RU2646135C1 - Method of storage of the piglet kidney for the production of monolayer of primary-tripsinated cells and using it in virusological studies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology. Invention is the method for storing kidneys of piglets at subnormal temperatures (0÷4°C) from 2 to 7 days in order to obtain the monolayer of primarily trypsinized cultures.

EFFECT: invention makes it possible to increase the storage time of the isolated kidney.

3 cl, 5 ex, 3 tbl, 5 dwg

Description

The invention relates to the field of biotechnology, and in particular to a method for storing a piglet’s kidney, intended for the further preparation of a monolayer of primary trypsinized cells used in scientific and practical work to determine the avirulence of vaccines, for setting up the neutralization and titration of viruses.

The main research and inventions in medical transplantology concern the creation of preservative solutions and media that help maintain the required level of organ conservation. An important role belongs to heparin (anticoagulant), buffer components (salt) and glucose, to ensure minimal energy metabolism [12-14]. The proposed methods for preserving rat liver using perfusates and containers that can withstand high pressure in order to avoid extra and intracrystallization of solutions and tissues are rather cumbersome and are still applicable only in laboratory conditions [15]. In all proposed inventions, the process of crystallization of organs is excluded [16].

Preservation of animal organs for their use in cell technology bypasses some of the problems associated with the aggregation (process of thrombosis) of red blood cells and white blood cells, as well as the full physiological state. In this case, it is important, after storage at subnormal temperatures, to obtain viable cells after trypsinization, which can adhere, divide and grow.

We have found out such an option as storing frozen piglet kidneys at a temperature from minus 4 ° C to minus 2 ° C (with crystallization of the organ). Trypsinization of the kidney of the piglet after thawing of the crystalline phase of the medium + kidney system was performed (i.e., the kidney was in ice and probably itself was partially crystallized). Organ trypsinization was the standard option, the resulting cell suspension was "seeded" in penicillin vials and plastic "mattresses" with an area of 25 cm ² . Cells that were not stained with trypan blue (concentration of 200,000 cells / ml) did not adhere and did not form a monolayer when inoculated, trypsinized cells were at the stage of apoptosis. It was finally concluded that any freezing with a crystallization phase without cryoprotectants negatively affects the survival of cells and tissues of organs in which about 90% of water [1, 8].

In medical practice and veterinary virology, no information was found on the use of isolated kidneys stored at low temperatures (about 0 ° C) to obtain cell cultures. In 1982, a method of cryopreservation of primary cell cultures and tissues for virological purposes was proposed. After using this method, cell viability decreased to 60% [11]. The method requires long preparation and recovery after cryopreservation. But there is an advantage, storage can be long, up to several months.

The main goal of developing a method for longer storage of kidneys (more than 2-4 hours) is to rationalize the process of obtaining primary cells, when the trypsinization period must be prolonged in time to obtain complete, not overgrown cell cultures suitable for virological studies.

The second goal is to study the safety of organs at subnormal temperatures in the laboratory for the further application of the obtained data in the field with the possibility of subsequent obtaining complete primary cells and subcultures. When a full monolayer of primary cells is obtained from trypsinized organs, it is also necessary to study their suitability for virological studies.

The primary cell culture obtained from the kidney of a piglet (SP) is used not only when working with foot-and-mouth disease virus, but also in the isolation of viruses that infect pigs, including African swine fever (ASF).

The authors have developed a method for storing a kidney of a piglet, capable of properly maintaining the initial state of an organ suitable for virological studies for a longer time than other known methods.

Our studies fill a gap in the longer use of donor organs for virological purposes.

To achieve these goals, experiments were carried out using medium 199, which is suitable for maintaining viability and specific cell functions. This medium is also used as the main component for storing human transplant kidneys. This medium contains 62 components of substances, including salts, amino acids, vitamins, nucleosides and glucose. A relatively low concentration of substances provides an insignificant metabolism and minimal accumulation of metabolites during storage [9]. Storage temperature ranged from 0 ° C to 4 ° C for 2-7 days.

Isolated kidneys from 3-5-week-old piglets were placed in a wide-necked vial with nutrient medium 199, in which, in addition to 100 units. penicillin and 100 units. streptomycin, 50 μg / ml gentamicin was added. The ratio of medium to gas phase was 2: 1. The vial was covered with a sterile Petri dish and placed in a household refrigerator. During storage, a temperature regime was chosen that excluded crystallization of the medium and organ.

