Method of estimating cell viability in microbioreactor by means of optical light guide

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biochemistry. Claimed is a method of evaluating cell viability in a microbioreactor by means of an optical light guide. The method includes the placement of cells into a membrane compartment of a replaceable cell unit of the microbioreactor, application of a working solution of a vital dye, introduction of the dye into the microbioreactor compartment. After the introduction incubation of the cells in the vital dye solution and removal of the vital dye solution, which has not bound with the cells, are performed. Removal is performed by the replacement of the incubation solution with a growth medium, which does not contain dye. The optical light guide, connected to a spectrometer, is brought into contact with an optically transparent material with the replaceable cell unit under the membrane compartment of the microbioreactor. After that, the support spectrum of a fluorescent signal is measured as an integral of the intensity of fluorescence on the membrane compartment of the microbioreactor, in which the cells to be analysed are absent. Also measured is the spectrum of the fluorescent signal as the integral of the intensity of fluorescence on the membrane compartment of the microbioreactor with the analysed cells. After that, the support spectrum of the fluorescent signal for the membrane compartment of the microbioreactor without the cells to be analysed is subtracted from the obtained spectrum of the fluorescent signal for the membrane compartment with the analysed cells. The quantity of the viable cells in the membrane compartment of the microbioreactor is calculated on the basis of the obtained value of the fluorescence signal intensity.

EFFECT: invention makes it possible to quickly determine viability of the cells under an impact of influencing factors in a real time mode in the microbioreactor.

6 cl, 3 dwg, 5 tbl, 5 ex

 

The invention relates to methods of research or analysis of materials using optical means, in particular using fluorescence, including devices to work with cells, tissues, animals or plants and / or crops viruses.

There is a method of assessing the viability of the cells in the cell microchip (microbioreactor), including the incubation of the cells, the introduction of an electrode in the form of a pipette with Ag/AgCl in a cell with cells, the electrochemical measurement signal (ion flow) using a Faraday cage, processing the electrical signal (1) (Pathak et al, 2011). The disadvantage of this method is the need for additional technical measures for grounding and eliminating noise, inaccuracy of measurement. The disadvantage of this method is also low specificity associated with the determination of the electrochemical signal which gives only indirect information about the viability of the cells.

There is a method of assessing the viability of the cells, including the introduction of cells into the cell microbioreactor, introduction to the culture medium of polytetrafluoroethylene, the evaluation of the dissipation of the mitochondrial transmembrane potential (?? m) as a marker of cell viability, in real time (2) (Wlodkowic et al, 2009). The disadvantage of this method is its limited applicability in the hinnon number of devices with a specific configuration (microbioreactor on the basis of cultural tablet), and lengthy sample preparation using a large number of chemical reagents, eliminating the possibility of conducting further experiments with this biological material.

There is a method of assessing the viability of the cells in microbioreactors, including the placement of cells in the cell microbioreactor, staining them with the vital dye (Trypanosoma blue), determining the number of living cells by eliminating painted Trifanova blue dead cells using an inverted light microscope (3) (Yang et al, 2008). The disadvantage of this method is an indirect assessment of the viability and loss of use of the method for microbioreactors complex design that includes a removable cell blocks.

There is a method of assessing the viability of the cells in microbioreactors, including the placement of cells in the cell microbioreactor, staining them with dye Trypanosoma blue, measurement of cell viability by the intensity of staining of cells in red light emitted from the semiconductor photodiodes (4) (Naoghare et al, 2007). The disadvantage of this method is that the dye tripney blue colors mostly dead cells, allowing only indirectly to determine cell viability. In addition, tripney blue colors not only Neji is incapable of cells, but the kernel of viable cells, which reduces the accuracy of the estimates.

Closest to the claimed method is assessing the viability of the cells in microbioreactors, including a room of normal or tumor cells in cell microbioreactor with the cultural environment, the preparation of a working solution of the vital dye suitable for use as a fluorescent marker of viable cells; introducing into a cell for cell culture of microbioreactor; incubation of the cells with a solution of the vital dye; removing unbound cells solution of the vital dye by replacing the solution incubation in growth medium that does not contain the vital dye; an assessment of the number of viable cells using a fluorescent microscope. As a vital dye used isothiocyanate fluorescein (5) (Meng et al, 2011). The disadvantage of this method is the need to move cells microbioreactor field observation under a fluorescent microscope, which requires interruption of the experiment, slows down the procedure for assessing the viability of the cells, does not allow it in real time. Another disadvantage of the prototype is that in the field fluorescent microscope it is impossible to assess cell viability in cells microbioreactor complex is th design, because it cell equipped with a membrane inserts for cell culture (hereinafter mebrane cells)are an integral part of a replaceable cell block, which includes also the camera Micronase valves Micronesia, the tank environment and other structural elements, and over the membrane cells are plugs that provide constant pressure for the implementation of the current environment and may not be removed during the experiment. Therefore, direct observation of cells in the membrane cell replacement cell block top under a microscope without disrupting the experiment impossible. A third disadvantage of the prototype is that used by the vital dye isothiocyanate fluorescein rigidly associated with the cells and not washed from the cells during subsequent cultivation, which makes it unsuitable for continuous monitoring of the viability of the cells.

