Method for in vitro recovery of experimental tuberculous granuloma in cultures


G01N1 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

FIELD: medicine.

SUBSTANCE: method for in vitro recovery of experimental tuberculous granulomas in culture involves induction of granulomatous inflammation by infecting experimental animals with BCG mycobacteria, recovery and mechanical disintegration of spleen tissues containing granulomas over a period adequate to form them in an animal's body, homogenisation of spleen tissue in a solution by agitation, purification of the homogenate from coarse tissue fragments by spontaneous deposition in a culture medium, removal of the deposits, recovery of the granulomas from a supernatant, washing in a new portion of the culture medium by centrifugation at acceleration 15-28 g at least three times and transfer of the deposited granuloma in the culture medium.

EFFECT: effective granuloma integrity and higher purity of the recovered granulomas.

4 cl, 3 ex

 

The invention relates to experimental biology and medicine, and in particular to methods of explantation and cultivation of granulomas in vitro, and can be used when developing and evaluating the effectiveness of the treatment of tuberculosis complicated with tuberculous granulomatous process (necrosis, fibrosis), as well as in the development of new immunotropic drugs that regulate the functional activity of cells of granulomas and enhance their anti-TB activity.

There is a method of allocating chistosortnykh granulomas, including the induction of granulomatous inflammation by intraperitoneal infection of experimental animals (mice CBA/J) cercariae schistosomes (Schistosoma mansoni), the selection of the liver over a period of time sufficient for the formation of granulomas (8 weeks), preparation of homogenate of liver tissue by mechanical disruption using a Waring blender at low speed, the laundering of the homogenate several times in buffer solution RPMI 1640 by spontaneous deposition, the allocation of granulomas from the homogenate by centrifugation cooled to +4°C. the homogenate at 350 g for 5 min, the location of sediment containing granulomas in a nutrient medium for cultivation of granulomas (1). The disadvantage of this method is its inapplicability to highlight tuberculous granulomas. This is due t the m unlike chistosortnykh granulomas, the cells are firmly linked together by collagen fibers, tuberculosis granulomas do not possess such strength and are destroyed when chopping liver tissue or captured together with areas of tissue with which they are closely linked.

There is a method of separating and explantation chistosortnykh granulomas in culture in vitro, including the induction of granulomatous inflammation by intraperitoneal infection of experimental animals (mice C57BL/6) cercariae schistosomes (Schistosoma japonicum), the selection of the liver over a period of time sufficient for the formation of granulomas (6 weeks), preparation of homogenate of liver tissue by mechanical disintegration within 15 seconds using a blender in 10 volumes of buffer Hanks solution, the laundering of granulomas in cold buffer Hanks solution, the selection of granulomas from the homogenate by centrifugation cooled homogenate at 200 g up until the supernatant becomes transparent, the location of granulomas in the nutrient medium for cultivation (2). The known method have the same shortcomings.

Closest to the claimed method is the selection of tuberculous granulomas, including the induction of granulomatous inflammation by vnutribruchinnogo infection of experimental animals (mice) the Mycobacterium is M. avium, removing the liver through a period of time sufficient for the formation of granulomas, mechanical disintegration of the liver tissue by gently rubbing it between two subject chilled glasses (between their polished surfaces), preparation of homogenate of liver tissue in buffered Hanks solution, cleaning homogenate from large pieces of fabric by spontaneous deposition, removal of sediment, the allocation of granulomas from the supernatant of the suspension by subsequent triple centrifugation at 150 g with sequential removal of the supernatant and dissolve the precipitate with granulomas in the new portion of the buffer Hanks solution; the location of the selected granulomas in the medium for cultivation (3). The disadvantage of this method is that using the liver as a source of granulomas does not allow to distinguish "pure granulomas without impurities. This is due to the significant integration of granulomas in the liver tissue, the high density of the liver that prevents the release of even the most durable and compact vibrazioni tubercular granuloma intact, because the process of disintegration must be very hard. For this reason, in the known method using glass slides with polished surface as a means of mechanical disintegration of tuberculous granulomas that PR is leads to significant structural damage granulomas, in terms of the violation of their integrity. In addition, due to the high degree of connection of hepatocytes of the liver among granulomas in their mechanical selection there is a large number of fragments of liver tissue, comparable in size with granulomas, which makes the process of fractionation of pure suspension of granulomas very problematic. Another disadvantage of this method is the presence of a dedicated granulomas impurities in the form of a large number of erythrocytes, lymphocytes and macrophages. This is because the centrifugation of the homogenate (after deposition of a large piece of fabric) at 150 g leads to the deposition of these cells together with granulomas, which makes to get a clean granuloma

The problem to which the invention is directed to a more efficient preservation of the integrity of granulomas, increasing the purity of their selection.

The solution of this problem is achieved by the fact that the selection of granulomas use tissue of the spleen, the supernatant suspension containing granulomas, centrifuged in a gradient accelerations 15-28 g; deposition of large fragments of material from the homogenate and breeding besieged granulomas after each centrifugation is carried out in a culture medium; a mechanical disintegration of the tissue of the spleen produced by grinding into fragments of size 0.5-1 mm; it is each centrifugation the supernatant suspension is carried out for 2-5 min; the selection of the spleen are carried out through 30-90 days after infection;

Description of the invention

The method of obtaining experimental tuberculous granulomas in vitro as follows.