We found that storing the kidneys under sterile conditions at temperatures from 0 ° C to 4 ° C does not lead to their crystallization, while at the same time, the vitality of histotypic cells is preserved, which, after trypsinization and obtaining a complete monolayer, are sensitive to various viruses, including including foot and mouth disease virus.

The use of the visualization control method of the invention (photographing and description of the obtained trypsinized cells grown in culture bottles) makes it possible to objectively evaluate and use the resulting cultures.

The essence of the invention is illustrated in the graphic materials:

FIG. 1 - monolayer of the primary culture of the joint venture obtained on the 5th day after trypsinization of the "fresh" kidney (0 hours of storage);

FIG. 2 - monolayer of the primary culture of the joint venture obtained on the 5th day after trypsinization of a 3-day kidney (72 hours of storage);

FIG. 3 - monolayer of the primary culture of the joint venture obtained on the 5th day after trypsinization of the 4-day kidney (96 hours of storage);

FIG. 4 - monolayer of the primary culture of the joint venture obtained on the 5th day after trypsinization of the 6-day kidney (144 hours of storage);

FIG. 5 - degeneration of a monolayer of primary SP cells (obtained from a kidney that was stored for 6 days) when interacting with foot and mouth disease virus.

DETAILED DESCRIPTION OF THE INVENTION

The methods of preserving human organs at subnormal temperatures (0–4 ° C) have been used in transplant surgery for several decades [7]. Thanks to fundamental studies conducted with animal organs, it was found that parenchymal organs (liver, kidneys) and heart can be transplanted within a day after removal from the donor, the cornea of the eyes for 3 days of storage [4, 7, 8]. After this period, irreversible biochemical and morphological changes in tissues occur that lead to organ rejection by the recipient [1, 6]. In most cases, irreversible changes occur in the vascular systems (blood clots form) and in stromal tissues, which ensure the survival of organs.

In veterinary virology, despite the recommended 2-4 hours of storage at 0 ÷ 4 ° C of internal organs and tissues [3], long-term storage of more than 2 days also has scientific and applied value. In literary sources, including patents, we did not find information regarding this problem in veterinary virology.

The primary trypsinized culture of the joint venture was obtained from the kidneys of piglets of 3-5 weeks of age using generally accepted methods for obtaining primary cell cultures [2, 4]. Storage of isolated kidneys was carried out at a temperature from 0 ° to 4 ° C in medium 199 with the addition of 100 units. penicillin, 100 units. streptomycin and 50 μg / ml gentamicin without serum. Kidney trypsinization was performed after 1-7 days of storage. As a result, we carried out work to study the adhesive ability of individual cells and their aggregations, the morphology of sedimentary cells, the timing of the formation of a monolayer, and sensitivity to foot and mouth disease virus [2, 3]. To grow the primary cells of piglet kidneys (SP), we used DMEM / F-12 nutrient medium with 0.1% GLA (lactalbumin hydrolyzate) from Sigma and fetal bovine serum from Bioclot. Cell cultivation was carried out in penicillin vials with an area of 3 cm ² and plastic bottles with an area of 25 cm ² .

The analysis of the morphology of the cells and the monolayer was carried out using an Olimpus CKX-41 phase contrast microscope. The productivity of primary cells was also calculated after the formation of a monolayer on days 5–7.

The sensitivity of the primary culture of SP cells was evaluated by titrating the working dose of the virus with the neutralization reaction. The same dilution of the culture of foot and mouth disease virus (CFJ) was titrated on the monolayer of the primary culture of SP cells grown immediately after trypsinization. After 2-3 days, the control of the virus infectivity was repeated on a SP cell culture grown after storage of a suspension of cells or whole piglet kidneys for 2-7 days at a temperature of 0 ° C to 4 ° C.

After kidney collection from 3-5-week-old piglets, the organs were placed in a wide-necked vial with nutrient medium 199, in which, in addition to 100 units. penicillin and 100 units. streptomycin, 50 μg / ml gentamicin was added. The ratio of the medium and the gas phase is 2: 1. The vial was covered with a sterile Petri dish and placed in a household refrigerator. A temperature close to zero was maintained in the refrigerator, preventing crystallization of the organ and the environment. The bottle with the medium in which the pig’s kidney was placed was not sealed with a plug so that passive gas exchange with the surrounding air took place. This storage condition was fulfilled in order to avoid acidification of the medium, since glycolysis occurs even in chilled organs [4]. When storing the kidney of a piglet (in 100 ml of medium 199) at a temperature close to zero for 96 hours, the pH of the medium increased from 7.0 to 7.1-7.2. In a 199 medium bottle where there was no organ, the pH increased to 7.6. The stabilization of the pH of the environment where the kidney is located indicates that the tissues of the body "breathe" by utilizing glucose and releasing carbon dioxide and metabolites.

