that P begins to decrease at the moment cell release occurs. Therefore, the decrease in the P value can be used as a basis for determining the timing of cell release.

[0073] In step S3 of this embodiment: the cells used in the experiment are zebrafish eggs with a diameter of 1 mm, and a micropipette with a size of 1.00 mm × 0.80 mm is selected to meet the inner diameter range in S1 and S2; microscopic vision is used as an auxiliary verification method in the experiment, and the microscopic view of the experimental process, the air pressure change curve, and the air pressure change rate curve calculated every three points are as follows: Figure 4 As shown. The specific operating procedure is as follows: apply negative pressure to the microtube; when the change in air pressure slope exceeds the threshold, it is considered that the cells have been successfully adsorbed to the tube opening, such as... Figure 4 (de); After completing the cell manipulation, positive pressure is applied to the microtube. When the change in the pressure slope exceeds a specific threshold, the cell is considered to have been successfully released, such as... Figure 4 (fg).

[0074] To verify the cell adsorption and release detection effect based on microtubule air pressure changes proposed in this embodiment of the invention, 30 zebrafish eggs were selected and the operation method of the embodiment was used for the experiment, and the success rate was statistically analyzed. The results are shown in Table 1. The manipulated cells and unmanaged cells were cultured separately, and their development was observed at 24h, 48h, and 72h after the experiment to verify that the proposed method does not affect normal cell development. The development results are shown in Table 1. Figure 5As shown in the figure, all manipulated cells develop normally, i.e., the development rate is 100%.

[0075] If no obvious morphological changes or structural alterations are observed in the cells, they are considered to be developing normally.

[0076]

[0077] Table 1

[0078] As shown in Table 1, the success rate of cell adsorption and release detection based on microtube pressure changes in the embodiments of the present invention reached 90%. The three zebrafish eggs that failed to be detected were smaller than normal fish eggs, which prevented the fish eggs from forming an effective seal with the microtube, making it impossible to form a significant pressure change before and after adsorption, ultimately leading to detection failure.

[0079] In summary, this invention realizes a cell adsorption and release detection method based on microtubule air pressure changes. This method does not rely on microscopic visual feedback and has a detection success rate of 90%.

[0080] Furthermore, it should be noted that the shapes and names of the parts and components described in the specific embodiments described in this specification may differ. All equivalent or simple variations made based on the concept, structure, features, and principles of this invention are included within the protection scope of this invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the structure of this invention or exceed the scope defined in these claims, all of which should fall within the protection scope of this invention.

Claims

1. A method for detecting cell adsorption and release based on microtubule pressure changes, characterized in that, Includes the following steps: S1: Establish a cell holding model by holding and releasing cells through microtubules; In step S1, the model occurs during cell adsorption, and the maximum inner diameter of the microtube capable of adsorbing solid and liquid cells is obtained based on the relationship between adsorption pressure and the length of the microtube into which the cell is adsorbed. S2: Modeling of intratubular pressure before and after cell adsorption and release in microtubules based on the Clapeyron equation; In step S2, the pressure change trend during the holding and releasing process is determined based on the established model and through calculation. In step S2, the deformation of the gas pipeline caused by temperature and pressure changes of the gas inside the pipe is ignored, and the relationship between the gas pressure and volume inside the pipe satisfies the Clapeyron equation. S3: Based on the above modeling results, cell adsorption and release are detected by detecting changes in microtubule air pressure; In step S3, the cell adsorption and release process is as follows: Step 1: Apply negative pressure to the microtube. When the change in air pressure slope is observed to exceed the threshold, the cell has been successfully adsorbed by the microtube opening. Step 2: After completing the cell manipulation, positive pressure is applied to the microtube. When the change in the pressure slope exceeds a specific threshold, the cell is successfully released from the microtube opening.

2. The cell adsorption and release detection method based on microtubule gas pressure changes according to claim 1, characterized in that, In step S1, the inner diameter of the microtube is smaller than the cell diameter, and a sufficiently large fluid force is generated to move the cell.

3. The cell adsorption and release detection method based on microtubule pressure changes according to claim 1, characterized in that, In step S2, the adsorption and release of the cell involves four processes: the cell is adsorbed and moves toward the tube opening, the cell is adsorbed and stuck at the tube opening, the cell is released and detaches from the tube opening, and the cell is released and detaches from the tube opening.

4. The cell adsorption and release detection method based on microtubule pressure changes according to claim 1, characterized in that: The zebrafish egg experiment was conducted through steps S1, S2, and S3, and the success rate was statistically analyzed.

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