CN103424350B - High throughput analysis system and counting method for low-order-of-magnitude mutation-induced cells - Google Patents

Abstract

The invention discloses a high throughput analysis system and a counting method for low-order-of-magnitude mutation-induced cells. The system comprises a PC (personal computer)/notebook computer, a cell culture box used for maintaining a cell growth environment, a high-throughput 96x microelectrode plate, normal cells, mutation-induced cells and a mutation inducer. According to the method, a growth curve of the mutation-induced cells is recorded by real-time cell monitoring software in the PC/notebook computer, dynamic features of a response curve of the mutation-induced cells are analyzed, a calculation operation is performed to obtain a reference value and a threshold value of the response curve of the mutation-induced cells, a relation equation (namely an estimation model) between the mutation-induced cell number and the growth time is built, an identification operation is performed by a nonlinear regression algorithm to obtain the parameters of the estimation model, and the low-order-of-magnitude mutation-induced cells to be measured are counted by the identified estimation model. The best advantage is that high-throughput detection can be achieved by non-invasive detection means without biomarkers.

Description

A kind of high throughput analysis systems of low order of magnitude mutagenized cell and method of counting

Technical field

The present invention relates to a kind of Cell counts technology and analytic system, particularly relate to a kind of low order of magnitude, the high flux statistical method of mutagenized cell and analytic system, belong to cytogene oxicity analysis field.

Background technology

Genotoxicity (Genotoxicity) refers to the characteristic that some materials have, this special performance compromise genes within cells information, destroys the integrality of inhereditary material in cell.In most of the cases, genotoxicity can make different cell and other body system produce mutation, thus causes the various diseases (as cancer, that is: the Growth of Cells out of hand in health) of biosome.

In physical environment, often there is a large amount of medicines or pollutant, these materials have certain genotoxicity, can make normal cell generation mutagenesis, produce mutagenized cell, cause biosome generation canceration.Therefore, by statistics mutagenized cell number, medicine or hazards of pollutants grade can be identified, thus prediction environmental contaminants produce the possibility of deleterious effect to health, that is: realize Human Health Risk assessment.But, mutagen concentration in physical environment is all very low, the mutagen generation mutagenized cell number of low concentration is also considerably less, the restriction of examined sensitivity, traditional method for cell count is difficult to the mutagenized cell number counting the low order of magnitude, such as: if during mutagenized cell number deficiency 100 in double dish, MTT colourimetry cannot realize detecting.

Patent " a kind of High-precision cell statistic technology and analytical equipment " [application number: 201210062508.5, publication number: CN102851208A] a kind of Cell counts method based on image analysis technology is proposed, by regulating microscopic imaging device, obtain cell microscopic sheet clearly, and a series of image manipulation is carried out to picture, obtain recognition result, and statistical study is carried out to result, obtain the number of detected cell, size and form etc.The method needs a set of high-precision micro imaging system, is usually used in normalized cell counts, and cannot realize high flux detection.

Patent " system and method for counting Cell and organism molecule " [application number: 200980121707.5, publication number: CN102089418A] sample of cell is contacted with fluorescent labeling reagent, and utilize fluorescent light beam, by checkout equipment imaging technique, be equipped with computer software, realize the counting to cell to be measured.The method is a kind of detection method of intrusive mood, needs fluorescent labeling reagent, and the activity of cell is subject to the impact of fluorescent labeling reagent, the infected chance of laboratory technician is larger, and its testing procedure is single uninteresting, laboratory technician easily tired, make mistakes, high flux cannot be realized and detect.

Summary of the invention

For above-mentioned the deficiencies in the prior art, the present invention proposes one in conjunction with the analytic system of the mutagenized cell of real-time cell analyser (Real Time Cell Analyzer, RTCA) and method of counting, and the high flux that can realize low order of magnitude mutagenized cell detects.

