Method for production of conditioning medium having colony-promoting activity

FIELD: medicine, in particular hematology.

SUBSTANCE: CBA/CaLac intact mouse spleen is homogenized in medium containing 95 % of RPMI-1640 and 5 % of ethyl silicate; filtered through nylon grid; washed by centrifugation with RPMI-1640. at 1500 rpm for 10-15 min; final concentration of living splenocites is adjusted to 2x106 colony/ml using media containing 10 % of ethyl silicate, 1 % of L-glutamine; 0.2 % of gentamycine, 0.05 % of mercaptoethanol, 89 % of RPMI-1640; obtained product is cultivated at 37°C for 6 days, centrifuged and supernatant containing target product is separated followed by storage at -20°C. Before filtering 1 ml of distilled water is added in sterile spleen homogenate and after 25 sec 10 ml of RPMI-1640 medium is added. Target product is stored for 1 month or less.

EFFECT: method of improved accuracy and validity.

1 tbl

 

The invention relates to medicine, specifically to Hematology and immunology, and the development of a method of producing a conditioned environment, with colony-stimulating activity.

Currently used in Hematology conditioned environment, with colony-stimulating activity are mainly recombinant hematopoietic growth factors. These substances are used, usually in two versions: purified recombinant growth factors intended for experimental research and has a high cost, or officinal drugs hematopoietic cells, which are due to the presence of additional substances (fillers) are unsuitable for use in the laboratory [5].

Known methods for producing conditioned medium with colony-stimulating activity by use of this purpose, conditioned media from immune cells (mostly splenocytes), treated with mitogens (concanavalin a, phytohemagglutinin (PHA)or lectin from lacunosa American (Pokeweed)).

Closest to the proposed method (prototype) is a method of obtaining a conditioned environment, with colony-stimulating activity: allocate the spleen from one intact mouse CBA/CaLac, homogenized ven the training environment the following composition: 95% RPMI-1640, 5% ETS, then filtered through nylon mesh and 2-fold washed by centrifugation at 1500 rpm for 10-15 minutes. After the last centrifugation count the total number of viable kariolou and bring the final concentration to 2×106the splenocytes/ml of medium of the following composition: 10% ETS, 1% L-glutamine, 0.2% gentamicin, 0.05% of 2-mercaptoethanol, 89% RPMI-1640 with the addition of PHA at the rate of 10 µg/ml of Cell suspension is left at 37°C, 100% humidity and 5% carbon dioxide for 6 days. At the end of the cultivation collect the supernatant, which was purified from cells by centrifugation and stored at -20°With 3-4 months at -80°With up to 2 years [2].

However, in these methods, together with the received conditioned environments with colony-stimulating activity in culture of hematopoietic cells are the remains of mitogen contained in the conditioned environment. The last influence on the processes occurring in the environment, in particular may be additional uncontrolled stimulation of colony [1].

In connection with this extremely important issue is the development of new available for use in everyday laboratory practice ways of getting effective conditioned environments with colony-stimulating activity, not providing additional is uncontrolled CSOs influence on the process of colony.

The problem solved by the invention, is to increase the reliability and accuracy of the method.

Set in the present invention this object is achieved by a method of obtaining a conditioned environment, with colony-stimulating activity by highlighting spleen intact mouse CBA/CaLac, its homogenization in the medium of the following composition: 95% RPMI-1640, 5% ETS, filtration through nylon mesh and 2-fold laundering by centrifugation for 10-15 minutes before the first laundering pour sterile homogenate, 1 ml of sterile distilled water and pipeinput within 25 seconds, bring to 10 ml of pure medium RPMI-1640 and after the second launder bring the final concentration to 2×106the splenocytes/ml of medium of the following composition: 10% ETS, 1% L-glutamine, 0.2% gentamicin, 0.05% of 2-mercaptoethanol, 89% RPMI-1640), the cultivation of suspension for 6 days at 37°and storing nadeshiko at -20°With no more than 1 month.