Stored 2-7 days at a temperature of 0 ° C to 4 ° C, the kidneys of the pig did not change their texture and color. The cortical layer was standardly ground with steel scissors, and it was also standardly dispersed with a warm trypsin solution. From 1 gram of renal tissue received up to 30 million cells not stained with trypan blue. As was revealed in previous works, trypsinized cells were polymorphic and belonged to different histotypic tissues. Not all cells produce colonies [2, 3].

In our case, cells obtained from kidneys stored at temperatures from 0 ° C to 4 ° C for 2-7 days were also polymorphic and subsequently had standard dynamics of sedimentation, adhesion, and proliferation. The first indicator of cell safety after trypsinization is the number of viable cells obtained from 1 g of renal tissue, and testing with trypan blue staining. The number of cells obtained from 1 g of kidneys, in all cases was equal to an average of 15-20 million.

In the process of conducting experiments on storing the kidneys by the proposed method at temperatures close to zero, the morphological characteristics of the primary cells depending on the "age" of the monolayer were refined.

As can be seen from the graphic materials, the formed monolayer in all cases is represented by polymorphic cells with a minimal amount of extracellular matrix. After storing the kidneys by this method for up to three days at a temperature close to zero, and after subsequent trypsinization of the tissues of the cortical layer (at the optimal seed concentration of cells), a monolayer is formed in 5 days (Fig. 1, table 2); when storing kidneys up to 5 days, a full-fledged monolayer is formed in 6 days (Fig. 3). During 6-day storage of the kidneys, the monolayer formed in 7 days with the predominance of normal polymorphic cells without extracellular matrix (Fig. 4).

According to our observations, the kidneys of piglets at a storage temperature close to zero do not change color, there are also no obvious signs of necrosis, and trypsin dispersion occurs in the standard mode. But after 5-day storage of the kidneys, the proliferative activity of surviving trypsinization cells is 15-20% lower. The dynamics of the formation of a monolayer is delayed by an average of 1 day. In all experiments, we did not observe contamination with banal microflora and received a primary sterile culture.

When conducting virological work, all variants of the obtained cellular material were suitable for determining the avirulence of semi-finished biological products, for carrying out the neutralization reaction, and for isolating foot and mouth disease virus.

The sensitivity of cells to foot and mouth disease virus was comparable to control. In our studies, the sensitivity to the FMD virus of primary cells obtained from stored kidneys by the proposed method was 7% higher than in the control (table 3).

The cytopathic effect of the foot and mouth disease virus on cells obtained after storage of the organ for up to 6 days was typical and intense (Fig. 5).

In the process of staging virologic reactions, we were able to analyze the dynamics and specifics of the cytopathic effect of VJ on primary cells. First of all, epithelial-like cells of a monolayer are affected. They become spherical, the process of deadhesion is accompanied by the appearance of many cytoplasmic outgrowths, after some time the surface of the cells becomes smooth, then granular, and then fragmentation occurs, i.e. destruction of the monolayer to detritus. In our opinion, the epithelial-like cells of the grown monolayer are the precursors of the epithelial tissues lining the surface of the baumen capsules, which are the first to take on contact with the viruses circulating in the piglet's body. The “descendants” of stromal tissues of blood vessels and organs - fibroblast-like cells, are last affected by a viral infection.

In literature, we did not find data on the storage conditions of animal kidneys to obtain primary cell cultures. Our research helps fill the gap in the area of longer-term use of donor organs. We found that storing the kidneys in sterile conditions at a temperature of 0 ° C to 4 ° C does not lead to crystallization of organs, at the same time, the vitality of histotypic cells is preserved, which, after trypsinization and obtaining a complete monolayer, are sensitive to foot and mouth disease virus.

It was determined that crystallization of the medium and kidneys during storage at temperatures from minus 4 ° C to minus 2 ° C leads to irreversible changes in trypsinized cells, which after inoculation into culture vessels do not adhere and do not give colonies.

As a result of the experiments, it was shown that when the primary trypsinized cell suspensions were stored in the refrigerator for two days at a temperature of 4 ÷ 6 ° C (table 2), partial death of the epithelial cells most sensitive to foot and mouth disease occurs. At the same time, storing the kidneys at subnormal temperatures (0 ÷ 4 ° C) from 2 to 7 days allows to obtain normal populations of primary cells with high sensitivity to the virus after trypsinization.