According to the object of the present invention, a kind of high throughput analysis systems of low order of magnitude mutagenized cell is proposed, this system comprises for monitoring Growth of Cells with the PC/notebook computer of its growing environment parameter of control, for maintaining the cell culture incubator (incubator) of Growth of Cells environment, a high-throughout 96x microelectrode plate (E-Plate), normal cell, mutagenized cell and mutagen; Wherein, PC/notebook computer is provided with the real-time cell software platform for SCADA of ACEA company exploitation, and this platform can control the environmental parameter of cell culture incubator, as: temperature, humidity, and gas concentration lwevel, thus keep the homogeneity of Growth of Cells environmental baseline; And the impedance change signal of microelectrode plate E-Plate can be detected in real time, and this signal is changed into cell index (Cell Index, CI) and be presented on screen, be also stored in PC/notebook computer hard disk simultaneously; 96x microelectrode plate is placed in cell culture incubator, and the environmental parameter (humiture, gas concentration lwevel) of cell culture incubator is controlled by real-time cell monitoring software, thus the environmental baseline remaining on whole mutagenic processes is constant; Normal cell is seeded in 96x microelectrode plate, and mutagenized cell and mutagen are added in the micropore of 96x microelectrode plate.

Wherein, normal cell is seeded in 96x microelectrode plate (E-Plate), cell meeting adherent growth, its number change can cause the impedance of the goldleaf sensor bottom E-Plate to change, attached cell on microelectrode is more, change in impedance value is larger, and this change is changed into cell index (CI) by real-time cell monitoring software, and display in systems in which.

Utilize the method for counting of above-mentioned analytic system, comprise the steps: that normal cell to be seeded in 96x microelectrode plate after 12 hours, mutagenized cell and the mutagen of varying number are added in different micropores (well), normal cell all kills by mutagen, mutagenized cell is allowed to grow, utilize the growth curve of the whole mutagenized cell of real-time cell software platform for SCADA record, and analyze the behavioral characteristics of this mutagenized cell response curve, calculate reference value and the threshold value of mutagenized cell response curve; On this basis, set up the relation equation between mutagenized cell number and growth time, i.e. estimation model, recycling nonlinear regression algo, identification obtains the parameter of this estimation model, utilizes the counting of the estimation model of identification realization to the low order of magnitude, mutagenized cell number to be measured.

Method for cell count of the present invention comprises the steps:

(1) normal cell is seeded in the micropore (well) of the microelectrode plate of 96x;

(2), after waiting for that normal cell stablizes 12 hours, in the different micropore of microelectrode plate, mutagenized cell and the mutagen of varying number are added;

(3) after mutagen kill normal cell, remaining mutagenized cell continued growth, real-time cell software platform for SCADA controls and records whole mutagenic processes (200 hours), records and stores the growth curve of different mutagenized cell;

(4) to the recorded smoothing process of mutagenized cell growth curve, the noise signal of sensor is removed;

(5) analyze the behavioral characteristics of mutagenized cell response curve, calculate reference value and the threshold value of varying number level mutagenized cell growth curve;

(6) maximal value of getting all growth curve reference values is final reference value, and the minimum value of getting all growth curve threshold values is final threshold value; Draw a horizontal linear respectively by final reference value and final threshold value, the growth curve of straight line and each mutagenized cell has two intersection points, and the time difference got between intersection point is the growth time of mutagenized cell;

(7) set up the power function equation (that is: estimation model) between mutagenized cell number and growth time, and obtain the parameter of this power function equation with nonlinear regression algo;

(8) power function equation (estimation model) is utilized to estimate the cell number of mutagenized cell sample to be measured.

The present invention's beneficial effect is compared with the prior art:

(1) adopt non-intrusion type, unmarked detection method, and the reading of cell index is non-invasive;

(2) adopt 96x microelectrode plate to carry out cell inoculation, the assessment of high flux mutagenized cell can be realized;

(3) the display data that employing real-time cell monitoring software can be continuous, real-time, record the growth course of whole mutagenized cell, complete cell benefit collection of illustrative plates can be obtained, instead of supposition cell be in certain suitable processing stage (end-point method), carry out cell analysis;

(4) this method of counting can realize the estimation of low order of magnitude cell.