The proposed method of producing a conditioned environment, with colony-stimulating activity, perform the following: identify the spleen intact mouse CBA/CaLac, its homogenized medium of the following composition: 95% RPMI-1640, 5% ETS, filtered through nylon mesh and 2-fold washed by centrifugation for 10-15 minutes, before the first laundering pour sterile 1 ml homogenate article is Riley distilled water and pipeinput within 25 seconds, bring to 10 ml of pure medium RPMI-1640 and after the second launder bring the final concentration to 2×106the splenocytes/ml of medium of the following composition: 10% ETS, 1% L-glutamine, 0.2% gentamicin, 0.05% of 2-mercaptoethanol, 89% RPMI-1640, suspension cultured for 6 days at 37°and store adosados at -20°With no more than 1 month.

New in the proposed method is that before the first laundering sterile spleen homogenate pour 1 ml of sterile distilled water, pipeinput within 25 seconds, bring to 10 ml of pure medium RPMI-1640.

Know the use of osmotic shock (treatment with distilled water) for cleaning the cell suspension from erythrocytes [2]. Information about how to use distilled water to produce a conditioned environment, with colony-stimulating activity in the analysed literature could not be found. This method of obtaining a conditioned environment, with colony-stimulating activity was tested in the Institute of pharmacology of RAMS Siberian branch, so it matches the criteria of the invention "industrially applicable".

To determine colony-stimulating properties of conditioned medium of splenocytes treated with distilled water, and comparison with known methods supplied samples in three versions. Studies performed on 28 mice-males if the AI CBA/CaLac at the age of 2-2 .5 months (7 per group). Animals 1 category (conventional linear mouse) obtained from the collection Fund of the laboratory of experimental biomedical modeling, Institute of pharmacology, Siberian branch of the Russian Academy of medical Sciences (certificate available). Before sampling, the mice were slaughtered by the method of dislocation of the cervical vertebrae under ether anesthesia.

Highlighted in the sterile environment of the spleen were subjected to homogenization in preparative environment (95% RPMI-1640, 5% ETS), filtered through nylon mesh, washed by centrifugation at 1500 rpm for 15 minutes in medium RPMI-1640, was decanted supernatant.

Sterile spleen homogenate from animals of the 1st group (control) resuspendable and counted (using Trypanosoma blue) the total number of viable kariolou. Drove the final cell concentration to 2×106cells/ml of medium of the following composition: 10% ETS, 1% L-glutamine, 0.2% gentamicin, 0.05% of 2-mercaptoethanol, 89% RPMI-1640.

Prototype method: sterile spleen homogenate from animals of the 2nd group resuspendable and counted (using Trypanosoma blue) the total number of viable kariolou. Drove the final concentration of splenocytes to 2×106cells/ml of medium of the following composition: 10% ETS, 1% L-glutamine, 0.2% gentamicin, 0.05% of 2-mercaptoethanol, 89% RPMI-1640 was added to PHA (Sigma, USA) at the rate of 10 mg/ml

Proposed method: in sterile the first of spleen homogenate from animals of the 3rd group were added to sterile distilled water at 25 sec., then immediately 10 ml of RPMI-1640 medium, filtered through nylon mesh and washed by centrifugation at 1500 rpm for 15 minutes. After centrifugation the supernatant was replaced by preparative medium (95% RPMI-1640, 5% ETS), the cell suspension resuspendable and counted (using Trypanosoma blue) the total number of viable kariolou. Drove the final cell concentration to 2×106cells/ml of medium of the following composition: 10% ETS, 1% L-glutamine, 0.2% gentamicin, 0.05% of 2-mercaptoethanol, 89% RPMI-1640.

The resulting mixture was incubated in each case within 6 days at a temperature of 37°C.

After 6-day incubation, conditioned medium of the cells was centrifuged, collected supernatant and used to stimulate growth of the seven day granulocytemacrophage colonies in vitro in a medium of the following composition: 80% RPMI-1640 medium ("Serva", Germany), 19% ETS ("Sigma", USA), 280 mg/l L-glutamine (Sigma, USA), 1% methylcellulose (Sigma, USA), 50 mg/l gentamicin ("Serva", Germany) supplemented with 10% from each supernatant of the 3 groups involved in the experiment [2].

