RU2558229C2 - Set and method for preparing multi-layered agarose blocks on surface of micro-slides for microscoly - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to field of biomedicine. Set and method are claimed for preparation of multi-layered agarose blocks on the surface of mini-slides. Set consists of first component, second and third components. First component represents plane-parallel plate, on which separating blocks, higher than thickness of mini-slides, are installed with equal spaced between them. Mini-slides are installed between blocks on 50-100 mcm. Second component represents plane-parallel plate, one side of which represents polished surface, on sides of which two lateral legs in form of 50±5 mcm thick blocks are welded. Third component represents plane-parallel plate, one side of which represents polished surface, on sides of which two lateral legs in form of 100±5 mcm thick blocks are welded. Method of preparing multi-layered agarose blocks on the surface of mini-slides is realised with application of claimed set.

EFFECT: inventions provide preparation of series of slides with agarose blocks of fixed volume with standard thickness of agarose layers.

2 cl, 4 dwg, 3 ex

Description

The invention relates to the field of biomedical and environmental studies and relates to a method for measuring DNA damage in individual non-proliferating cells induced by the action of anthropogenic physical, chemical and biological environmental factors, the comet test method (electrophoresis of individual cells in agarose gel, method DNA comets, Comet assay).

Prevention of contact of living organisms with potential factors capable of exerting a genotoxic effect seems to be the most constructive way of protecting against the effects of induced mutagenesis. However, a total test of potential agents for genotoxicity is impossible due to the huge amount of research. To reduce the cost and speed up the work on genotoxic screening, studies are carried out using simple and fast-performing methods using microorganisms or cultures of mammalian cells as a test object. To assess the mutagenic properties of chemical compounds to date, many different test systems have been proposed, many of which are well developed and have been widely used. However, to date there is no universal test system that could detect all the main types of genetic damage and would be free from a number of fundamental shortcomings.

Some studies require preliminary isolation of DNA from the cell homogenate, which makes it impossible to individually analyze the cell suspension. In a heterogeneous cell suspension, the level of damage can vary greatly, and there is a chance of screening a small population of severely damaged cells (capable of initiating carcinogenesis and other diseases) against the background of the remaining slightly damaged. A number of methods include the stages of intensive manipulations with cells, which often leads to a significant increase in the level of additional DNA damage. Some highly sensitive methods for assessing the degree of DNA repair, such as unplanned DNA synthesis, are not able to assess the level of direct DNA damage, such as single-stranded and double-stranded DNA strand breaks. Methods for determining chromosomal aberrations and sister chromatid exchange are alternative methods for detecting DNA damage, however, only metaphase cells can be analyzed, they have a long analysis time and laboriousness. The micronuclear test is widely used to analyze DNA fragmentation in bone marrow cells and has a very limited use for many other types of cells. Mass spectrometry methods require a long time for analysis and expensive equipment, and immunohistochemical studies can only determine double-stranded DNA chain breaks.

Of the methods currently available in the arsenal of genotoxicology for solving these problems, the most promising method is the comet test (electrophoresis of individual cells of agarose gel or the method of DNA comets). The method is designed to detect direct DNA damage and evaluate the effectiveness of the repair systems in the tissue cells of living organisms and plants under the action of a wide range of damaging agents of chemical, physical and biological nature. The method allows the detection of single-stranded and double-stranded breaks in the DNA chain, alkaline labile sites, cross-links between DNA and proteins, and oxidized purine and pyrimidine bases in combination with enzymatic treatment of the preparations. The advantage of the method is simplicity, a small amount of the required experimental material, profitability, speed of obtaining results, high sensitivity and productivity.

The comet test method was first proposed in 1984 by Ostling and Johanson to evaluate radiation-induced DNA damage in mammalian cells. Later, Singh et al. Proposed a modification of the method with electrophoresis under alkaline conditions, which, due to its high sensitivity, was most recognized by researchers. The method is based on the registration of various DNA motility and possible DNA fragments of lysed cells in a constant electric field. In this case, the DNA migrates to the anode, forming an electrophoretic trace that visually resembles the “comet tail”, the parameters of which depend on the degree of DNA damage to the studied cells.

The protocol for performing studies using the alkaline version of the "comet test" includes the following steps: obtaining a cell suspension, preparing preparations (slides) with cells immobilized in agarose, cell lysis, alkaline DNA denaturation, alkaline electrophoresis in neutral conditions, alkali neutralization, staining DNA fluorescence dye, microscopy of preparations with image capture and digital analysis of DNA distribution, statistical processing and assessment of the degree of DNA damage to the studied object. One of the most important stages is the stage of preparation of preparations with cells immobilized in agarose for subsequent procedures with these cells, which determines the duration of the entire study and its quality.