The problem to which the invention is directed, is to quickly determine the viability of the cells under the influence of the influencing factors in real time in microbioreactor complex design that includes a removable cell blocks.

The solution of this problem is achieved by the fact that the cells are placed in a membrane cell replacement cell unit micro is ioreactor; optical fiber connected to the spectrometer, is brought into contact with the optically transparent material removable cell block directly under the membrane cell replacement cell block microbioreactor, then measure a reference spectrum of the fluorescent signal as the integral of the intensity of fluorescence in the corresponding vital dye in the wavelength range on the membrane cell replacement cell block microbioreactor that lacks the investigated cells, and measure the spectrum of the fluorescent signal as the integral of the intensity of fluorescence in the corresponding vital dye in the wavelength range on the membrane cell replacement cell block microbioreactor with the test cells; obtained from spectrum fluorescent signal for membrane cell replacement cell block with the test microbioreactor cells subtract the reference spectrum of the fluorescent signal for membrane cell replacement cell block microbioreactor without the analyzed cells; calculate the number of viable cells in the cell membrane replacement cell block microbioreactor on the basis of the value measured fluorescence intensity; the working solution of the vital dye is prepared by cultivation of the vital dye in dimethyl sulfoxide to a concentration which AI 10 mmol/l; to the resulting solution add culture medium not containing serum to achieve a concentration of the vital dye 1-5 µmol/l; as a vital dye used CellTracker™ Green or CellTracker™ Red; the detection of fluorescent signals is carried out in the wavelength range 500 nm-540 nm and 600 nm-640 nm, respectively.

This technical result allows you to quickly assess the viability of the cells in microbioreactor without removal from the system and conduct a minimum of manipulation.

Disclosure of inventions

A method of evaluating the viability of the cells in microbioreactor as follows. Place cells in the cell membrane replacement cell block (SKB) microbioreactor. Prepare the working solution with a vital dye suitable for use as a fluorescent marker of viable cells. Make it in the cell membrane replacement cell block microbioreactor. Incubated cells with a working solution of the vital dye, remove unbound cells, the solution of the vital dye by replacing the solution incubation in growth medium containing no dye. The optical fiber is brought into contact with the optically transparent material SKB directly under the membrane cell, and then measured reference spectrum fluorescent is ignal as the integral intensity of the fluorescence signal in the corresponding vital dye in the wavelength range on the membrane cell replacement cell block microbioreactor, without the tested cells, and measure the spectrum of the fluorescent signal as the integral of the intensity of the fluorescence signal in the corresponding vital dye in the wavelength range on the membrane cell replacement cell block microbioreactor with the test cells. Then from the obtained spectrum fluorescent signal for membrane cell replacement cell block microbioreactor with the test cells subtract the reference spectrum of the fluorescent signal for membrane cell replacement cell block microbioreactor without the investigated cells. On the basis of the received intensities of the fluorescence signal to determine the number of viable cells in the cell membrane replacement cell block microbioreactor.

For implementing the method using the cell culture of various organs and tissues, such as skin cells of the intestine.

As a vital dye used CellTracker™ Green or CellTracker™ Red that do not affect cell viability and allow to repeatedly check for long-term cultivation of cells. When using the vital dye CellTracker™ Green detection of fluorescent signals is carried out in the wavelength range 500 nm-540 nm. When using the vital dye CellTracker™ Red detection fluo ascentage signal is carried out in the wavelength range 600 nm-640 nm.

The working solution of the vital dye is prepared according to the standard procedure in accordance with the Protocol of the manufacturer (6). The manufacturer's recommended concentration of 1 µmol/l is the lowest possible to guarantee results with minimal exposure of the dye to the cells and reagent consumption, but to implement the inventive method may use a concentration in the range of 1-5 µmol/L.

The standard procedure for the preparation of working solution of the vital dye in accordance with the Protocol of the manufacturer (6) as follows. Dried vital dye dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mmol/l; on its basis, prepare the working solution of the vital dye set concentration in the culture medium not containing serum.

Staining of the investigated cells produce membrane cell replacement cell block microbioreactor with the test cell, the second membrane cell replacement cell block that does not contain the investigated cells, are used for testing. From the membrane cell SKB containing the tested cells, remove the culture medium. Cells are washed with Hanks solution (HBBS) three times. Make a working solution with a vital dye in the cell membrane for cell culture SKB. Volume doba is tion of the dye in the standard staining procedure is chosen on the basis of the geometrical parameters of the cell SKB of microbioreactor thus, to all of the cells were covered with a working solution with the dye within the recommended time of incubation. For example, when the diameter of the membrane cell SKB 4.26 deaths mm and surface area for cell culture 0,143 cm2quite injection of 50 µl of the working solution with dye.

