RU2308281C1 - Method for in vitro cloning of regional pancreas stem cells - Google Patents

Abstract

FIELD: biology, medicine.

SUBSTANCE: claimed method includes dissociation of laboratory animal pancreas tissue by homogenizator. Substance with antiprotease activity, such as contrical is added into solution for homogenization. Obtained cell elements are placed in cultural medium containing DMEM medium 80 %; non-inactivated embryo calf serum 20 %; glucose 10 g/l; L-glutamine 500 mg/l; gentamicin 50 mg/l; heparin 5000 IU/l; insulin 50 mg/g; stem cell growth factor 10 ng/ml; epidermal growth factor 30 ng/ml; leucosis-inhibiting factor 10 ng/ml; interleukin-6 10 ng/ml; fibroblast growth factor 10 ng/ml; and incubated in CO₂-incubator at 37°C, 5 % CO₂ and air humidity of 100 % for 14-21 days.

EFFECT: new method for cloning of regional pancreas stem cells.

1 ex, 2 dwg

Description

The invention relates to the field of experimental biology and medicine, and relates to a method for in vitro cloning of regional pancreatic stem cells.

There are a large number of methods for in vitro culturing the cellular elements of many tissues in culture media of various compositions, with various additives, most often using incubation of cellular material in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity [1 , 2].

However, a drawback of the existing methods is that their use does not make it possible to clone regional pancreatic stem cells in vitro.

The development of this method is an urgent task, the solution of which is necessary for scientific purposes: to conduct basic research on the proliferative and differentiation balance of regional stem cells in situ in the pancreas and the patterns of their functioning and vital functions, as well as to search for and create drugs that can influence the functional activity of this fraction of resident clonogenic cellular elements of glandular tissue in order to increase the efficiency of the treatment of pancreas left, and, first of all, diabetes mellitus of various origins.

The problem solved by this invention is to provide a method for in vitro cloning of regional pancreatic stem cells.

The task is achieved in that the dissociation of pancreatic tissue of laboratory animals is carried out using a homogenizer, and a substance with antiprotease activity is added to the solution. Contrical is used as a substance with antiprotease activity. The obtained cell elements are placed in a culture medium and incubated in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity for 14-21 days. The culture medium has the following composition that we offer: 80% DMEM medium, 20% uninactivated fetal calf serum, 10 g / l glucose, 500 mg / l L-glutamine, 50 mg / l gentamicin, 5000 IU / l heparin, 50 mg / l of insulin, 10 ng / ml of stem cell growth factor, 30 ng / ml of epidermal growth factor, 10 ng / ml of leuko-inhibiting factor, 10 ng / ml of interleukin-6, 10 ng / ml of fibroblast growth factor.

New in the proposed method is the use of substances with antiprotease activity during mechanical dissociation of pancreatic tissue and the composition of the culture medium.

Diseases of the pancreas are very common throughout the world and occupy a significant place in the structure of morbidity and disability of the population of our country. Of particular danger to the health and social significance of the pathology of this organ is diabetes. Moreover, despite the fact that there is a significant amount of medications for the treatment of this disease, today there are no ways to radically cure its various forms. Patients have to constantly, throughout their lives, take replacement therapy or other hypoglycemic drugs. In addition, it is very difficult and not always successful in the treatment of exocrine pancreatic insufficiency. At the same time, thanks to the modern development of cell technologies and the associated detection of resident multipotent stem cells in various organs (the purpose of which is tissue regeneration in response to physiological loss of cells or their death caused by a damaging factor) [3], it became possible to search for new ways development of pathogenetically substantiated methods of treating pancreatic diseases [4], based on stimulation of stem cell functions. However, as described above, to develop such methods, a method for cloning regional pancreatic stem cells is needed, which allows in vitro studies of the effects of various drugs and substances on them.

The fact of the use of the culture medium from the components used and their concentration, including the addition of the specified set of multifunctional cytokines to the medium, which are early acting growth factors with multidirectional mechanisms of action on the functional state of cellular elements, and the use of substances with antiprotease activity during mechanical dissociation of pancreatic tissue with the achievement of a new result: the creation of a method for in vitro cloning of regional stem cells of the pancreas oh gland, for a specialist is not obvious.

The method for dissociating pancreatic tissue used in the present invention and the composition of the medium in the aggregate are optimal for in vitro cloning of regional pancreatic stem cells.

The claimed essential features in the aggregate showed new properties that are not obvious to a specialist and not arising explicitly from the prior art in this field. New features make it possible to clone regional pancreatic stem cells in vitro and the present invention can be used in experimental biology and medicine. An identical set of features was not found in the study of the state of the art in patent and medical literature.

Based on the foregoing, the claimed technical solution should be considered relevant criteria: "Novelty", "Inventive step", "Industrial applicability".

The invention will be clear from the following description and the accompanying drawings.

In figure 1 A - acinus-like formations on the background of heterogeneous cellular elements that grew on the 14th day of cultivation (native preparation), uv. 250 x; figure 1 B - acinus-like formation that grew on the 21st day of cultivation (native drug), uv. 600 x

Figure 2 - acinus-like formation that grew on the 21st day of cultivation, okr. azur II-eosin, uv. 600 x

The method is as follows.

