CN111751522B - Cell detection analyzer and detection method of cell proliferation information - Google Patents
Cell detection analyzer and detection method of cell proliferation information Download PDFInfo
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- CN111751522B CN111751522B CN201910237323.5A CN201910237323A CN111751522B CN 111751522 B CN111751522 B CN 111751522B CN 201910237323 A CN201910237323 A CN 201910237323A CN 111751522 B CN111751522 B CN 111751522B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Abstract
The application discloses a method and a device for monitoring and analyzing the whole process of cell proliferation, saturation and death in real time. The method mainly comprises the steps of amplitude value ranges of a first area, a second area and a third area, and a mode and a rule for analyzing data: generating a first segmentation range according to the value range of the first region, and determining the amplitude change in the range according to the first segmentation range; based on the range of amplitude trends, the method used to classify and calculate the data is selected to establish the number and rate of cell proliferation. And determining the amplitude stability according to the value range of the second area, thereby establishing the cell saturation state. Generating a second segmentation range according to the value range of the third region, and determining the amplitude change in the range according to the second segmentation range; the method used to classify and calculate the data is selected based on the range of amplitude trends, thereby establishing the number and rate of cell death.
Description
Technical Field
The application relates to the field of experimental data analysis, in particular to a novel method for real-time and quantitative detection and analysis of cell proliferation information.
Background
The whole process of cell proliferation, saturation and death reveals the whole process of cell life, and has important significance and effect on the research of life bodies. Cell proliferation is one of the important physiological functions of cells, and is an important vital sign of organisms. Proliferation of cells is the basis for the growth, development, propagation and inheritance of organisms. Once living cells are stimulated from the outside, proliferation may be affected. When the body is exposed to environmental factors such as radiation, poisons, etc., the proliferation of normal cells of the body may change. Cancer cells are a class of cells with unlimited proliferation capacity, whose proliferation characteristics are markedly different from those of normal cells. Stem cells are a class of pluripotent cells with self-replicating capacity, with potentially great advantage in applications for restoring cells damaged by major diseases. In the induction of stem cell differentiation, the condition of cell proliferation is an important factor to be monitored. Based on the significance of cell proliferation, with the rapid development of biomedicine, cell proliferation assay is of great interest. Cell death refers to the cell reaching the end of life in an irreversible state. The matrix can regulate normal function by cell death, maintaining intracellular homeostasis. Cell death is also closely related to the occurrence of diseases, and once the cell death regulatory mechanism is disturbed, it causes the occurrence of related diseases including autoimmune diseases, neurodegenerative diseases, cancers and the like.
Whether to detect the clinic drug motor reagent, induce stem cell proliferation differentiation or analyze cell growth regulating factor, the detailed cell activity state detection is necessary, so the comprehensive and accurate cell proliferation evaluation method is important.
The cell proliferation detection method commonly used at home and abroad at present is based on the influence of drugs on cells, and is generally used for detecting the number of cells in division or the change of cell populations, and is mainly divided into four categories: DNA synthesis assay, metabolic activity assay, cell proliferation-related antigen assay and ATP concentration assay. Cell proliferation is assessed by cell marker recognition, quantitative statistics, time-point analysis methods, such as: MTT method, brdu detection method, hydroxy fluorescein diacetate succinimidyl ester (CFSE) detection method, etc., but the chemical staining agent or fluorescent indicator used in these measurement methods can cause permanent damage to cells, and the measurement result at the instant time point can not meet the requirement of cell proliferation stage investigation, so that the whole cell proliferation process can not be observed and detected in real time, accurately, continuously and effectively.
Disclosure of Invention
To overcome the shortcomings of the traditional cell proliferation detection method: 1) Counting the number of cells based on a cell staining method, wherein the result is not objective; 2) Trace methods using conventional optical indicators can cause permanent damage to cells; 3) The invention discloses a device and a method for real-time, objective and quantitative detection and analysis of the whole process of cell proliferation, saturation and death, aiming at realizing continuous, long-time, real-time, quantitative and multi-mode dynamic monitoring by adopting a time point analysis method only.