Traditionally, such preparations with one or two agarose blocks are prepared on ordinary microscope slides (76 × 26 mm) or specially prepared slides are used. The preparation of agarose blocks on standard glasses for microscopy is carried out by applying a drop of molten agarose to the glass surface and applying a standard size coverslip (24 × 24 mm) onto it. The glass slide is cooled until the agarose solidifies and the coverslip is carefully removed. A drop of a mixture of low-melting agarose with cells is applied to the resulting gel. Again, cover with a coverslip and refrigerated until the agarose solidified. After gel formation, the coverslip is removed and the next layer of low-melting agarose is formed on the gel surface in a similar manner. However, this method of forming an agarose block has several disadvantages.

First, only two blocks can be formed on the surface of a standard microscope glass. Secondly, as a result of the action of surface tension forces, the coverslip is deformed, and the agarose layer with cells has an identical thickness and an uneven distribution of cells inside the gel.

Known specialized glass company Trivigen (USA). These glasses are standard microscope slides coated with a special hydrophobic composition of a certain thickness. On the surface of these glasses there are circular areas (windows) that are not covered by such a composition (maximum three windows). Suspension of cells in low-melting agarose is dug into such windows and placed in a refrigerator for solidification of agarose. However, these glasses can be used only once, which leads to a significant increase in the cost of research.

A device is known described in the application for invention US 2012277118 A1, which allows to prepare an agarose matrix with cells for subsequent procedures with them in order to analyze damage and DNA repair. An advantage of the invention is the possibility of immobilization of a large number of cells in agarose in a strictly predetermined order of their placement in the matrix and subsequent manipulations with the entire matrix or its part, which leads to an increase in the analysis performance. However, time-consuming and lengthy procedures for preparing a cell cassette, including the preparation of an agarose matrix material with micro-cavities for cells, placing cells in cavities, coating the cells with an upper layer of agarose, do not guarantee the safety of the cells, their uniform distribution inside the matrix and can lead to the loss of large parts of biological material. Very stringent requirements are imposed on the geometric dimensions of the recesses in the matrix, since with their small size difficulties are created when cells are placed in them, with their large size there is a high probability of several cells entering the cavity, which is unacceptable for analysis. Given the different sizes of cells even in one population (for example, the sizes of cells in the total fraction of white blood leukocytes may differ by more than 5 times), the preparation of a matrix with cells seems to be a rather difficult task, the solution of which requires additional time (several hours) and procedures. All this leads to a significant increase in the cost of analysis. The patent does not describe special procedures for preparing a matrix with cells to ensure its plane parallelism and a certain fixed volume, as well as coating this matrix with an additional layer of agarose with the same required properties. The procedures for processing the cassette with cells with various solutions do not guarantee the maximum complete removal of the previous solution and the reagents contained in it. As a result of diffusion, which the authors do not take into account, contamination of reagents in unknown concentrations can occur, which will cause possible artifacts and distortions of the analysis results.

A device is known, described in the application for invention GB 2466816 A, which allows you to distribute agarose gel with cells immobilized in it according to the type of multi-well plate in order to analyze DNA damage. An advantage of the invention is the ability to automate a number of DNA comet method procedures, which leads to an increase in analysis performance. However, when cells are formed by a matrix substrate and a gasket with a series of channels, additional difficulties arise in creating a waterproof contact, the violation of which during the procedure of the method can lead to an uneven distribution of solutions across the cells and, consequently, to non-standard processing of cells with reagents and, as a result, large variability of the analysis results . When distributing a mixture of agarose with cells over the formed cells, it is very difficult to achieve uniform distribution of the suspension along the bottom of the cell due to the formation of a meniscus (for example, the authors declare the suspension volume for one cell of 96-well cuvettes as 20 μl, while the recommended minimum volume should be not less than 25 μl), uncontrolled formation of air bubbles in the gel is also possible. Considering that in the process of solidification of agarose, cells float in it, forming a saturated upper layer, the result is a high heterogeneity of the distribution of cells in solidified agarose with a maximum concentration on the agarose-air interface. The subsequent procedures of the method (lysis, denaturation and electrophoresis) will lead to a distortion of the nucleoid shape of such cells and, as a result, to a distortion of the analysis results.