The pancreas of laboratory animals (mice) is placed in a homogenizer with a nutrient medium containing 5 U / ml of contracal and homogenized until a homogeneous cell suspension is obtained, which, after filtration through a nylon mesh, is placed in a culture medium of the following composition: 80% DMEM, 20% uninactivated fetal calf serum, 10 g / l glucose, 500 mg / l L-glutamine, 50 mg / l gentamicin, 5000 IU / l heparin, 50 mg / l insulin, 10 ng / ml stem cell growth factor, 30 ng / ml epidermal growth factor, 10 ng / ml leuko-inhibiting its factor, 10 ng / ml of interleukin-6, 10 ng / ml of fibroblast growth factor and incubated in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity for 14-21 days.

The proposed method was studied in experiments on mice of the CBA / CaLac line (category 1 animals, conventional linear mice) in an amount of 27 pcs. weighing 18-20 g. Animals were obtained from the nursery of the experimental biomedical modeling department of the Research Institute of Pharmacology, Siberian Branch of the Russian Academy of Medical Sciences (certificate is available).

Example 1

The method is as follows. In a CBA / CaLac mouse weighing 20 g, euthanized by dislocation of the cervical vertebrae under ether anesthesia, the pancreas was removed under sterile conditions (box equipped with laminar). The pancreas was washed from red blood cells in physiological saline. Then it was placed in a homogenizer with DMEM nutrient medium containing 5 U / ml of contrycal and homogenized until a homogeneous cell suspension was obtained at room temperature. After this, the cellular material was filtered through a nylon mesh to remove large aggregates, washed twice by centrifugation at 1500 rpm for 5-10 minutes, and the total number of nuclears was counted, determining their viability using trypan blue. Then the cells were placed in the medium of the following composition: 80% DMEM (Serva, Germany), 20% uninactivated ETS (Sigma, USA), 10 g / L glucose, 500 mg / L L-glutamine (Sigma, USA ), 50 mg / l gentamicin (Serva, Germany), 5000 IU / l heparin (Biochemie, Austria), 50 mg / l pork multicomponent insulin (Novo Nordisk, Denmark), 10 ng / ml growth factor stem cells ("Sigma", USA), 30 ng / ml of epidermal growth factor ("Sigma", USA), 10 ng / ml of leuko-inhibiting factor ("Sigma", USA), 10 ng / ml of interleukin-6 ("Sigma" , USA), 10 ng / ml fibroblast growth factor (Sigma, USA). The concentration of cellular elements was adjusted to 200 thousand / ml and 3 ml of the prepared suspension was placed in plastic 6-well plates (Costar, USA). The culture was incubated for 14-21 days in a CO ₂ incubator (Jouan, France) at 37 ° C, 5% CO ₂ and 100% air humidity. After incubation using a MBS-9 binocular microscope (SW 56x), the number of multicellular (more than 30 cells) acin-like formations was counted (Fig. 1). Incubated cellular material in the plate was fixed with methanol and stained with azure P-eosin for 30-40 minutes, after which the preparations were studied under immersion (figure 2). Moreover, incubation for 14 days only leads to the beginning of the formation in the culture of individual, mainly incompletely formed acinus-like structures. Extending the incubation up to 21 days leads to the formation of not only fully formed acini, but also often allows them to receive drain growth.

The proposed method allows in vitro to clone regional pancreatic stem cells and obtain, in addition to the growth of heterogeneous cellular elements, organ-specific growth - acinus-like multicellular formations, which are structural and functional units of glandular tissue.

Literature

1. Goldberg E.D., Dygay A.M., Shakhov V.P. Methods of tissue culture in hematology. - Tomsk: Publishing house of TSU, 1992. - 272 p.

2. Gritti A., Bonfanti L., Doetsch F., Caille I., Alvarez-BuyIla A., Daniel A. Lim, Galli R., Jose Manuel Garcia Verdugo, Daniel G. Herrera, Vescovi A.L. Multipotent Neural Stem Cells Reside into the Rostral Extension and Olfactory Bulb of Adult Rodents // J. Neuroscience. - 2002. - V.22. - P. 437-445.

3. Goldberg E.D., Dygay A.M., Zhdanov V.V. Modern views on the problem of stem cells and the possibilities of their use in medicine // Cellular technologies in biology and medicine. - 2005. - No. 4. - S.184-199.

4. Orlic D., Kajstura J., Chimenti S., Anversa P. Mobilized bone marrow cells repair the infarcted heart, improving function and survival // Natl. Acad. Sci. USA - 2001. - V.98. - P.10344.

Claims (1)

The method of in vitro cloning of regional pancreatic stem cells, which consists in incubating cells placed in a culture medium in a CO ₂ incubator at 37 ° C, 5% CO ₂ and 100% air humidity, characterized in that upon dissociation of pancreatic tissue a substance with antiprotease activity is added to the solution of laboratory animals using a homogenizer, which is used as a contracal, then the obtained cell elements are placed in a culture medium and incubated for 14-21 days, and as a culture A mixture of the following composition is used in the medium: 80% DMEM medium, 20% uninactivated fetal calf serum, 10 g / l glucose, 500 mg / l L-glutamine, 50 mg / l gentamicin, 5000 IU / l heparin, 50 mg / l insulin 10 ng / ml stem cell growth factor, 30 ng / ml epidermal growth factor, 10 ng / ml leuko-inhibiting factor, 10 ng / ml interleukin-6, 10 ng / ml fibroblast growth factor.

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