The inventor of the invention utilizes a mechanical vibration system to monitor amplitude change in real time, and realizes real-time, objective and quantitative monitoring of cell states through the relationship between cell adhesion and activity thereof, thereby achieving the purpose of analyzing cell proliferation, saturation and death in the whole process.
Most mammalian cells grow both in vivo and in vitro by attachment to a substrate, which may be other cells, collagen, glass or plastic, etc. Cultured cells are generally spheroid-like before being unattached to a substrate; when attached to a substrate, the cells gradually expand to form a certain shape, such as a fibroblast-like or epithelial cell-like shape. Important growth characteristics of in vitro cultured cells are: attachment and extension, and density limitations of contact inhibition and growth. Attached and stretched are the basic growth characteristics of most in vitro cultured cells.
The inventors of the present invention have ingeniously utilized the relationship between cell status and cell and shake table adhesion. The number of cells on the platform increases due to cell proliferation, and the amplitude (amplitude) of vibration increases at this time; while as the cells die, adhesion decreases and the number of cells on the platform decreases, at which time the amplitude decreases. The stronger the factor affecting the proliferation or death of the cell, the larger the amplitude change trend, and the degree of the factor can be obtained through quantitative analysis of the data.
An aspect of the present invention provides a cell detection analyzer including at least a data acquisition unit, a data analysis unit, and a data processing unit;
the data acquisition unit comprises a vibration platform and a recording component, cells are attached to the vibration platform and vibrate together with the vibration platform, and the recording component records that the phase deviation generated by the vibration platform is the vibration amplitude A;
the data analysis unit calculates the rate of change of the cell number on the platform based on the rate of change of the amplitude;
the data processing unit determines the whole process of cell proliferation, saturation and death according to the calculation result of the analysis unit.
In a preferred embodiment, the vibration platform applies vibration excitation to the cells to be measured attached to the vibration platform, and the generated excitation signal is amplified and recorded as the amplitude a by the recording means.
In a preferred embodiment, the data acquisition unit comprises a first region, a first segmentation limit, a second region, a second segmentation limit, and a third region;
the first region includes at least one time period of a region of 0.ltoreq.Δa=a/t <10% ×a;
the first division range includes ΔA>10%. Times.A, and t n+1 >t n And A (t) n+1 )>t( n ) At least one time period of the region of (2);
the second region includes at least one time period of 0.ltoreq.ΔA;
the second division range includes ΔA<0, and t n+1 >t n And A (t) n+1 )<t( n ) Is at least one time period of (1);
the third region includes at least one time period of 0.ltoreq.Δa=a/t <10% ×a.
In a preferred embodiment, the rate of change of amplitude ΔA is selected>10%. Times.A, and t n+1 >t n And A (t) n+1 )>t( n ) Generating a first segmentation range, wherein a starting point of the first segmentation range is a first rate change point, and an end point of the first segmentation range is a last rate change point;
the first segmentation limit includes cell proliferation information.
In a preferred embodiment, the second area is from the end of the first division range to the end of the second division range, the area selects the amplitude change rate 0.ltoreq.Δa <10% ×a, and the start point of the second division range is the end of the first division range;
the second segmentation limit includes cell saturation information.
In a preferred embodiment, the third area is from the end of the second division range to the end of the third division range, the area selects the amplitude change rate Δa <0, and the start point of the third division range is the end of the second division range;
the third split range includes cell death information.
In a preferred embodiment, the rate of change of amplitude Δa is indicative of a cellular process related to:
when Δa= >10% ×a, a cell proliferation process is indicated;
when 0.ltoreq.ΔA < 10%. Times.A, the cell proliferation process is indicated
When Δa <0, the cell death process is indicated.