A device is known described in the application for the invention US 2012058467 A1, which allows to distribute agarose gel with cells immobilized in it into a multi-well plate with electrodes placed in the wells for electrophoresis to analyze DNA damage. An advantage of the invention is the ability to automate a number of DNA comet method procedures, which leads to an increase in analysis performance. However, the formation of a plane-parallel agarose layer with cells is difficult due to the formation of a meniscus when the suspension is placed in the cells and the cells float in agarose. As a result of distortion of the shape of nucleoids located at the agarose-air interface, in the course of subsequent method procedures, there is a high probability of obtaining great variability and incorrect analysis results. The electrophoresis procedure using electrodes placed directly in an agarose gel with cells can lead to additional uncontrolled DNA damage due to secondary redox processes near electrodes during electrophoresis (active forms of oxygen and nitrogen, hypochlorites, etc.), especially under alkaline conditions.

High-performance techniques proposed by the authors of the above applications are necessary only for screening studies and are redundant and extremely expensive in most cases, the use of the comet test method in biomedicine.

The objective of the proposed invention is the creation of such a device that allows for the formation of a three-layer agarose block with an area of 10 × 10 mm and agarose layers of calibrated thickness on the surface of a glass slide measuring 25 × 10 mm.

The method of preparation of agarose blocks (slides) using the proposed device allows you to prepare calibrated multilayer agarose blocks, in which the agarose layer of cells is closed on top of an additional layer of agarose, which prevents the exit of the DNA molecule loops from the gel, because all of them are in the thickness of agarose. Coating the agarose layer with cells with the upper (third) layer of agarose allows the analysis of all cells contained in the slide, which removes the problem of "edge effects". The technique allows you to prepare individual mini-preparations, further work with which can occur both individually and in large quantities using special substrates that fix the mini-glass. This allows the necessary processing of cells with reagents on any number of mini-glasses simultaneously and a controlled change of solutions with reagents. The calibrated thickness of the agarose layers and, accordingly, the calibrated total volume of agarose blocks allows you to accurately calculate the required concentration of the reagent or dose of exposure.

The technical result that can be obtained by implementing the invention is to create three-layer agarose blocks with a calibrated layer thickness on the surface of mini-glasses, reducing the time required to prepare preparations, significantly reducing the consumption of chemicals, using standard equipment, and reducing environmental pollution with chemical reagents and increase the productivity of the researcher.

The specified technical result is achieved by the fact that the proposed device provides the preparation of a series of slides with agarose blocks of a fixed volume with a standardized thickness of agarose layers.

The device is illustrated in the figure, which shows a general view of the device assembly with two mini-glasses (Figure 1). The device consists of three parts, differing in design, and mini-glasses, on the surface of which three-layer agarose blocks are formed.

Part 1 (Figure 2) is a plane-parallel glass plate (1) on which dividing blocks (2) of a given thickness are fixed at regular intervals. The height of the blocks exceeds the thickness of the mini-glasses (3) by 50-100 microns.

Detail 2 (Figure 3) is a plane-parallel glass plate (4), one side of which is a polished surface, along the edges of which two side legs are welded in the form of glass blocks (5) with a thickness of 50 ± 5 μm.

Detail 3 (Figure 4) is similar to detail 2, it is a plane-parallel glass plate (6), but the thickness of the blocks (7) is 100 ± 5 μm.

Parts 1, 2 and 3 can be made of both glass and Teflon, metal or other materials that are resistant to concentrated solutions of organic acids and alkalis.

The device is used as follows.

Mini-glasses (3) are fixed in part 1 between the blocks (2) by gluing them to a drop of 1% normal agarose. Then, on top of part 1, part 2 is placed with the side not containing blocks (5). An agarose solution (concentration from 0.5 to 1%) is introduced into the lumen formed between the mini-glasses and part 2 using a semi-automatic pipette. The lumen is filled due to surface tension forces. After the agarose has solidified, part 2 is removed from part 1 by plane-parallel displacement of part 2 relative to part 1. On the gels formed in this way, 10 μl of a suspension of the studied cells in low-melting agarose is applied to each gel. On part 1, part 2 is placed with the polished side on an agarose solution containing cells. Then the assembly is placed in a refrigerator (4-8 ° C) for 5-7 minutes, for solidification of the agarose gel. After the specified time, the assembly is removed from the refrigerator and part 2 is removed from part 1 by plane-parallel displacement. In the next step, 10 μl of a solution of low-melting agarose is applied to the surface of the formed gels, and part 1 is covered with part 3 placed on the legs (7). The assembly is again placed in the refrigerator for 5-7 minutes. After this time, the assembly is removed from the refrigerator. Part 3 is removed from part 1 by plane-parallel displacement. Mini-glasses with agarose blocks formed on their surface are removed from part 1.