Cells for the test system was prepared as follows: bone marrow was isolated from the femur of a mouse 0.5 ml preparative environment (95% RPMI-1640, 5% ETS), suspended and counted the total number of myelokaryocytes, determining their viability. Then deleted the adgesiruta cell PU is eat inactivated 1 ml of cell suspension with cellularity 10 10/l for 1 hour in plastic Petri dishes (37°C, 100% humidity). Non-stick items brought to 2.0×105/ml, and 0.5 ml of the prepared suspension was placed in a 24-hole plastic plates ("Costar, USA) and were incubated for 7 days in CO2incubator ("Jouan, France) at 37°C, 5% CO2and 100% humidity. After incubation counted the number of colonies. Under the colony meant formed in the culture hearth of hemopoiesis, containing more than 50 cells. Counting colonies were made with a binocular microscope MBS-9 (Uvh). The number of CFU-GM was calculated from 105neadgezii of myelokaryocytes.

The results were processed by the method of variation statistics using t-student criterion [4].

The results of the study

The experiments showed that conditioned medium from splenocytes treated with distilled water, not inferior in activity to the use of conditioned medium from splenocytes, preliminary processing phytohemagglutinin (experimental group) and has a marked stimulating effect on the growth of progenitor cells of granulomatous type CFU-GM in comparison with the growth of the colonies under the effect of conditioned medium from splenocytes, not subjected to any treatment (control group). Thus, the significant difference between the performance of the control and experimental groups were detected in all series of experiments (table 1). The values of indicators of colony (CFU-GM) in groups using conditioned media from splenocytes treated with distilled water and PHA, on average 2-3 times higher than the figure in the group, where as a stimulant used the environment from the intact splenocytes. It should be emphasized that at the same time, the use of distilled water was the most effective. The number of CFU-GM in the above-mentioned method of stimulation exceeded on average 2 times the figures in the group to stimulate growth granulocytemacrophage colonies were used conditioned medium from splenocytes treated with PHA. The main positive effect of using the proposed method was higher purity of the experiment, the accuracy and reliability of the obtained results, due to the fact that in culture of hematopoietic cells are the remains of mitogen contained in conditioned environment, which affect the processes occurring in the environment, thereby reducing the accuracy and reliability of the method.

Thus, studies have shown that the use of conditioned medium from splenocytes, pre-treated with distilled water, the ability to stimulate growth granulocytemacrophage colonies in vitro exceeds prototype method, rezultati, obtained in this way, more accurate and reliable.

Table 1
The number granulocytemacrophage colonies (CFU-GM×105the nuclears), grown from bone marrow cells of intact mice of CBA/CaLaC depending on the means of stimulating the growth of colonies in vitro (X±m; P)
StimulantA series of experiments
123
Without stimulant2,33±0,82

**
3,33±0,821,50±0,55

**
Phytohemagglutinin5,00±0,89

*
4,17±0,757,00±0,89

*
Distilled water9,67±0,82

* **
11,00±1,26

* **
14,33±0,82

* **
* - reliability of differences with the control (without application of the stimulator) ** - reliability of differences with group 2 (stimulation of colony growth by phytohemaglutinin)

Sources of information

1. Galaktionov VG Immunology. - M.: Medicine, 1998. - 479 C.

2. Goldberg ED, Digi A. M., Shah V.P. Methods of tissue culture in Hematology. - Tomsk: Publishing house of Tomsk state University, 1992. - 272 S. - prototip.

3. Goldberg ED, Digi A.M., Sherstoboev TRIGGER Mechanisms of local regulation of hematopoiesis. - Tomsk: STT, 2000. - 148 C.

4. Lakin GF Biometrics. - M.: Higher school. - 1973. - 215 S.

5. Mashkovsky PPM Medicines. In two parts. 4.2. - 12th ed., revised and enlarged extra - M.: Medicine, 1993. - 736 S.

6. Petrov R.V. Immunology. - M.: Medicine, 1983. - 368 S.

A method of obtaining a conditioned environment, with colony-stimulating activity by homogenizing the spleen intact mouse CBA/CaLac in the medium of the following composition: 95% RPMI-1640, 5% ETS, filtering through a nylon mesh, launder by centrifugation medium RPMI-1640 at 1500 rpm for 10-15 minutes, bringing the final concentration of viable splenocytes to 2×106cells/ml of medium of the following composition: 10% ETS, 1% L-glutamine, 0.2% gentamicin, 0.05% of 2-mercaptoethanol, 89% RPMI-1640, culturing at 37°C for 6 days, centrifugation and separation of the supernatant containing the target product, and then stored it at -20°C, characterized in that before filtering add to sterile spleen homogenate, 1 ml of distilled water at 25 C and then immediately 10 ml of RPMI-1640 medium, and the target product store no more than 1 month.



 

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