In another aspect, the present invention provides a method for calculating and analyzing cell proliferation, saturation and death information using the above cell detection analyzer, the method at least comprising the steps of:
applying vibration to cells to be detected through the vibration platform, and recording amplitude A at each detection time point in real time through the recording part in the whole process;
calculating the rate of change of the amplitude Δa by the data analysis unit;
and determining the whole process of cell proliferation, saturation and death by the data processing unit according to the calculation result of the data analysis unit.
In a preferred embodiment, the data analysis unit calculates the rate of change of the amplitude ΔA and calculates the rate of change of the amplitude ΔA according to a function Ae (Bx) Or the Sigmoid function simulates and calculates the proliferation rate of the cells;
dividing the data acquisition unit into a first area, a first dividing range, a second area, a second dividing range and a third area;
the first region includes at least one time period of a region of 0.ltoreq.Δa=a/t <10% ×a;
the first division range includes ΔA>10%. Times.A, and t n+1 >t n And A (t) n+1 )>t( n ) At least one time period of the region of (2);
the second region includes at least one time period of 0.ltoreq.ΔA;
the second division range includes ΔA<0, and t n+1 >t n And A (t) n+1 )<t( n ) Is at least one time period of (1);
the third region includes at least one time period of 0.ltoreq.Δa=a/t <10% ×a.
In yet another aspect of the present invention, there is provided at least one application of the above cell detection analyzer and the above method in calculating cell proliferation and death rate, tracking stem cell differentiation, discriminating cancer cell growth factor, analyzing effect of stimulating cancer cell growth factor, analyzing toxicity of drug, biosafety assessment, and nano drug effect analysis.
The beneficial effects that this application can produce include:
1) According to the real-time quantitative detection and analysis method for the whole cell proliferation, saturation and death process, the change of the cell number on the vibration platform can be calculated through the change of the vibration amplitude of the vibration platform, and the change of the cell activity and the change of the adhesiveness are recorded in the whole process.
2) The rate of cell proliferation is calculated in the methods of the present application by modeling the increasing function, reflecting the effect of the cell agonist and the regulatory mechanism of the growth factor.
3) The method can track the whole differentiation process of the stem cells in real time in the whole process, and can also track the quality change of the markers in the stem cells.
4) The method in the application distinguishes the cancer cell growth factor through a whole-course real-time recording mode and analyzes the action mechanism of the cancer cell growth factor.
5) The method calculates the toxicity of the related drugs in terms of cell death, evaluates the biological safety and analyzes the effect of the nano drugs in a whole-course real-time recording mode.
6) The method in the application monitors in real time through the whole process and quantitatively calculates the maximum amount of the medicine taken by the cells in different time periods.
Drawings
FIG. 1 is a schematic diagram of a cell proliferation assay.
Fig. 2 is a schematic diagram of an apparatus according to an embodiment of the invention.
FIG. 3 is a schematic diagram of a first zone for detecting the proliferation of cells.
Fig. 4 is a schematic diagram of detection of the first region.
FIG. 5 is a schematic flow chart of a process for analyzing cell proliferation data in the data analysis area.
Detailed Description
The application provides a device and an analysis method capable of detecting cell proliferation in real time. The cell to be measured is attached to the upper surface of the vibration platform. The number of cells on the platform increases due to cell proliferation, and the amplitude (amplitude) of vibration increases at this time; while as the cells die, adhesion decreases and the number of cells on the platform decreases, at which time the amplitude decreases. The stronger the factor affecting the proliferation or death of the cell, the larger the amplitude change trend, and the degree of the factor can be obtained through quantitative analysis of the data.
The device of the invention controllably provides vibration, and the high-speed detection equipment in the device can record the change of the cell number in real time, and the change information is presented in a vibration waveform within the recording range.
