Single cell quality detection method
By using biomicroscope and trypsin digestion, adherent cells are converted into suspended cells, their diameters are measured and their quality is calculated, which solves the problem that existing single-cell quality detection methods require expensive instruments and poor stability, and achieves fast, simple and efficient single-cell quality detection.
Patent Information
- Application Number
- CN202411989679.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-15
AI Technical Summary
The existing single-cell quality detection methods require expensive and complex instruments, which lead to difficult detection implementation and poor stability.
Using a biological microscope that must be equipped for cell culture, adherent cells are digested into suspended spherical cells by trypsin digestion, the diameter of the suspended cells is measured, and the mass of a single suspended living cell is calculated based on the diameter.
The mass of a single suspended living cell is achieved in the existing cell culture device, the detection value is close to the most advanced "single-cell balance" results, and the mass of 500 cells can be measured per minute, with a significantly higher efficiency than traditional methods.
Smart Images
Figure CN119827381A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for detecting single cell mass. Background Art
[0002] All organisms in nature are composed of cells, which are the primary subject of biological science research. However, existing common cell biology techniques primarily target the average results of a given change in a group of cells, lacking effective means for capturing changes at the single-cell level. Single-cell mass measurement can help understand the mechanisms of cell growth regulation under various chemical-mechanical stimuli, providing an important foundation for understanding and treating diseases and developing new drugs. Furthermore, single-cell mass data can provide correction factors for determining the bioequivalent dose of boron neutron capture therapy (BNCT).
[0003] Existing single-cell mass determination methods are mainly divided into three categories based on their detection principles: microcantilever resonator method, optical method and photoelectrodynamic microfluidic platform method.
[0004] The microcantilever resonator is a resonant device with a microfluidic channel in the micromechanical method. The cell buoyancy mass is obtained by changing the resonant frequency of the microcantilever with adsorbed cells. f0 is the resonant frequency, and κ is the elastic coefficient. There are three main types of microcantilever resonators: suspended microchannel resonators, microelectromechanical system resonators, and base resonant sensors. The suspended microchannel resonator is currently the mainstream device for measuring the mass of single cells. It consists of a microcantilever with a fluid channel embedded in a vacuum-encapsulated cantilever. When the cell to be measured passes through the microfluidic channel, the cell's mass will change the vibration frequency of the cantilever. The mass of the single cell can be obtained by measuring the rate of change of the cantilever's vibration. A major representative example is the "single-cell balance," the world's first instrument that can directly and accurately measure the mass of a single living cell, developed by the Institute of Biophysics at the Swiss Federal Institute of Technology in Zurich. It can measure the mass of a single cell to 2-3 nanograms with a measurement accuracy of one trillionth of a gram. However, because the resonant frequency is extremely susceptible to environmental factors, this measurement method has relatively poor stability.
[0005] Quantitative phase microscopy, as an optical method, can be used to determine the mass of individual cells. It uses four-wave lateral shearing interferometry to measure phase changes in living cells in real time, enabling quantitative analysis of their mass. Telight has launched the Q-Phase holographic microscope, which features quantitative cell phase imaging. Through image acquisition, Q-Phase can directly measure cell mass. Other optical methods for measuring individual cell mass, including quantitative phase tomography and digital holographic microscopy, require complex optical path designs, limiting their application.
[0006] The photoelectric dynamics (PDO) microfluidic platform for measuring the mass of individual cells is based on a combination of sedimentation theory, computer vision, and particle manipulation techniques. A simple, time-controlled projected light pattern illuminates a selected area on the platform's microfluidic chip, allowing the cells to "free fall" into the selected area. Computer vision algorithms accurately track the cell's movement, linking its trajectory with sedimentation theory to rapidly determine the mass of each cell. PDO microfluidic platforms require both microfluidics and optoelectronic chip systems, and their computational complexity hinders their widespread and effective application.
[0007] In summary, in addition to their respective technical deficiencies, current single cell quality detection methods all require expensive and complex instrumentation to implement their detection, making the implementation of single cell quality detection very difficult. Summary of the Invention
[0008] The present invention provides a method for detecting the mass of a single cell. By using a biological microscope, which is essential for cell culture, the diameter of a single suspended living cell is obtained, and the mass of the single suspended living cell is calculated based on the diameter. The detection method is stable, simple, rapid, and easy to promote.
