UA127428U - METHOD OF OBTAINING CULTURE OF AORT OF LABORATORY ANIMALS - Google Patents

Abstract

A method of obtaining aortic explants culture in laboratory animals involves in vitro cultivation by aortic tissue fragments explants. The adventitia is removed from the aorta with micro-tools.

Description

The useful model belongs to experimental medicine, in particular to the field of cell and tissue technologies, and can be used to obtain a culture of aorta explants of laboratory animals for the purpose of researching the regenerative potential of stem cells and further developing new methods of treating diseases of the cardiovascular system in humans.

The use of tissue-specific progenitors from main vessels is considered a promising approach for accelerating the regeneration of ischemic tissue damage accompanied by endothelial dysfunction. Obtaining in a short period of time a pure population of cells with the necessary morphological and functional characteristics to achieve the maximum therapeutic effect in the pathology of the cardiovascular system is an urgent task of modern biotechnology and regenerative medicine.

In recent years, new methods of cultivating tissue-specific stem and progenitor cells from various sources, including main vessels, have been actively implemented in Ukraine and the world. Several approaches are used to obtain cell cultures from the vessel wall in experiments and clinics.

In particular, there is a known method of obtaining a culture of smooth muscle cells from the aorta (Pat. 2529947

Russian Federation IPC StT2M 5/071, S12M 55/02, S12M 5/077, publ. 10.10.2014. pErulymlmi lire.gu|. The method consists in cutting out a fragment of a vessel, chopping it into pieces no bigger than 2 mm, and incubating in a 0.1 95 collagenase solution for 30 minutes. and cultivated in a culture flask with scratches previously applied to the bottom of the flask.

However, this method concerns obtaining cultures, first of all, of smooth muscle cells and only in humans, as it involves the selection of the ascending fragment of the thoracic aorta during coronary artery bypass surgery.

There is also a well-known method of culturing IP myo endothelial cells from the aorta of rats (Pat. 104673744А SM, IPC St2M 5/071; applicant and patent owner Mapa dipia (SM); publ. 06/03/2015 pOrz/Lmopamide. ezrasepei.soti|. According to this method at the stage of incubation in collagenase, the excised aorta is pre-filled with a nutrient medium containing heparin.

The ends of the vessel are sutured with a sterile needle and thread and incubated in CO? incubators

However, the use of this approach requires mastering the vascular overlay technique

From the seam and ensuring its tightness and sterility during incubation and when the medium is changed daily. Also, this technique cannot be used in laboratory mice due to the significantly smaller size of the aorta compared to the corresponding vessels in the rat.

A method for isolating endothelial cells from mouse aorta is also known from literary sources

IA bitrie Meinoady oi Izoyaipd Moise Aopis Epdoipeiia! Seyiv / M. Kobrauavni, K. Iposhye, E. UMagabi, Yei a. // the children of Ashegozsiegov apa (pgotrbrozviv. - 2005. - Moi. 12, Me 3. - R. 138-42). According to the publication, this method includes filling the aorta through a cannula with a solution of collagenase type HER for 45 minutes. followed by washing out the isolated cells with a nutrient medium. The obtained suspension is washed and cultivated in the medium for endothelial cells, and the aorta is cut into fragments with a side of 2-3 mm and cultivated by the explant method to isolate smooth muscle cells.

However, this method requires additional stages of tying the distal end of the aorta, washing it with a nutrient medium, and using a special coating of vials for cultivation, which increases the time and financial costs of its implementation.

As a prototype, the authors took the method of obtaining and cultivating endothelial cells from the thoracic aorta of a mouse (Pat. 106591217А СМ, МПК С12М 5/071; applicant and owner of the patent Opim

Zisptsap (SM); published 26.04.2017. пР/Амопамиде.езрасепей.соти|, which involves preliminary cutting of the aorta into rings and their cultivation by the method of explants on the Mayide matrix! in a special environment for endothelial cells.

However, this method does not prevent contamination of the culture with fibroblasts from the outer lining of the aorta and requires significant financial costs for growth factors and cover for culture dishes.

The basis of this useful model is the task of improving the method of obtaining the culture of explants of the aorta of laboratory animals, which would be reliable and effective and allow obtaining a pure culture of smooth muscle or endothelial cells from the inner membranes of the aorta, which is not contaminated by stromal cells from the outer connective tissue membrane, which additionally will reduce the time and cost of culturing the required cell population.