The number of cells in the non-proliferative phase is stable, and the number of cells separated by shaking gradually increases as the cells undergo a change from the non-proliferative phase to the proliferative phase. Likewise, when the proliferation of cells is completed and a change from the proliferation period to the non-proliferation period is experienced, the number of cells separated by vibration is gradually reduced to be kept stable, and the high-speed detection apparatus also records these change information in the form of vibration waves. The high-speed detection device has a function of providing vibration, capturing, amplifying, and recording information. The high-speed detection device has an operating rate (i.e., the processing capacity of the detection) of more than 1M/s, where M represents the mass of the collection of cells to be detected.
The data acquisition unit comprises a first area, a first segmentation range, a second area, a second segmentation range and a third area.
The amplitude change detected by the high-speed detection device is very small when the cells are in the non-proliferative phase. The non-proliferation period of the cells lasts for a certain time, and is reflected to the amplitude, namely, a period of silence with very small amplitude change, and the amplitude changes after the period of silence.
The high-speed detection device can sensitively capture the amplitude change and record the deltaa and deltat values when the cells change from the non-proliferation phase to the proliferation phase. The first region includes 0.ltoreq.ΔA=A/t<At least one time period of 10% x a zone. The first division range includes DeltaA>10%. Times.A, and t n+1 >t n And A (t) n+1 )>t( n ) At least one time period of the region of (a). The second region includes at least one time period of 0.ltoreq.ΔA. The second division range includes delta A<0, and t n+1 >t n And A (t) n+1 )<t( n ) Is set, is provided for a time period of at least one of the time periods. The third region includes 0.ltoreq.ΔA=A/t<At least one period of 10% x a. The first segmentation limit contains cell proliferation information.
The high-speed detection device records deltaa and Δt values as the cells change from proliferative to non-proliferative. The second area is from the end point of the first dividing range to the end point of the second dividing range, the amplitude change rate is 0-10% x A, and the start point of the second dividing range is the end point of the first dividing range. The second segmentation limit contains cell saturation information.
The high-speed detection device records deltaa and Δt values when the cells enter the death phase. The third area is from the end point of the second division range to the end point of the third division range, the area selects amplitude change rate delta A <0, and the starting point of the third division range is the end point of the second division range. The third split range contains cell death information.
Accurate calculation of the amplitude variation law will be based on the function Ae (Bx) Or Sigmoid function simulation, judging the cell number according to the specific amplitude magnitude variable delta A, and simultaneously simulating the situation according to the ground gain functionAnd judging the magnitude of the increment coefficient, thereby judging the proliferation rate of the cells.
In use of Ae (Bx) When calculating the cell proliferation rate, wherein A represents the theoretical maximum amplitude, B represents the function change coefficient (increasing or decreasing trend), and x represents the strain value.
When the cell proliferation rate is calculated using the Sigmoid function simulation, the function S (x) =1/(1+ae-Bx), where a represents the theoretical maximum amplitude, B represents the trend of the function change, and x represents the strain value.
The invention adopts a detection principle similar to an atomic force microscope, namely, when the signal acquisition is carried out on a vibration system, the vibration frequency is kept unchanged, and only the amplitude signal is monitored.
The present application is described in detail below with reference to examples, but the present application is not limited to these examples.
Fig. 1 shows a flow chart of the invention, reflecting the inventive concept as a whole.
Example 1
Isolation and culture of bone marrow mesenchymal stem cells
According to the method provided in the literature (Chinese tissue engineering study 2018, 22 (01), 1-6), C57BL/6 mice were sacrificed by cervical dislocation cervical diversion, soaked in 70% ethanol for 5min, femur and tibia were removed, and soaked in PBS containing 5% diabody for 30min. The bone marrow cavity was then exposed with the instrument and bone marrow was flushed from the bone marrow cavity with a 1ml syringe containing alpha-MEM medium, with gentle reduction of mechanical damage to cells throughout the process. The cell suspension was centrifuged at 1500r/min for 5min to form a cell pellet, and the supernatant was discarded. Cells were incubated with alpha-MEM medium at 37℃with 5% CO 2 Is cultured in a cell culture incubator. And transmitting to the third generation for standby.