Thus, the claimed device allows for the preparation of mini-slides with three-layer agarose blocks on their surface, standardize the thickness of the agarose layers, obtain uniform distribution of cells in the agarose gel, significantly save the consumption of chemicals and increase the productivity of the researcher.

The claimed design was tested in model experiments on isolated cells of laboratory animals, as well as during genotoxic blood tests of healthy donors and patients undergoing postoperative outpatient chemotherapy for breast cancer, and has shown its high efficiency.

Example 1

The device was used in comparative experiments to identify the causes of inter-laboratory differences in the recorded level of DNA damage in mouse bone marrow cells. Using the device allowed for electrophoresis in smaller chambers. For example, for 24 samples prepared using standard glass slides, it was necessary

use an electrophoretic chamber of the type Sub-Cell Model 192 (Bio-Rad, USA) with an electrophoretic solution volume of 2.1 l. While for 24 glasses prepared using the inventive device, a SE-1 camera (Helikon LLC, Russia) with a volume of electrophoretic solution of only 0.25 liters was enough. From this example it can be seen that the consumption of the electrophoretic solution is reduced by almost 10 times, thereby reducing the cost of the analysis and the level of environmental pollution. The use of a cheaper electrophoretic camera also leads to a reduction in the cost of the analysis.

Example 2

The device was used to study the damaging effects of x-rays on the leukocyte DNA of whole mouse blood. Using six similar instances of the device, 24 preparations (slides) with leukocytes immobilized into agarose were prepared, while agarose was mixed with 6 times diluted whole blood and applied to slides. The study required 20 μl of whole blood and 800 μl of low-melting 0.5% agarose. The total preparation time was less than 30 minutes. As a result of the studies, the dose dependence of the damaging effect of x-ray radiation (doses of 0, 0.1, 0.2, 0.5, 1, 2, 5, and 10 Gy) on the leukocyte DNA of whole blood of a selected laboratory animal was determined using three repeated preparations (slides) for each experimental point, which allowed to obtain statistically reliable results. Using the device allowed to accelerate the preparation time of preparations and, therefore, accelerate the analysis, reduce the amount of biological material required for analysis and reduce the consumption of expensive chemically modified low-melting agarose.

Example 3

On preparations prepared using the inventive device and containing mouse splenocytes irradiated with x-ray at a dose of 8 Gy, the distribution of cell nucleoids over the entire surface of the slide was evaluated. Not only the level of DNA damage was recorded, but also the coordinates of individual nucleoids. Analysis of the results showed a uniform distribution of nucleoids on the surface of the preparation when scanning in one optical plane and the absence of significant differences in the level of DNA damage between nucleoids located in the central part of the preparation and at the edges. Thus, the use of the device allows you to get a uniform distribution of nucleoids on the slide, allows you to analyze all the cells contained in the slide, and removes the problem of "edge effects".

Claims (2)

1. A set for the preparation of multilayer agarose blocks on the surface of mini-glasses, consisting of the first part, which is a plane-parallel plate (1), on which dividing blocks (2) are fixed at regular intervals with a height exceeding the thickness of mini-glasses (3), installed between the blocks (2), at 50-100 microns; the second part, which is a plane-parallel plate (4), one side of which is a polished surface, on the edges of which two side legs are welded in the form of blocks (5) with a thickness of 50 ± 5 μm; the third part, which is a plane-parallel plate (6), one side of which is a polished surface, on the edges of which two side legs are welded in the form of blocks (7) with a thickness of 100 ± 5 μm; the first, second and third parts can be made of glass, Teflon, metal or other materials that are resistant to concentrated solutions of organic acids and alkalis. 2. A method of preparing multilayer agarose blocks on the surface of mini-glasses using the kit according to claim 1, characterized in that the mini-glasses (3) are fixed in the first part between the blocks (2) by gluing them onto agarose, while on top of the first part place the second part with the side containing no blocks (5), and the formed gap between the mini-glass and the second part is filled with agarose solution, after the hardening of the agarose gel, the second part is removed from the first part for application to the formed agarose gels ratios of the studied cells in low-melting agarose, after applying the cells, the second part with the polished side is placed on the first part containing agarose solution with the studied cells, after the agarose gel has solidified, the second part is removed from the first part and the low-melting agarose solution is applied to the surface of the formed gels, then the first part is covered placed on the legs of the third part, after solidification of the agarose gel, the third part and mini-glasses with formed on their surface are removed from the first part agarose blocks.

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