[0009] To achieve the above objectives, the present invention adopts the following technical solutions:
[0010] A method for detecting single cell quality comprises the following steps:
[0011] S1: Adherent cells were digested into suspended spherical cells using trypsin digestion;
[0012] S2: Measure the diameter of suspended cells using a biological microscope with image processing capabilities;
[0013] S3: Calculate the volume of each single cell and use the spherical volume mass formula to calculate the mass of each single cell.
[0014] In the above steps, S1 specifically includes the following steps: when the growth density of the cultured cells reaches 80-90%, discard the culture medium, gently wash the cells with PBS, add 0.25% trypsin for digestion, and add complete culture medium to terminate the digestion when the cells are observed to be in a round floating state under a microscope; gently blow the cells down, collect the cell suspension in a centrifuge tube, centrifuge at 1000 rpm for 3-5 minutes, discard the supernatant, add 8 ml of complete culture medium to the cell pellet, and blow evenly to obtain a single cell suspension.
[0015] In S2, the diameter of a single cell is the average of the maximum and minimum diameters of the measured cells.
[0016] In S3, the volume of a single cell is obtained according to the spherical volume formula, that is,
[0017] v=4 / 3π(d / 2) 3 ,
[0018] Where d is the diameter of a single cell;
[0019] Since 95% of cells are made up of water molecules, the density of water is used as the density of cells, and the mass of a single cell is the product of density and volume.
[0020] Beneficial Effects: The present invention provides a method for measuring single-cell mass. Adherent cells are digested into suspended spherical cells using trypsin digestion. Advanced image processing technology from a biological microscope, essential for cell culture, is used to directly measure the diameter of the suspended cells. The volume of each single cell is then directly calculated, and finally, the mass of each single cell is calculated. Single-cell mass measurement can be achieved using existing cell culture equipment, eliminating the need for specialized, expensive single-cell mass detection equipment. The single-cell mass values measured by the present method (3.11±1.81ng for U251 glioma cells, 6.30±2.53ng for U87 glioma cells, and 2.73±0.81ng for GL261 glioma cells) are very close to the 2-3ng value measured by the most advanced "single-cell balance." Furthermore, the present method can measure the mass of 500 suspended live cells per minute, significantly exceeding the detection efficiency of the "cell balance." Therefore, the present invention provides a stable, simple, rapid, and easily scalable method for measuring the mass of single suspended live cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is an outline image of U251 glioma cells in the BZ-X800 analysis application in an embodiment of the present invention;
[0022] Figure 2 This is an outline image of U87 glioma cells in the BZ-X800 analysis application in an embodiment of the present invention. DETAILED DESCRIPTION
[0023] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments:
[0024] The biological microscope with image processing function used below is a phase contrast inverted microscope.
[0025] In this example, the single cell mass of three types of glioma cells, U251, U87, and GL261, was measured using a Keyence BZ-800X phase-contrast inverted microscope. The specific steps are as follows:
[0026] 1. Preparation of Glioma Cell Single-cell Suspension
[0027] When the growth density of the three glioma cell cultures, U251 / U87 / GL261, reached 80-90%, the culture medium was discarded, the cells were gently washed with PBS, and 0.25% trypsin was added for digestion. When the glioma cells were observed to be floating in a round shape under a phase-contrast inverted microscope, complete culture medium was added to terminate the digestion. The glioma cells were gently pipetted and collected in a centrifuge tube. The tube was centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and 8 ml of complete culture medium was added to the cell pellet, which was pipetted evenly to obtain a single-cell suspension of glioma cells.
[0028] 2. Determination of Single Glioma Cell Diameter
[0029] Place 10cm culture dishes containing three types of glioma single-cell suspensions, U251, U87, and GL261, on the stage of a phase-contrast inverted microscope. Click "Lens" to change to a 20x objective lens. Enable the hybrid cell counting mode in the BZ-X800 analysis application and click "Hybrid Cell Count." After selecting an image, the program automatically sets the brightness threshold to be extracted. Based on the brightness of the image, a preliminary standard is set to distinguish cells from background. This standard can then be adjusted as needed to allow the program to more accurately extract cells and avoid counting background or other irrelevant parts as cells. Then, click the "Measure Result" button in the measurement result area. Click "Save Result" in the dialog box that appears. The measurement results will directly display the number of cells in the measurement area and the maximum and minimum diameters of each single cell. The diameter of a single cell is the average of the long and short diameters in the measurement results.