Incubation of the cells in the solution of the vital dye carried out according to the Protocol of the manufacturer of the dye (6) for 15-30 minutes at 37°C. to Remove unbound cells, the solution of the vital dye by replacing the solution incubation in growth medium not containing dye; optimal replacement produces twice.

The number of cells in the membrane cell replacement cell block is calculated based on the intensities of the fluorescence signal using a calibration curve constructed conventional method on samples with a known number of viable cells obtained by using multiple dilutions of cell suspension, for example, according to the method described in (7). To construct the calibration curves used stained with the vital dye cells are placed in 96-well your tablet or cell replacement cell block microbioreactor, so that the number of cells in each subsequent cell was two times less than in the previous (fill 7 cells in one repeat). Measure the value of interest is cunosti of the fluorescence signal in each cell according to the claimed method. Produce a count of cells in each cell using the camera Goryaeva. Construct a calibration curve by plotting on the x-axis the number of viable cells determined using the camera Goryaeva, and the ordinate axis represents the intensity of the fluorescence signal. The calibration curve is built for each specific cell type, type of dye, the concentration and quantity of working solution with a vital dye introduced into the cell membrane SKB, and then use whenever implementing the inventive method when appropriate this calibration curve conditions (cell type, type of dye, the concentration and the amount entered in the cell of the working dye solution). When implementing the inventive method, the number of viable cells in the cell is determined using a calibration curve by comparing the intensities of the fluorescence signal in the cell with the corresponding value of the number of viable cells on the calibration curve.

For measuring the intensity of the fluorescence signal passing through an optically transparent material microbioreactor (in particular, polydimethylsiloxane), the optical fiber is brought into contact with the optically transparent material removable cell block directly below the cell with the cells. When the permanent works is one microbioreactors optical fiber can be permanently mounted in the composition of microbioreactor. For high-throughput analysis of the viability of the cells under different conditions, when using a large number of replacement blocks of microbioreactor, the optical fiber is fixed in such a way that it is possible to put a removable cell block microbioreactor, then remove and set the following.

For implementing the method can be used microbioreactor with flow (Fig.1) or closed (Fig.2) circuit current environment.

Replacement cell block composed of microbioreactor is a system for culturing cells in constant current mode environment in conditions close to physiological temperature, composition, cultural and gas environment, etc. Complex design microbioreactor, provides long-term cultivation of cells with minimal external influence (simulation of a living organism).

In Fig.1 shows a removable cell block microbioreactor with a flowing path of liquid culture or other medium). Replacement cell block microbioreactor is a holder with membrane cells for cells 1, in which the pipe arrives culture or other medium from the reservoir 2 to enter the environment; camera Micronase 3, providing through its valves 4 promoting environment through channels to embranes cells for cells 5 and the drain port protection 6, valves to control the current environment 7, 8, 9.

In Fig.2. the scheme replaceable cell block microbioreactor closed loop current environment, including the holder with membrane cells for cells 1, in which the pipe arrives culture or other medium from the reservoir to enter the environment 2; camera Micronase 3, providing through its valves 4 promotion of environment on membrane cells for cells 1.

In Fig.3 shows a diagram of the optical system for measurements performed using an optical fiber. The optical system includes a radiation source 10, the light emitting wavelength λ1,coming through the connector 11 to the optical fiber 12 in the cell membrane for cells 1, the emitting light wavelength λ2because of the coloring of the investigated cells with the vital dye; optical fiber 13 is connected at one end with the membrane cell cell 1 cell replacement unit microbioreactor with a flowing path of liquid or removable cell block microbioreactor closed loop current environment. The other end of the optical fiber 13 is connected through a connector 14 to the spectrometer 15. For implementing the method can be used, for example, the spectrometer CCS100 CCD-spectrometer (Thorlabs, Newton, NJ, USA).

List of figures, drawings and other materials

Fig.1. Scheme replaceable cell BC is ka microbioreactor flow path environment.

Fig.2. Scheme replaceable cell block microbioreactor closed loop environment.

Fig.3. Functional diagram measurement by optical fiber.

The implementation of the invention

Example 1. Assessment of cell viability skin model.

Key parameters:

The cellular model of the skin

Vital dye CellTracker™ Green CMFDA

The concentration and the number entered in the cell of a working solution of the vital dye: 1 umol/l, 50 μl

Duration of cultivation with the vital dye: 15 min

Microbioreactor closed loop current environment

Cellular skin model, obtained using primary fibroblasts and cell lines of the keratinocytes Nast, were cultured in a membrane cell for cell culture SKB of microbioreactor closed current environment (Fig.1) in culture medium DMEM containing 5% fetal bovine serum (Gibco), 200 mm L-glutamine and 0.1% antibiotic penicillin/streptomycin (Penicillin Streptomycin, Gibco).