The task is solved by the fact that in the method that includes the cultivation of ip mygo by the method of explants of fragments of aorta tissue, according to this useful model, the adventitia is removed from the aorta beforehand with the help of micro-instruments.

The technical result, which is achieved when applying this method, is that thanks to the preliminary removal of the adventitia in the culture of ip migo from explants of the aorta, cells migrate only from its inner membranes. The absence of adventitia and para-aortic adipose tissue in explants of aorta fragments prevents contamination of the culture by fibroblasts and multipotent stromal cells, which rapidly proliferate and reduce the efficiency of migration of the target population of cells from the inner membranes. With this method, the time and costs of nutrient media and materials for cultivating the required population of cells are reduced. This method does not require significant investment of time and the use of expensive high-tech equipment.

The method is carried out as follows.

Animals are subject to euthanasia by the method of cervical dislocation after intraperitoneal administration of 2.5 95 solution of avertin at a dose of 400 mg/kg. Under sterile conditions, aortas are prepared and transferred to Petri dishes with ARMI-1640 medium, which contains 1 95 of a mixture of antibiotics

Repeigter Under a stereomicroscope, the aortic cavity is washed with a sterile medium

ARMI-1640, fragments of para-aortic adipose tissue are removed and the outer layer of the aorta (Shpisa echiegta, adventitia) is dissected from the middle (Shpisa tedia) and inner (Shpisa ipiita) aorta. The selected internal membranes are washed in ARMI-1640 medium, crushed into fragments about 1 mm in size: and incubated in a 0.1 95 solution of type I collagenase for 40 minutes at a temperature of 437 "C, after which they are washed in ARMI-1640 medium with 1 95 of a mixture of antibiotics Repoiger.The crushed elements of the inner lining of the aorta are transferred to cups

Petri dishes with a diameter of 35 mm with complete nutrient medium MEM Aoteaia with the addition of 10 95 fetal bovine serum.

Cultivation of all cell samples is carried out under standard conditions in a CO2 incubator at a temperature of 37 "С and a humidified atmosphere with a CO concentration of 595. The first change of the medium is carried out 10 days after the start of cultivation. Further replacement of the nutrient medium is carried out every three days. Subcultivation is carried out at reaching 80 95 confluency of the monolayer with the help of 0.25 95 trypsin solution.

Example.

The source of progenitor cells from main vessels was the aorta of male EVM mice

From the age of 5 months. Cultures of cells obtained from the adventitia and inner membranes of the aorta isolated by the proposed method were compared.

Attached adventitia explants maintained adhesion to plastic throughout the culture time until the first passage. The migration of cells from the explants of the adventitia of the mouse aorta began on the 7-10th day of cultivation. At early passages, the culture was heterogeneous in terms of morphological characteristics: cells were predominantly elongated spindle-shaped with a clear visualization of a round or oval nucleus and nucleoli. Some of the cells acquired a polygonal shape and formed long processes that contacted neighboring cells, forming clusters of a confluent monolayer. Cytoplasmic vacuolation was detected in some of the cells, in a short time they acquired a rounded shape and detached from the surface of the culture dishes, which indicates their apoptosis.

According to the morphological characteristics, the culture of cells from the inner membranes of the aorta of mice was represented by a homogeneous population of polygonal cells, which were in close contact with each other and formed characteristic monolayer islands of the "cobblestone" type. In the dynamics of cultivation, the islands merged with each other, forming a confluent monolayer. At the same time, the cytoplasm of the cells was light, homogeneous, without signs of vacuolation. Nuclei and nucleoli kept the correct rounded shape and were located in the center of the cells. At the early stages of cultivation, single giant flattened cells were present, and spindle-shaped cells were concentrically located around them, which were not detected during further passage.

As a result, from the explants of the aorta of mice, from which the adventitia was removed, a homogeneous cell culture was obtained, which in terms of morphological characteristics is similar to endothelial cells, without preservation of stromal elements.

Thus, this method is affordable, does not require expensive reagents and equipment, provides a pure cell culture, not contaminated by fibroblasts and stromal cells, and can be used in experimental medicine.

Claims (1)

USEFUL MODEL FORMULA The method of obtaining the culture of aorta explants of laboratory animals, which includes the cultivation of the ip migo by the method of explants of fragments of aorta tissue, which differs in that that beforehand, the adventitia is removed from the aorta with the help of micro-instruments.