[0030] 3. Determination of Single Glioma Cell Quality
[0031] According to the spherical volume formula v=4 / 3π(d / 2) 3 The volume of a single cell is obtained (d is the diameter of a single cell). Since 95% of cells are composed of water molecules, the density of water (1g / cm 3 ) as the cell density, the mass of a single glioma cell is the product of density and volume. Based on the diameters of the obtained glioma cells, the mass of each glioma cell was obtained and the average value was taken. As shown in Table 1, the average mass of 99 U251 glioma cells was 3.11±1.81 ng; as shown in Table 2, the average mass of 87 U87 glioma cells was 6.30±2.53 ng; and as shown in Table 3, the average mass of 125 GL261 cells was 2.73±0.81 ng. These results are similar to the 2-3 nanograms of single cell mass measured by the "single cell balance," the world's first instrument capable of directly and accurately measuring the mass of a single living cell, developed at the Institute of Biophysics at ETH Zurich, Switzerland.
[0032] Table 1 U251 glioma cell diameter and corresponding single cell mass
[0033]
[0034]
[0035]
[0036] Table 2 Diameter of U87 glioma cells and corresponding cell mass
[0037]
[0038]
[0039]
[0040] Table 3 GL261 glioma cell diameter and corresponding cell mass
[0041]
[0042]
[0043]
[0044] The above method can also be performed using other types of biological microscopes. As long as the biological microscope has an image processing function, it can be used in the method of the present invention.
[0045] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principles of the present invention. These improvements should also be regarded as the scope of protection of the present invention.
Claims
1. A method for detecting single cell quality, characterized in that: The following steps are involved: S1: Use trypsin digestion to digest adherent cells into suspended spherical cells; S2: The diameter of suspended cells was measured using a biological microscope with image processing function; S3: Calculate the volume of each single cell based on the measured diameter, and then calculate the mass of each single cell.
2. The method for detecting single cell mass according to claim 1, characterized in that: S1 specifically includes the following steps: when the growth density of the cultured cells reaches 80-90%, the culture medium is discarded, the cells are washed with PBS, 0.25% trypsin is added for digestion, and when the cells are observed to be in a round floating state under a microscope, complete culture medium is added to terminate the digestion; the cell suspension is collected to obtain a single cell suspension.
3. The method for detecting single cell mass according to claim 2, characterized in that: After terminating the digestion, the cells were blown off, the cell suspension was collected, and centrifuged at 1000 rpm for 3-5 minutes. The supernatant was discarded, and 8 ml of complete culture medium was added to the cell pellet, and the mixture was blown evenly to obtain a single cell suspension.
4. The method for detecting single cell mass according to claim 1, characterized in that: In S2, the diameter of a single cell is obtained by taking the average of the maximum and minimum diameters of the measured cells.
5. The method for detecting single cell mass according to claim 1 or 4, characterized in that: In S2, the diameter of the suspended cells was measured using a phase contrast inverted microscope.
6. The method for detecting single cell mass according to claim 5, characterized in that: Measuring the diameter of suspended cells using a phase contrast inverted microscope specifically includes the following steps: Place the culture dish containing the single-cell suspension on the stage of the phase contrast inverted microscope, click lens to change to the 20x objective lens, enable the hybrid cell counting mode from the analysis application of the phase contrast inverted microscope, click Hybrid Cell Count, select the image type, the program automatically sets the brightness threshold to be extracted, shape the extraction threshold as needed, then click the Measure Result button in the measurement result area, click Save Result in the displayed dialog box, the measurement result will directly display the number of cells in the measurement area and the maximum and shortest diameters of each single cell. The diameter of a single cell is the average of the long and short diameters in the measurement result.
7. The method for detecting single cell mass according to claim 6, characterized in that: The extraction threshold is shaped as needed: the program sets a preliminary standard according to the brightness of the image to distinguish cells from the background, and then the standard can be adjusted according to actual needs, so that the program can extract cells more accurately and avoid counting the background or other irrelevant parts as cells.
8. The method for detecting single cell mass according to claim 1, characterized in that: In S3, the volume of a single cell is obtained according to the spherical volume formula, namely: v=4 / 3π(d / 2) 3 , Where d is the diameter of a single cell.
9. The method for detecting single cell mass according to claim 1 or 8, characterized in that: Since 95% of cells are made up of water molecules, the density of water is used as the density of cells, and the mass of a single cell is the product of density and volume.