To construct the calibration curve used a cell model of the skin and vital dye CellTracker™ Green CMFDA in a concentration of 1 µmol/l in a volume of 50 μl, the duration of cultivation 15 minutes

Assessment of the viability of the cells was performed according to the claimed method. Used working solution of the vital dye CellTrackerTM Green CMFDA in concentration micromole/l and a volume of 50 μl per membrane cell for cell culture SKB. Culturing cells with a working solution of the vital dye was carried out at 37°C for 15 minutes the Amount of fluorescence intensity was calculated in the wavelength range from 500 nm to 540 nm. Assessment of cell viability was performed on the 1st and 14th days of culturing skin cells. For implementing the method used spectrometer CCS100 CCD-spectrometer (Thorlabs, Newton, NJ, USA). The results are shown in table 1.

Table 1
Assessment of viability of skin cells on the 1st and 14th days of cultivation
The cell typeDay of culturing cellsThe intensity of the fluorescence signalThe number of viable cells, thousand
Skin cells1198±711,4±0,6
14172±119,7±0,4

Thus, the analysis of the viability of skin cells on the claimed method using an optical fiber showed that after 14 days of cultivation in microbioreactor closed loop environment inespo the institutional capacity of the cells is reduced by 15%.

Example 2. Assessment of cell viability skin model.

Key parameters:

The cellular model of the skin

Vital dye CellTracker™ Red

The concentration and the number entered in the cell of a working solution of the vital dye: 1 umol/l, 50 μl

Duration of cultivation with the vital dye: 15 min

Microbioreactor closed loop current environment

Cellular skin model, obtained using primary fibroblasts and cell lines of the keratinocytes Nast, were cultured in a membrane cell for cell culture SKB of microbioreactor closed current environment (Fig.1) in culture medium DMEM containing 5% fetal bovine serum (Gibco), 200 mm L-glutamine and 0.1% antibiotic penicillin/streptomycin (Penicillin Streptomycin, Gibco).

To construct the calibration curve used a cell model of the skin and vital dye CellTracker™ Red at a concentration of 1 µmol/l in a volume of 50 μl, the duration of cultivation 15 minutes

Assessment of the viability of the cells was performed according to the claimed method. Used working solution of the vital dye CellTracker™ Red at a concentration of 1 µmol/l and a volume of 50 μl per membrane cell for cell culture SKB. Culturing cells with a working solution of the vital dye was carried out at 37°C for 15 minutes the value of the intensity of halogen with exceptiona the fluorescence was calculated in the wavelength range from 600 nm to 640 nm. Assessment of cell viability was performed on the 1st and 14th days of culturing skin cells. For implementing the method used spectrometer CCS100 CCD-spectrometer (Thorlabs, Newton, NJ, USA). The results are shown in table 2.

Table 2
Assessment of viability of skin cells on the 1st and 14th days of cultivation
The cell typeDay of culturing cellsThe intensity of the fluorescence signalThe number of viable cells, thousand
Skin cells1221±1010,7±0,5
14188±129,1±0,6

Thus, the analysis of the viability of skin cells on the claimed method using an optical fiber showed that after 14 days of cultivation in microbioreactor closed loop environment, the cell viability is reduced by 15%.

Example 3. Assessment of viability of cell lines the intestine.

Key parameters:

Cell line of intestine

Vital dye CellTracker™ Red

Concentration is the number entered in the cell of a working solution of the vital dye: 1 umol/l, 50 µl

Duration of cultivation with the vital dye: 15 min

Microbioreactor closed loop current environment

Cellular model of intestinal cell-based line SASO-2 were cultured in microbioreactor closed loop environment for 14 days in MEM medium containing 20% fetal bovine serum, 2 mm glutamine, 1% nonessential amino acids, 1 mm pyruvate, 0.1% antibiotic.

To construct the calibration curve used a cell line intestines and vital dye CellTracker™ Red at a concentration of 1 µmol/l in a volume of 50 μl, the duration of cultivation 15 minutes

Assessment of the viability of the cells was performed according to the claimed method. Used working solution of the vital dye CellTracker™ Red at a concentration of 1 µmol/l and a volume of 50 μl per membrane cell for cell culture SKB. Culturing cells with a working solution of the vital dye was carried out at 37°C for 15 minutes the Amount of fluorescence intensity was calculated in the wavelength range from 600 nm to 640 nm. Assessment of cell viability was performed on the 1st and 14th days of culturing skin cells. For implementing the method used spectrometer CCS100 CCD-spectrometer (Thorlabs, Newton, NJ, USA). The results are shown in table 3. As can be seen from table 3, the evaluation of the viability of the bowel lines SASO-2 declared with the person using optical fiber showed that after 14 days of cultivation in microbioreactor closed loop environment, the cell viability is reduced by 10%.