Our proposed method for storing the kidneys of piglets makes it possible to more efficiently use donor organs to obtain primary cultures used in virology. At the same time, this method of storing organs for preserving and transporting organs obtained in the field from domestic and wild animals can be proposed.

The invention is illustrated by examples of its implementation and use.

Example 1. With the selected physical parameters of kidney storage and with free gas exchange, the pH of the medium remains at the level of physiological values - 7.1-7.2, which indicates the functional activity of the organ and suitability for trypsinization. In the control medium (without kidney), the pH increased to 7.6.

Example 2. Obtaining a primary trypsinized culture of SP from kidneys stored for different days is illustrated in Table 1. The monolayer of grown cell cultures obtained from stored kidneys retains proliferative activity, but the formation period of the confluent layer is increased by 1-2 days. Primary cell polymorphism is maintained (Fig. 1-4).

Example 3. In the process of staging virologic reactions, we were able to analyze the dynamics and specifics of the cytopathic effect of VJ on primary cells. First of all, the epithelial-like cells of the monolayer are affected by the virus (Fig. 5). The sensitivity to the virus of primary cells obtained from stored kidneys was 7% higher than in the control (table 3).

Example 4. Cells obtained from kidneys stored at 0-4 ° C for 2-7 days were polymorphic and subsequently had standard dynamics of sedimentation, adhesion and proliferation. The first indicator of cell safety after trypsinization is the number of viable cells obtained from 1 g of renal tissue and tested with trypan blue staining. The number of cells obtained from 1 g of kidneys, in all cases was equal to an average of 15-20 million.

Example 5. In the process of long-term storage of the kidneys, after trypsinization, epithelial-like cells prevail in the suspension, which are primarily affected by the foot and mouth disease virus. The cytopathic effect is manifested in the degradation of cells, their transformation into spherical structures with a permeable membrane and further lysis of the monolayer.

The proposed method for storing the kidneys of piglets at 0 ÷ 4 ° C for 2-7 days provides a full-fledged cell preparation for scientific research: determining the avirulence of biological products, staging the neutralization reaction and isolation of foot and mouth disease virus.

Information sources

1. Kirpatovsky V.I. Kudryavtsev Yu.V. Cryopreservation of rat and rabbit kidneys. // II international conference "Successes of modern cryobiology". - Kharkov, 1992. - p. 82-83.

2. Manin B.L., Koropova N.V. The influence of artifacts of the biological status of piglets on the cultivation of the primary culture obtained from the kidney // Tr. Federal Center for Animal Health. - Vladimir, 2012 .-- T. 10 .-- S. 238-245.

3. Overnight V.T. Comprehensive standardization of the production of primary cell cultures. "Cytology", M .; 1999 T. 41, p. 298-299.

4. Troshina V.P. The functional state of isolated tissues at a temperature close to zero. West. LSU, 1957, 3, ser. Biol., 1, p. 111-121.

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6. Yurchenko T.N., Zhulikova E.P., Govorukha T.P. Structural and functional parameters of liver cells at the stages of preparation for cryopreservation. // Problems of cryobiology. - 1991. - No. 1. - S. 8-16.

7. Yangson R.-M .. Surgery. What and why does the surgeon do? Minsk "Popuri" 1997 - 592 s.

8. Jeske A.N., Fontebs A.M., Karow A.M. Functional preservation of the mammalian kidney. III. Ultrastructural effects of perfusion with DMSO / - Cryobiology, 1974, 11, No. 2, p. 170-181.

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Claims (3)

1. The method of storage of a piglet kidney isolated under sterile conditions, namely, that medium 199 is used as a nutrient medium with the addition of 100 units / ml penicillin, 100 μg / ml streptomycin, 50 units / ml gentamicin; to optimize the chemical homeostasis of the storage medium, the organ is placed in a container with a wide neck, with a ratio of medium and air phase of 2: 1, and the container is covered with a Petri dish; stored at a temperature close to zero for 7 days, preventing crystallization of the medium and organ, for the further production of primary trypsinized cells and the cultivation of a complete monolayer of the primary culture of piglet kidney cells (SP). 2. The method according to p. 1, characterized in that the kidney of the piglet is stored at a temperature of from 0 ° C to 4 ° C. 3. The method according to p. 1, characterized in that the resulting monolayer of the primary culture of the cells of the kidney of the piglet is suitable for determining avirulence, the neutralization reaction and for isolation of foot and mouth disease virus.

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