Accompanying drawing explanation

Fig. 1 is the structure principle chart of mutagenized cell analytic system; Wherein, 1-normal cell, 2-microelectrode plate, 3-mutagen, 4-mutagenized cell, 5-cell culture incubator, 6-PC machine;

Fig. 2 is the schematic flow sheet of low order of magnitude mutagenized cell counting;

Fig. 3 is low order of magnitude mutagenized cell growth curve, and calculate reference value and threshold figure;

Fig. 4 is mutagenized cell estimation model curve map.

Fig. 5 is low order of magnitude mutagenized cell estimated result figure.

Embodiment

Refer to the 1st figure, it is for the present invention is for the analytic system structure principle chart of low order of magnitude mutagenized cell high flux counting, as shown in the figure, the present invention includes one for monitoring the PC 6 of Growth of Cells and its growing environment parameter of control, one for maintaining the cell culture incubator 5(incubator of Growth of Cells environment), a high-throughout 96x microelectrode plate 2(E-Plate), normal cell 1, mutagenized cell 4, mutagen 3.

Wherein, be provided with the real-time cell software platform for SCADA of ACEA company exploitation in PC 6, this platform can control the environmental parameter of cell culture incubator, as: temperature, humidity, and gas concentration lwevel, thus the homogeneity keeping Growth of Cells environmental baseline; And the impedance change signal of microelectrode plate 2 can be detected in real time, and this signal is changed into cell index (Cell Index, CI) and be presented on screen, be also stored in PC hard disk simultaneously.

Wherein, 96x microelectrode plate 2 is placed in cell culture incubator 5, and the environmental parameter (humiture, gas concentration lwevel) of cell culture incubator 5 is controlled by real-time cell monitoring software, thus the environmental baseline remaining on whole mutagenic processes is constant.

Wherein, normal cell 1 is seeded in 96x microelectrode plate 2(E-Plate) in, cell meeting adherent growth, its number change can cause the impedance of the goldleaf sensor bottom E-Plate to change, attached cell on microelectrode is more, change in impedance value is larger, and this change is changed into cell index (CI) by real-time cell monitoring software, and display in systems in which.

Wherein, after normal cell 1 is seeded in microelectrode plate 2 upper 12 hour, mutagenized cell 4 and the mutagen 3 of varying number are added in different micropores (well), normal cell can all kill by these mutagen 3, mutagenized cell 4 is allowed to grow, the growth course of the whole mutagenized cell 4 of real-time cell monitoring software record.

Method of counting of the present invention: the growth curve first utilizing real-time cell monitoring software record mutagenized cell, and analyze the behavioral characteristics of this mutagenized cell response curve, calculate reference value (Baseline) and the threshold value (Threshold) of mutagenized cell response curve, on this basis, set up relation equation between mutagenized cell number and growth time (that is: estimation model), recycling nonlinear regression algo, identification obtains the parameter of this estimation model, utilizes the counting of the estimation model of identification realization to the low order of magnitude, mutagenized cell number to be measured.

As shown in Figure 2, method of counting of the present invention specifically comprises following steps:

1st step: normal cell 1 is seeded in the microelectrode plate 2 of 96x;

2nd step: after waiting for that normal cell 1 stablizes 12 hours, add the mutagenized cell 4M of varying number in the different micropore of microelectrode plate 2 _j(M _j∈ 512,256,128,64,32,16,8,4,2,1}, j=1,2 ..., 10) and mutagen 3;

3rd step: after mutagen 3 kill normal cell, remaining mutagenized cell 4 continued growth, real-time cell monitoring software controls and records whole mutagenic processes (200 hours), records and stores different mutagenized cell M _jat not cell index CI in the same time _j[i] (i=1,2 ..., 200, that is: real-time cell monitoring software sampling in an every 1 hour point), as shown in Figure 3, wherein the implication of each curve representative is as follows: a-is without mutagenized cell for its result; B-1 mutagenized cell; C-2 mutagenized cell; D-4 mutagenized cell; E-8 mutagenized cell; F-16 mutagenized cell; G-32 mutagenized cell; H-64 mutagenized cell; I-128 mutagenized cell; J-256 mutagenized cell; K-512 mutagenized cell; L-reference value CI ^b; M-threshold value CI ^t.