Table 3
Assessment of viability of cell lines intestine SASO-2
The cell typeDay of culturing cellsThe intensity of the fluorescence signalThe number of viable cells, thousand
Cells of intestine1205±1011,8±0,6
14180±710,4±0,6

Example 4. Assessment of viability of cell lines the intestine.

Key parameters:

Cell line of intestine

Vital dye CellTracker™ Red

The concentration and the number entered in the cell of a working solution of the vital dye: 5 umol/l, 50 μl

Duration of cultivation with the vital dye: 15 min

Microbioreactor closed loop environment

Cellular model of intestinal cell-based line SASO-2 were cultured in microbioreactor closed loop credif for 14 days in medium MEM, containing 20% fetal bovine serum, 2 mm glutamine, 1% nonessential amino acids, 1 mm pyruvate, 0.1% antibiotic.

To construct the calibration curve used a cell line intestines and vital dye CellTracker™ Red at a concentration of 5 µmol/l in a volume of 50 μl, the duration of cultivation 15 minutes

Assessment of the viability of the cells was performed according to the claimed method. Used working solution of the vital dye CellTracker™ Red at a concentration of 5 µmol/l and a volume of 50 μl per membrane cell for cell culture SKB. Culturing cells with a working solution of the vital dye was carried out at 37°C for 15 minutes the Amount of fluorescence intensity was calculated in the wavelength range from 600 nm to 640 nm. Assessment of cell viability was performed on the 1st and 14th days of culturing skin cells. For implementing the method used spectrometer CCS100 CCD-spectrometer (Thorlabs, Newton, NJ, USA). The results are shown in table 4.

Table 4
Assessment of viability of cell lines intestine SASO-2
The cell typeDay of culturing cellsThe intensity of the fluorescent signal is AI The number of viable cells, thousand
Cells of intestine1263±1312,5±0,6
14238±1711,3±0,8

As can be seen from table 4, the evaluation of the viability of the bowel lines SASO-2 according to the claimed method using an optical fiber showed that after 14 days of cultivation in microbioreactor closed loop environment, the cell viability is reduced by 10%.

Example 5. Assessment of cell viability skin model.

Key parameters:

The cellular model of the skin

Vital dye CellTracker™ Green CMFDA

The concentration and the number entered in the cell of a working solution of the vital dye: 1 umol/l, 50 μl

Duration of cultivation with the vital dye: 30 min

Microbioreactor flow path environment

Cellular skin model, obtained using primary fibroblasts and cell lines of the keratinocytes Nast, were cultured in a membrane cell for cell culture SKB of microbioreactor flow path of the medium (Fig.2) in culture medium DMEM containing 5% fetal bovine serum (Gibco), 200 mm L-glutamine and 0.1% antibiotic interest is Illin/streptomycin (Penicillin Streptomycin, Gibco).

To construct the calibration curve used a cell model of the skin and vital dye CellTracker™ Green CMFDA in a concentration of 1 µmol/l in a volume of 50 μl, the duration of cultivation 30 minutes

Assessment of the viability of the cells was performed according to the claimed method. Used working solution of the vital dye CellTrackerTM Green CMFDA in a concentration of 1 µmol/l and a volume of 50 μl per membrane cell for cell culture SKB. Culturing cells with a working solution of the vital dye was carried out at 37°C for 30 minutes the Amount of fluorescence intensity was calculated in the wavelength range from 500 nm to 540 nm. Assessment of cell viability was performed on the 1st and 14th days of culturing skin cells. For implementing the method used spectrometer CCS100 CCD-spectrometer (Thorlabs, Newton, NJ, USA). The results are shown in table 5. Thus, the analysis of the viability of skin cells on the claimed method using an optical fiber showed that after 14 days of cultivation in microbioreactor flow path environment cell viability is reduced by 15%.

Table 5
Assessment of viability of skin cells on the 1st and 14th days of cultivation
The cell typeDay of culturing cellsThe intensity of the fluorescence signalThe number of viable cells, thousand
Skin cells1201±1110,9±0,6
14177±99,3±0,5

Sources of information

1. Pathak P, Zhao H, Gong Z, Nie F, Zhang T, Cui K, Wang Z, Wong ST, Que L. Real-time monitoring of cell viability using direct electrical measurement with a patch-clamp microchip // Biomed Microdevices. 2011 Oct;13(5):949-53.

2. Wlodkowic D, Falev S, Skommer J, McGuinness D, Cooper JM. Biological implications of polymeric microdevices for live cell assays // Anal Chem. 2009 Dec 1;81(23):9828-33.

3. Yang ST, Zhang X, Wen Y. Microbioreactors for high-throughput cytotoxicity assays // Curr Opin Drug Discov Devel. 2008 Jan; 11(1):111-27.

4. Naoghare PK, Kwon HT, Song JM. An automated method for in vitro anticancer drug efficacy monitoring based on cell viability measurement using a portable photodiode array chip // Lab Chip. 2007 Sep;7(9):1202-5.