Experimental animals such as mice, intravenous Mycobacterium BCG vaccine for the induction of systemic tuberculosis inflammation. After the formation of experimental tuberculous granulomas (after 30-90 days after infection) are slaughtered in compliance with the rules of work with experimental animals with the use of anesthesia. Remove the spleen observing aseptic conditions and verify a sample of the spleen in the presence of induced granulomas, for example, by fluorescent or histochemical analysis. This is followed by mechanical disintegration of the tissue of the spleen by grinding into fragments of size 0.5-1 mm using a cutting tool such as a scalpel. Transfer minced tissue of the spleen in the medium for cultivation and homogenized by shaking, for example, on a shaker, within 1-3 minutes Spend cleaning homogenate from large fragments of tissue spleen by spontaneous sedimentation within 1-2 min in culture medium. Remove sediment. To highlight granulomas obtained supernatant, the suspension is centrifuged at an acceleration in the drop 15-28 g for 2-5 minutes The precipitate, containing granulomas, resuspension in a new piece of culture medium, repeat the procedure with the specified mode centrifugation at least two times. This allows you to clear granulomas from impurities of different cell types (erythrocytes, lymphocytes, excess macrophages). The precipitate, containing granulomas, diluted in medium for cultivation, cultivated under standard conditions at 37°C in atmosphere containing 5% CO2.

The advantage of using the spleen as an organ for excretion of granulomas is that granulomas are less integrated in the tissue of the spleen, which gives the opportunity to avoid their destruction at the stage of extraction and mechanical disintegration of the tissue of the spleen. In addition, the use of spleen allows you to select BCG-induced granuloma at all stages of their development, including prior to the beginning of fibrosis.

The selection of granulomas appropriate conduct through 30-90 days after infection. Early (30 day) in the spleen fully formed granulomas, in this period of adhesive contacts between cells granuloma sufficient to preserve the integrity of granulomas when you select them (they are not "crumble"). The increase in the period of formation of granulomas can be carried out to study the morphogenesis and development of granulomas in the later stages of vplo the ü to the stage of fibrosis.

Replacement homogenization by grinding between the glass (see the description of the prototype) on the mechanical grinding into fragments of size 0.5-1 mm contributes to the preservation of granulomas and increased purity of their allocation, because, on the one hand, their sizes from 60 to 125 μm, significantly smaller than these pieces of cloth; on the other hand, chopping into chunks of a specified size allows the release of granulomas from large pieces of tissue of the spleen.

The choice of gradient acceleration 15-28 g and duration of centrifugation experimentally chosen so as to precipitate granuloma of the homogenate earlier than in the sediment will fall in the homogenate of erythrocytes, lymphocytes and macrophages, the latter can be removed with the supernatant liquid, and does not precipitate with granulomas, as in the method prototype, which significantly increases the purity of the selection of granulomas. The speed of rotation of the centrifuge rotor when the same acceleration depends on the model of a centrifuge, or rather the radius of the rotor, and is determined by the well-known formula G=(2·π·f)2·R, where R is the radius of rotation of the rotor or the orbit of the body), f is the rotation speed in revolutions per second. For example, when using a centrifuge rotation speed corresponding to the acceleration 15 g, 400 rpm.

Method for assessing the viability of the cells based on p is jiznennoi colouring Trifanova blue it is shown that 99.5% of the cells in the granulomas remain viable. According to a computer morphometry digital photos granulomas, explanted into culture, it is shown that the culture of granulomas does not contain red blood cells and lymphocytes, and the number of macrophages is very small.

The number of free macrophages in suspension granulomas when released by the claimed method varies from 2-3 to 10-15 per selected granuloma or from 20 to 150 macrophages in 1 ml of the suspension depends on the number of ottavani granulomas by centrifugation in a solution of culture medium and the individual characteristics of the animal. Increasing the number of these procedures it is possible to achieve almost complete absence of macrophages in the composition of the granulomas. However, the presence of a small number of macrophages contributes to the improvement of culture conditions granulomas and increases the likelihood of their acceptance in the culture.

Using the inventive method culture medium (instead of buffer solution Hanks in the prototype) at the stage of spontaneous deposition of large fragments of material from the homogenate and on stage centrifugation promotes the viability of granulomas.

Another advantage of the claimed method is the possibility of its implementation without the use of expensive reagents, which makes extensive use of the ü it in practice research immunopathological mechanisms granulomatosa in the development of tuberculosis, the study of the immunological mechanisms that regulate the formation and evolution of granulomas in infectious granulomatous inflammatory processes, search, development and study of new anti-TB drugs.