Testing the Effect of osteoprotegerin single chain antibody on proliferation of bone marrow mesenchymal Stem cells
The mesenchymal stem cells of a certain concentration were tested using the cell detection analyzer shown in fig. 2. The concentration of the cells does not affect the experimental results, but the initial concentration values must be kept consistent each time a comparative experiment is performed using the apparatus of the present invention.
When the amplitude is observed to increase to a stable value in real time throughout the course, namely, after Δa=t <10% ×a, a single-chain sclerostin antibody (Scl-sc Fv) with a certain concentration is injected slowly and constantly, and at this time, the amplitude is observed to increase suddenly and then to be stable. Jump-up occurs at Δa >10% ×a, and after the amplitude region stabilizes, the recovery is again to Δa=t <10% ×a. The amplitude is then significantly increased due to the effect of Scl-sc Fv on bone marrow mesenchymal stem cell proliferation. The number and growth rate of cell proliferation and differentiation were obtained by analyzing the change in amplitude of the data.
The effect of the single-chain antibody of the osteoprotegerin on the proliferation of the mesenchymal stem cells can be calculated through calculation.
Example 2 cell proliferation assay
Embodiments of the present invention will be described in detail with reference to fig. 3 to 5. Mesenchymal stem cells proliferate under the influence of Scl-sc Fv, the number of cells increases significantly, the vibration amplitude increases, and the rate of increase ΔA>10% x A, data acquisition was determined to be divided into a first region, a first segmentation limit, and a second region as described above. According to a function Ae (Bx) Or comparing the sigmoid function with the amplitude increasing curve, and performing simulation calculation on the target curve in the first segmentation range to obtain an optimal B value which can be used for explaining the cell proliferation rate; when the cells are saturated, the growth rate is 0.ltoreq.DeltaA=A/t<10% x a and maintained in the second zone. When the cells begin to die, the cell number is obviously reduced, the vibration amplitude is reduced, and the amplitude reduction rate delta A is reduced>10% x A, data acquisition was determined to be divided into a second region, a second segmentation limit, and a third region as described above. According to a function Ae (Bx) Or comparing the sigmoid function with the amplitude reduction curve, and performing simulation calculation on the target curve in the second segmentation range to obtain an optimal B' value which can be used for explaining the cell death rate. Thus, the whole process of cell proliferation, saturation and death is determined by reflecting the stress response of the cells after different stimuli by the data processing unit.
The idea of the invention is to use the relation between the cell state and the adhesion of the cells to the vibrating platform. As the cells proliferate, the number of cells on the platform increases, the amplitude of the vibration increases, while as the cells die, the adhesion decreases, and the number of cells on the platform decreases, the amplitude decreases. The stronger the factor affecting cell proliferation or death, the greater the trend in amplitude change. The degree of influence factors can be obtained through quantitative analysis of the data. The qualitative analysis of cancer cell proliferation, the degree of normal cell proliferation, and the like can be realized based on the monitoring of the cell proliferation process, so the cell detection analyzer and the analysis method of the present invention are also applicable to such related studies.
The foregoing description is only a few examples of the present application and is not intended to limit the present application in any way, and although the present application is disclosed in the preferred examples, it is not intended to limit the present application, and any person skilled in the art may make some changes or modifications to the disclosed technology without departing from the scope of the technical solution of the present application, and the technical solution is equivalent to the equivalent embodiments.