5. Meng Q, He Z, Zhang L, Zhao L, Li E, Zhang O, Zhang X, Yang D. Zou L, Gao Z, Wang Q Development of a double-layer microfluidic chip with flow medium for chemotherapy resistance analysis of lung cancer // Electrophoresis. 2011 Nov;32(23):3446-53.

6. ER-Tracker™ Dyes for Live-Cell Endoplasmatic Reticulum Labeling. Product Information

http://tools.lifetechnologies.com/content/sfs/manuals/mp12353.pdf).

7. Blaheta, R. A., Franz, M., Auth M. K. H., H. J. Wenisch C., Markus B. H. A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation //J. Immunol. Methods, 1991. 142. P. 199-206.

1. A method of evaluating the viability of the cells microbioreactor using an optical fiber, including the introduction of cells into the cell for cell culture of microbioreactor, preparation of solution of the vital dye suitable for use as a fluorescent marker of viable cells; introducing into a cell for cell culture of microbioreactor; incubation of the cells in the solution of the vital dye; removing unbound cells solution of the vital dye by replacing the solution incubation in growth medium containing no dye, characterized in that the cells are placed in a membrane cell replacement cell block microbioreactor; an optical fiber connected to the spectrometer, is brought into contact with the optically transparent material removable cell block directly under the membrane cell replacement cell block microbioreactor after which measure a reference spectrum of the fluorescent signal as the integral of the intensity of fluorescence in the corresponding vital dye in the wavelength range on the membrane cell replacement cell block microbioreactor that lacks the investigated cells, and measure the spectrum of the fluorescent signal as the integral of the intensity of fluorescence in the corresponding vital dye in the wavelength range on the membrane cell replacement cell block microbioreactor with ASCS is edamame cells, then from the obtained spectrum fluorescent signal for membrane cell replacement cell block microbioreactor with the test cells subtract the reference spectrum of the fluorescent signal for membrane cell replacement cell block microbioreactor without the analyzed cells; calculate the number of viable cells in the cell membrane replacement cell block microbioreactor on the basis of the received intensities of the fluorescence signal.

2. The method according to p. 1, characterized in that the working solution of the vital dye is prepared by cultivation of the vital dye in dimethyl sulfoxide to a concentration of 10 mmol/l; in the resulting solution add culture medium not containing serum to achieve a concentration of the vital dye 1-5 μm/L.

3. The method according to p. 1, characterized in that as a vital dye used CellTracker™ Green.

4. The method according to p. 1, characterized in that as a vital dye used CellTracker™ Red.

5. The method according to p. 3, characterized in that when using the vital dye CellTracker™ Green detection of fluorescent signals is carried out in the wavelength range 500 nm-540 nm.

6. The method according to p. 4, characterized in that when using the vital dye CellTracker™ Red detection of fluorescent signals is carried out in the range is the area of wavelength 600 nm-640 nm.



 

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3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to surgery, and can be used in cholecystectomy in patients with cholelithiasis. That is preceded by determining a patient's body weight index (BWI), glycaemia, glucosuria; blood pressure is measured; spinal osteochondrosis and gonarthrosis are detected. The derived results are assessed and scored. If the BWI equals to 28-30 kg/m2,10 points are assigned. If the BWI equals to 30-35 kg/m2,15 points are assigned. If the BWI is more than 35 kg/m2,20 points are assigned. If glycemia is more than 5.5 mmole/l, 3 points are assigned. If the patient suffers from glucosuria, 5 points are assigned. Arterial hypertension of more than 140/190 mm Hg requires assigning 3 points. The detected spinal osteochondrosis is assigned with 3 points, diagnosed gonarthrosis is assigned as 3 points. The derived points are summed up. If the total score is 23, a cholecystectomy with a biliopancreatic diversion is performed. If the total score is 14 to 22 points, laparoscopic cholecystectomy is performed. If the derived value makes 13 points, conventional cholecystectomy is performed.

EFFECT: invention provides selecting the type of cholecystectomy taking into account the metabolic status and a degree of obesity and as a consequence, normalizing the body weight and compensating the components of the metabolic syndrome.

2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: enzyme immunoassay is used to determine blood serum leptin in the boys at the age of 11-12 years old; the body weight index (BWI) is calculated; and leptin is related to the derived BWI. If the calculated numerical value is more than 0.4, enzyme immunoassay is used to measure dihydrotestosterone (DHT) and anti-Mullerian hormone (AMH); if the derived relation value makes 30.86 and less, delayed puberty is predicted in the prepubertal boys.

EFFECT: higher accuracy.

3 ex

FIELD: medicine.