4th step: the smoothing process of varying number level mutagenized cell growth curve to recorded:

$\left\{\begin{array}{c}{y}_j\left[k\right]=\frac{\left(\underset{i=k-\mathrm{Num}}{\overset{k}{\mathrm{\&Sigma;}}}{\mathrm{CI}}_j\right[i]-\max \left({\left\{{\mathrm{CI}}_j\right[i\left]\right\}}_{i=k-\mathrm{Num}}^k\right)-\min \left({\left\{{\mathrm{CI}}_j\right[i\left]\right\}}_{i=k-\mathrm{Num}}^k\right))}{\mathrm{Num}-2};k\&GreaterEqual;\mathrm{Num}\\ y_j\left[k\right]\frac{\underset{i=1}{\overset{k}{\mathrm{\&Sigma;}}}{\mathrm{CI}}_j\left[i\right]}{k};k<\mathrm{Num}\end{array}\right.---\left(1\right)$

Wherein, Num is the length of gliding smoothing forms, gets Num=6 here; K is sampling instant k=1,2 ..., 200; y _j[k] is M _jcell index value after mutagenized cell smoothing processing.

5th step: calculate varying number level mutagenized cell M _jthe reference value of growth curve and threshold value.For obtaining rational reference value and threshold value, the rate of change B of cell index first to be calculated _j[k]:

$B_j\left[k\right]=\frac{y_j[k+1]-y_j[j-1]}{2}---\left(2\right)$

The rate of change B of cell index _j[k] directly reflects the growth rate of mutagenized cell at sampling instant k, if negative value, then represents cell death, otherwise then represents Growth of Cells; Greatly, cell more has vigor to the higher expression cell index change of absolute value.

On this basis, the cell index rate of change B of varying number level mutagenized cell is calculated _jwhen [k] first time is greater than 0 value, and the cell index CI obtaining this moment is the reference value of this order of magnitude mutagenized cell

${\mathrm{CI}}_j^b={\mathrm{CI}}_j\left[k_j^b\right];j=\mathrm{1,2},...,10$

$\left\{\begin{array}{c}{k}_j^b=\underset{k=\mathrm{1,2},...,200}{\min }\left(k\right)\\ s.t.B_j\left[k\right]>\mathrm{\&delta;}\end{array}\right.---\left(3\right)$

Wherein, δ is very little numerical value, here for avoiding the impact of system noise, gets δ=10 ^-4(close to 0).

In addition, the cell index rate of change B of varying number level mutagenized cell is calculated _jduring [k] maximal value value, and the cell index CI obtaining this moment is the threshold value of this order of magnitude mutagenized cell

${\mathrm{CI}}_j^t={\mathrm{CI}}_j\left[k_j^t\right];j=\mathrm{1,2},...,10$ （4）

$s.t.k_j^t=\underset{k=\mathrm{1,2},...,200}{\arg \max }\left(B_j\right[k\left]\right)$

6th step: the reference value of getting all order of magnitude mutagenized cells maximal value be final reference value CI ^b:

${\mathrm{CI}}^b=\max \left({\left\{{\mathrm{CI}}_j^b\right\}}_{j=1}^{10}\right)---\left(5\right)$

Get the threshold value of all order of magnitude mutagenized cells minimum value be final threshold value CI ^t:

${\mathrm{CI}}^t=\min \left({\left\{{\mathrm{CI}}_j^t\right\}}_{j=1}^{10}\right)---\left(6\right)$

7th step: with final reference value CI ^bdraw a horizontal datum, as shown in Figure 3, the growth curve of this datum line and each mutagenized cell has an intersection point k _j, the horizontal ordinate minimum value of getting this intersection point is evaluation time starting point t (k ^s), that is:

$k^s=\min \left({\left\{k_j\right\}}_{j=1}^{10}\right)$ （7）

$s.t.k_j=\underset{k=\mathrm{1,2},...,200}{\arg \min }(k{|\mathrm{CI}}_j\left(k\right)={\mathrm{CI}}^b)$

Draw a level thresholds line with final threshold value CIt, as shown in Figure 3, the growth curve of this threshold line and each mutagenized cell has individual intersection point k _j, get the time value t (k of this intersection point _j) and evaluation time starting point t (k ^s) difference be this order of magnitude mutagenized cell M _jgrowth time t _j:

$t_j=t\left(k_j\right)-t\left(k^s\right);j=\mathrm{1,2},...,10$ （8）

$s.t.k_j=\underset{k=\mathrm{1,2},...,200}{\arg \min }\left(k\right|{\mathrm{CI}}_j\left(k\right)={\mathrm{CI}}^t)$

8th step: set up mutagenized cell number M _jwith growth time t _jbetween power function equation (that is: estimation model):

$t_j=a_1{\left(M_j\right)}^{a_2}+a_3---\left(9\right)$

Wherein a ₁, a ₂, a ₃for estimation model parameter,

Nonlinear regression algo is adopted to obtain the parameter of this power function equation, as shown in Figure 4, in this embodiment: a ₁=255.1, a ₂=-0.99, a ₃=104.8.

9th step: with the growth curve of step 1 to the disposal methods mutagenized cell sample to be measured of step 7, obtain the growth time t of mutagenized cell to be measured, utilize estimation model (9) that the cell number of mutagenized cell sample to be measured can be estimated

$\hat{M}={\left(\frac{t-a_3}{a_1}\right)}^{\frac{1}{a_2}}---\left(10\right)$

For implementation result of the present invention is described, the mutagen 3 of 0.2uL are added in mutagenized cell sample to be tested, and this testing sample is inoculated in 96x microelectrode plate 2, altogether need 16 micropores occupying 96x microelectrode plate 2, the growth curve of these 16 micropores is as shown in Figure 5.Can be calculated by formula (10), in mutagenized cell sample to be measured, mutagenized cell number is 25.Under this result and microscope, the result of artificial counting is basically identical, illustrates that the present invention can realize the counting of low order of magnitude mutagenized cell, and has higher precision.

Claims (2)

1. a method of counting for the high throughput analysis systems of low order of magnitude mutagenized cell, is characterized in that, comprise the steps:

(1) normal cell is seeded in the micropore of microelectrode plate of 96x;

(2), after waiting for that normal cell stablizes 12 hours, in the different micropore of microelectrode plate, mutagenized cell and the mutagen of varying number are added;

(3) after mutagen kill normal cell, remaining mutagenized cell continued growth, real-time cell software platform for SCADA controls and records whole mutagenic processes, records and stores the growth curve of different mutagenized cell;

(4) to the recorded smoothing process of mutagenized cell growth curve, the noise signal of sensor is removed;

(5) analyze the behavioral characteristics of mutagenized cell response curve, calculate reference value and the threshold value of varying number level mutagenized cell growth curve;

(6) maximal value of getting all growth curve reference values is final reference value, and the minimum value of getting all growth curve threshold values is final threshold value; Final reference value and final the threshold value respectively horizontal linear at place and the growth curve of each mutagenized cell have two intersection points, and the time difference got between intersection point is the growth time of mutagenized cell;

(7) set up the power function equation between mutagenized cell number and growth time, i.e. estimation model, and obtain the parameter of this estimation model with nonlinear regression algo; Described estimation model is: wherein a ₁, a ₂, a ₃for estimation model parameter, M _jfor mutagenized cell number, t _jfor the growth time of mutagenized cell; J represents the sequence number of varying number mutagenized cell, and its span is j=1, and 2,3,4,5,6,7,8,9,10;

(8) estimation model is utilized to estimate the cell number of mutagenized cell sample to be measured.

2. method of counting according to claim 1, is characterized in that, the whole mutagenic processes of described step (3) is 200 hours, real-time cell software platform for SCADA sampling in an every 1 hour point.