Examples of specific performance

Example 1

The mice BALB/c mice were injected intravenously Mycobacterium BCG vaccine (0.5 mg in 0.25 ml saline). 30 days after injection in mice was induced by chronic systemic inflammation, simulating generalized tuberculous inflammation. The mouse was scored after light ether anesthesia method of cervical dislocation. The spleen was removed, observing aseptic conditions for obtaining biological tissue samples for cultivation (in a laminar box "Clinic-572"), and were placed in a sterile plastic Petri dish (diameter 40 mm), which was pre-made and 1 ml of culture medium, namely the medium of alpha-MEM containing 5% serum of cow embryos.

Small fragments of spleen (1-2 mm) were cut with a scalpel for subsequent morphological analysis. Typical BCG-induced granulomas in the spleen was verified by fluorescent analysis (on the microscope AxioI-mager Z1, Zeiss) fragment spleen stained with acridine orange, as well as in the subsequent analysis of the sample villages is the eye the standard method of histological analysis (according to the traditional scheme, including formalin fixation, obtaining paraffin sections, dewaxing, painting with hematoxylin and conclusion in balm).

The contents of the spleen were released from the capsules and conducted preliminary mechanical homogenization of the splenic tissue directly in the Petri dish in 1 ml of the specified environment for cultivation by grinding with a scalpel into fragments of a size of 0.5-1.0 mm Further pre gomogenizirovannogo tissue of the spleen was placed into the flask with a working volume of 20 ml, was made in 9 ml of the specified environment for the cultivation and were shaken on a shaker at a frequency of 20 Hz for 1 min. Then the homogenate in the form of a suspension containing red blood cells, splenocytes, granulomas and larger fragments negozinegoziodisnay splenic tissue was transferred into a centrifuge tube 10 ml, left 1 min for deposition of large fragments negozinegoziodisnay splenic tissue. The supernatant suspension containing red blood cells, splenocytes, granulomas in the volume of 10 ml was centrifuged at acceleration 15 g for 5 minutes the Supernatant was decanted, the precipitate was reasponsible in a new portion of the specified culture medium. The procedure was repeated twice, and the duration of the second centrifugation was 5 min, the third - 3 minutes the precipitate containing granulomas, bred in an environment e is I cultivation and made in plastic tablets for cultivation, in which pre-placed glass cover. The cultivation was carried out on the basis of standard procedures: at a temperature of 37°C in atmosphere containing 5% CO2.

Dimensions isolated from spleen granulomas, explanted in culture, varied from 50 to 70 microns in diameter, reflecting the various stages of the granulomatous process.

The number of granulomas in the suspension amounted to a value of 19 granulomas/ml without morphological signs of violation of their integrity (integrity - 100%), while erythrocytes and lymphocytes were absent, and the content of macrophages was small (85 cells per 1 ml of suspension).

Example 2

The claimed method was carried out analogously to the description in example 1 with the difference that the spleen was isolated after 90 days after infection. Each centrifugation was performed three times when starting acceleration 28 g for 2 minutes

Method for assessing cell viability, based on the in vivo colouring Trifanova blue, shows that 99.5% of the cells in the granulomas remain viable. According to a computer morphometry digital photos granulomas, explanted into culture, sizes isolated from spleen granulomas, explanted in culture, varied from 60 to 125 microns in diameter, reflecting the various stages of the granulomatous process.

The number of granulomas in suspense and amounted to the value of 21 granulomas/ml, the share of granulomas without morphological signs of integrity - 86%, the content of macrophages - moderate (175/ml suspension).

Example 3

The claimed method was carried out analogously to the description in example 1 with the difference that the spleen was isolated in 60 days after infection. Each centrifugation was performed 4 times during acceleration 15 g for 5 minutes It was possible to isolate a pure granuloma, almost not containing macrophages, invalid splenocytes and other related cells, which may be necessary for future research in the study of factors that produced the actual granulomas.

The number of granulomas in the suspension amounted to a value of 20 granulomas/ml suspension, the share of granulomas without morphological signs of integrity violation - 90%, the content of macrophages is very low (15/ml suspension).

Sources of information

1. The method of obtaining experimental tuberculous granulomas in vitro culture, including the induction of granulomatous inflammation by infection of experimental animals with Mycobacterium BCG, selection and mechanical disintegration of the tissues of the body, containing granulomas, through a period sufficient for their formation in the animal body, homogenization of tissue in the solution by shaking, cleaning homogenate from the available fragments of tissue spontaneous deposition in a physiologically acceptable solution, removal of sediment, the allocation of granulomas from the supernatant suspension and laundering in a physiologically acceptable solution by centrifugation at least three times; the transfer besieged granulomas in the environment for cultivation, characterized in that the selection of granulomas use tissue of the spleen, the supernatant suspension containing granulomas, centrifuged at acceleration 15-28 g; deposition of large fragments of material from the homogenate and breeding besieged granulomas after each centrifugation is carried out in a culture medium.

2. The method according to claim 1, characterized in that the mechanical disintegration of the tissue of the spleen is produced by grinding the fragments of 0.5-1 mm.

3. The method according to claim 1, characterized in that each centrifugation the supernatant suspension is carried out for 2-5 minutes

4. The method according to claim 1, characterized in that the selection of the spleen carried out in 30 to 90 days after infection.



 

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