SUBSTANCE: invention represents a differential diagnostic technique for respiratory sarcoidosis and tuberculosis in males by biological fluid analysis; the technique involves measuring an amount of progesterone in the primary examination, and determining a probability coefficient P by formula Р=1е(3,2439420,8692291×Х1)+1, wherein P is the probability coefficient showing if a binary response takes on a value of 1; e is a base of the natural logarithm equal to approximately 2.718282; 3.243942 is a shifting coefficient that we have obtained; (-0.8692291) is a slope coefficient that we have obtained; X1 is the progesterone level in the specific patient, nmole/l; if P is more than 0.61, the patient is referred to a group of those suffering from active tuberculosis; if P is below 0.61 - to the group of patients suffering from respiratory sarcoidosis.

EFFECT: more reliable and objective diagnosis for the purpose of prescribing timely treatment and carrying out the adequate complex of therapeutic measures, simplifying the differential diagnosis of tuberculosis and respiratory sarcoidosis.

2 ex

FIELD: medicine.

SUBSTANCE: invention represents a method for assessing a severity of endogenous intoxication in the patients suffering from acute abdominal diseases by blood examination, differing by the fact of determining the following basic structural-functional values of haemoglobin daily starting from the first day of admission to hospital and after a surgical intervention: relative blood haemoglobin, relative ligand-releasing power of haemoglobin and pyrrole ring oscillations; if the first and third values tend to increase in relation to the norm by 18.5 and 15.1% respectively, while the second one decreases by 17.9%, moderate endogenous intoxication is stated; an increase of the first and third values by 35.1 and 28.2% respectively and an decrease of the second value by 26.8% show severe endogenous intoxication.

EFFECT: invention provides the accurate and adequate assessment of the severity of endogenous intoxication in the patients suffering from acute abdominal diseases that enables taking timely measures for the therapeutic correction.

3 ex, 4 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to application of bis(2,4,7,8,9-pentamethyldipyrrolylmethen-3-yl)methane dihydrobromide as fluorescent zinc (ii) cation sensor.

EFFECT: invention will make it possible to increase fluorescent activity of heterocyclic organic compound with respect to zinc (II) ion in presence of other ions of metals.

1 tbl, 40 ex

FIELD: machine building.

SUBSTANCE: invention refers to the field of DNA sequencing, in particular, to the DNA sequencing with the use of time-controlled fluorescence determination for identification of DNA bases The device comprises a field of embedding for containment of the sequencing reaction components, light sources featuring a capability of emitting the light pulse of a definite wave length, detector pixel, detector, output featuring a capability of transmitting an electric signal from the detector pixel, gating means for detector gating, at that, the detector pixel comprises additionally the first and the second storage batteries The first storage battery is provided with a capability of accumulating electric signal from the detector in response to the first light pulse, while the second storage battery is provided with a capability of accumulating electric signal from the detector in response to the second light pulse.

EFFECT: increased rate of receiving sequencing results.

12 cl, 7 dwg

FIELD: chemistry.

SUBSTANCE: sensor includes semiconductor nanocrystals (quantum dots), imbedded into a near-wall layer of track pores of polyethylene terephthalate membranes, with the pores remaining empty. If ammonia vapours are present in an air sample, ammonia molecules bind with quantum dot surfaces, which results in decrease of quantum dot luminescence.

EFFECT: invention solves the tasks of increasing sensitivity, accuracy of determination of ammonia vapour concentration, terms of exploitation and simplification of the sensor manufacturing.

5 dwg, 1 ex

FIELD: instrumentation.

SUBSTANCE: mix of test gases is forced through test cell. Fluorescent radiation is excited therein by readjustable solid-state lasers with wavelengths corresponding to lines with maximum absorption by isotopes 129I and 127I and nitrogen dioxide. Concentration of said isotopes 129I and 127I and nitrogen dioxide are defined in analysed mix by formulae that allow for the composition of buffer gases.

EFFECT: higher sensitivity of determination.

2 cl, 2 dwg

FIELD: ecology.

SUBSTANCE: method comprises placing the fluorescent test-objects in the control and analyzed samples, irradiation with the excitation light, definition of fluorescence characteristics, by which change the toxicity of the controlled environment is assessed. Microalgae of species Scenedesmus apiculatus are used as test-objects, which are previously isolated from environmentally safe areas of the test water reservoirs.

EFFECT: use of the claimed method enables to assess quickly and accurately the toxicity of water and bottom sediments of the Azov and Black Seas.

6 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biophysics. Claimed are methods of determining space-time distribution of proteolytic enzyme activity in heterogeneous system, in accordance to which: provided is system in vitro, which contains sample of blood plasma, whole blood, water, lymph, colloidal solution, crystalloid solution or gel, and proteolytic enzyme or its precursor; fluorogenic, chromogenic or luminescent substrate for said enzyme id added; space distribution of signal of released substrate label is registered at specified time moments and space-time distribution of proteolytic enzyme activity is obtained by solving reverse problem of type "reaction-diffusion-convection" taking into account label binding with medium components. Also described is device for realisation of methods in accordance with claimed invention and method of diagnosing hemostasis disorders, based on their application.

EFFECT: invention can be further applied in the study of blood coagulation system and diagnostics of diseases associated with blood coagulation disorders.