Claims (4)
1. The cell detection analyzer is characterized by at least comprising a data acquisition unit, a data analysis unit and a data processing unit;
the data acquisition unit comprises a vibration platform and a recording component, cells are attached to the vibration platform and vibrate together with the vibration platform, and the recording component records that the phase deviation generated by the vibration platform is the vibration amplitude A, delta A=A/t;
the data acquisition unit comprises a first area, a first segmentation range, a second area, a second segmentation range and a third area;
the first zone comprises at least one first period of time for a zone of 0.ltoreq.ΔA < 10%. Times.A;
the first division range includes ΔA > 10%. Times.A, and t n+1 ＞t n And A (t) n+1 )＞A(t n ) At least one first segmentation period of the region of (2);
selecting amplitude change rate delta A > 10%. Times.A, and t n+1 ＞t n And A (t) n+1 )＞A(t n ) A kind of electronic deviceThe method comprises the steps of generating a first segmentation range, wherein a starting point of the first segmentation range is a first rate change point, and an end point of the first segmentation range is a last rate change point;
the first segmentation limit comprises cell proliferation information;
the second region includes at least one second period of time for a region of 0.ltoreq.ΔA;
the second division range includes ΔA <0, and t n+1 ＞t n And A (t) n+1 )＜A(t n ) At least one second segmentation period of the region of (2);
the second area is from the end point of the first segmentation range to the end point of the second segmentation range, the amplitude change rate of the second area is more than or equal to 0 and less than 10 percent of A, and the starting point of the second segmentation range is the end point of the first segmentation range;
the second segmentation limit comprises cell saturation information;
the third zone comprises at least one third time period of a zone of 0.ltoreq.ΔA < 10%. Times.A;
the third area is from the end point of the second division range to the end point of the third division range, the amplitude change rate delta A is selected by the third area to be less than 0, and the starting point of the third division range is the end point of the second division range;
the third split range contains cell death information;
wherein the first time in the first time period < the first dividing time in the first dividing time period < the second time in the second time period < the second dividing time in the second dividing time period < the third time in the third time period;
the data analysis unit calculates the rate of change of the cell number on the platform based on the rate of change of the amplitude;
the data processing unit determines the whole process of cell proliferation, saturation and death according to the calculation result of the analysis unit;
the rate of change of amplitude Δa describes the following cellular process:
when DeltaA is more than or equal to 10 percent multiplied by A, the proliferation process of the cells is indicated;
when delta A is more than or equal to 0 and less than 10 percent multiplied by A, the cell saturation process is represented;
when ΔA <0, the cell death process is indicated.
2. The cell detection analyzer according to claim 1, wherein the vibration platform applies vibration excitation to the cells to be measured attached to the vibration platform, and the generated excitation signal is amplified and recorded as the amplitude a by the recording means.
3. A method of calculating, analyzing cell proliferation, saturation and death information using the cell detection analyzer according to any one of claims 1 to 2, characterized in that the method comprises at least the steps of:
applying vibration to cells to be detected through the vibration platform, and recording amplitude A at each detection time point in real time through the recording part in the whole process;
calculating, by the data analysis unit, a rate of change of the amplitude Δa, Δa=a/t;
and determining the whole process of cell proliferation, saturation and death by the data processing unit according to the calculation result of the data analysis unit.
4. A method according to claim 3, characterized in that the data analysis unit calculates the rate of change of the amplitude Δa, Δa=a/t and according to a function Ae (Bx) Or the Sigmoid function simulates and calculates the proliferation rate of the cells;
dividing the data acquisition unit into a first area, a first dividing range, a second area, a second dividing range and a third area;
the first zone comprises at least one first period of time for a zone of 0.ltoreq.ΔA < 10%. Times.A;
the first division range includes ΔA > 10%. Times.A, and t n+1 ＞t n And A (t) n+1 )＞A(t n ) At least one first segmentation period of the region of (2);
the second region includes at least one second period of time for a region of 0.ltoreq.ΔA;
the second division range includes ΔA <0, and t n+1 ＞t n And A (t) n+1 )＜A(t n ) At least one second segmentation period of the region of (2);
the third zone comprises at least one third time period of a zone of 0.ltoreq.ΔA < 10%. Times.A;
wherein the first time in the first time period < the first dividing time in the first dividing time period < the second time in the second time period < the second dividing time in the second dividing time period < the third time in the third time period.
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| EP1457781A1 (en) * | 2003-03-12 | 2004-09-15 | F. Hoffmann-La Roche Ag | Simultaneous determination of cell proliferation inhibition activity and toxicity using flow cytometry |
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