22 cl, 6 dwg

FIELD: chemistry.

SUBSTANCE: invention refers to medicine, particularly to medical diagnostics, and may be used for two- and three-dimensional (tomographic) fluorescent imaging of a diagnosed object. A device comprises a fluorophore absorption band probing emitter provided with a fibre output, an emission receiver in the form of a CCD camera, an object scanning system with the emitter in a projection configuration, as well as a data processing and visualisation system. The device comprises the fluorophore emission band probing emitter provided with the fibre output, fluorophore absorption and emission band omnidirectional emitters in a reflection configuration, a second emission receiver with the fibre output in the form of a photomultiplier, the object scanning system of the photomultiplier in a projection configuration in relation to the probing emitter, as well as a scanning control unit. The data processing and visualising system is provided with original software for implementing methods for surface imaging, projection visualisation and diffuse fluorescent tomography.

EFFECT: device is characterised by simplicity and low measurement time.

2 cl, 3 dwg

FIELD: measurement equipment.

SUBSTANCE: invention relates to the field of monitoring of natural and process waters and is designed to determine partial concentrations of physical-chemical forms of uranium (VI) in aqueous solutions, which is necessary, in particular, for optimisation of the process of uranium extraction by method of underground leaching. The method consists in radiation of the volume of the investigated sample with nanosecond pulses of laser radiation in ultraviolet range and subsequent registration of dependence of intensity of a signal of mixture fluorescence on intensity of laser radiation and time of delay of receiver strobe relative to the laser pulse. The source of the laser radiation may be a AIG:Nd laser with conversion of the radiation frequency into the fourth harmonics (wave length 266 nm) with maximum energy in the pulse of at least 1 mJ. The system for registration of the fluorescence signal may be a CCD-chamber strobed by nanosecond pulses and connected to a spectral device (polychromator).

EFFECT: invention provides for increased accuracy of detection.

5 cl, 6 dwg

FIELD: agriculture.

SUBSTANCE: method relates to the field of agriculture, in particular fruit growing and selection. The method comprises the freezing of annual shoots in the dormant period in the environmental chamber. At that the evaluation of damaged shoots is carried out not visually but according to the size of the maximum quantum efficiency of photochemical reactions of the photosystem II and the relative velocity of the electron transport by the photosystem II in the cambium tissue and buds, which are determined by the microbial cell adsorption reaction fluorometer. The minimum level of fluorescence and changes in this index under the action of actinic light with density of 190 mcmol/(m2s) are recorded, and after exposure to the object of high intensity light pulse (10 000 mcmol/(m2s), 450 nm).

EFFECT: method enables to accelerate the evaluation of damage of fruit plants with frost.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to laboratory diagnostics and may be used for diagnosis and monitoring of the treatment of various diseases. The method for monitoring of the treatment of the disease involves a fluorescence centre excitement of a biological fluid sample by the exposure to an emission of at least two wavelengths, and recording at least two spectra of the emission generated by the sample, respectively. The presence, degree and nature of the disease are identified by determining the spectral characteristics of the emission generated by the sample as compared to the respective reference (health) spectra and typical spectra of various diseases with the spectra compared within the range involving a laser emission dispersion line. The group of inventions also refers to a device for implementing the above method involving the lasers with various working wavelengths, fibre optic lines collected from the side of the sample into a bundle with a common tip, a spectrometer, a control unit and a computer to process the fluorescence spectra. The spectrometer comprises a collimator with removable optical light filter, a diffraction grating and a charge-coupled device matrix coupled with a signal pre-processor. The control unit regulates laser switching off/on and placing into a collimator of the optical light filter related to the switched-on laser.

EFFECT: group of inventions enables higher rate and accuracy of obtaining the analysis results.

7 cl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method for estimating the bifidus bacteria and lactic acid bacilli in experimental animal's gastrointestinal tract involves preparing bacterial mutants on dense nutrient media with increasing rifampicin concentrations from 10 mcg·ml-1. The grown spontaneous mutants are selected, and the dense nutrient rifampicin medium is re-inoculated in the concentration of up to 160 mcg·ml-1. The mutants characterised by hereditarily stable rifampicin resistance are selected for administration into laboratory animals in the concentration of 150-160 mcg·ml-1 in the nutrient medium. The amount of the administered mutants is calculated. After keeping the animals, the selected faeces are suspended in isotonic sodium chloride. A supernatant is selected, and the dense nutrient medium containing rifampicin 110 mcg·ml-1 is inoculated in a Petri dish. It is incubated for 72 h in the microaerophilic environment at 37°C. The plate count is measured, and the growth-positive bacteria count is re-calculated per 1 g of faeces. A portion of the growth-positive microorganisms of the administered count is determined and used to state the survival.

EFFECT: invention enables effectively determining the portion of the positive-growth bifidus bacteria and lactic acid bacilli passed through the animal's gastrointestinal tract.

2 dwg, 3 tbl, 3 ex

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