UA146819U - METHOD OF LONG-TERM CULTIVATION OF HEMOPOETIC STEM CELLS - Google Patents

Abstract

Method of long-term cultivation of hematopoietic stem cells using diffusion chambers, into the cavity of which the investigated cells are introduced, including stages of manufacturing sealed chambers by stamping from polyacrylamide gel due to its polymerization, introduction into cell cavity of cell material complete nutrient medium in vitro or in vivo, removal of the chambers in due time from the appropriate nutrient medium and examination of cultured cells directly in the chambers under an inverted microscope, or removal of cells from the chamber, fixation on slides, staining and examination under a light microscope. At the stage of manufacturing the chambers create a common two-layer gel plate for several chambers, formed by superimposing a flat gel plate on the gel plate with holes, and seal the formed internal cavities due to the final polymerization of gel components, the resulting sealed two-layer plate is washed. , with 3-4 times change of physiological solution, the chambers are stamped out of it, which are further stored in 40% ethyl alcohol solution, and the day before culturing cells the chambers are moved from alcohol solution to physiological solution of 0.9% NaCl, then in the inner cavity of the chamber is injected with suspensions of test cells in the nutrient medium using a syringe through a puncture of its wall and seal the puncture site by introducing through the same needle semi-liquid agar during the slow removal of the syringe from the puncture site

Description

The useful model belongs to cell biology, biotechnology, toxicology and regenerative medicine, is related to the support in conditions closest to natural ones, the proliferation and differentiation of hematopoietic cells ip miigo and ip mimo, and can be involved in hematological and immunological studies. The stated solution can be used for growing tissues and forming a potential transplant and assessing its quality.

The main goal of the implementation of cell technologies is to create optimal conditions outside the body in which any cells could develop, proliferate and differentiate to the amount necessary for future transplantation to the patient. When searching for the conditions closest to physiological ones, the primary role is given to the method that achieves the expansion of viable, functionally directed cells in the necessary direction.

Stem cells and among them the most promising - hematopoietic stem cells are an attractive object for the accumulation of cells and the study of cellular interactions.

A well-known method of detecting stem cells is the method of placing cellular material in glass or plastic Petri dishes filled with nutrient medium with a semi-permeable filter, followed by cultivation of ip miigo (11.

One of the significant disadvantages of the method is the impossibility of creating in Petri dishes, vials, culture mattresses, tablets, etc., conditions that are closest to natural ones, although many nutrient media, additives, growth factors, cytokines and their combinations are currently offered to support the cell culture of i.p. miigo. But, unfortunately, there is still no method that would completely imitate the physiological conditions for a living cell, which are optimal for its life support.

In recent years, works have appeared in which researchers associate the reason for the extinction of hematopoiesis in culture with the special nature of the rigidity of the base that forms the microenvironment. Selection of ratios of components of gels of such rigidity, which would allow in the experiment to obtain long-term hematopoiesis with the accumulation of stem cells and progenitor cells in culture, is the task of today.

The choice of the basis of the determined stiffness optimal for its characteristics for cell expansion remains the main task in the development of bioengineering structures.

Currently used as a coating of polystyrene or glass Petri dishes, both natural and

Synthetic materials from collagen, alginate, chitosan, gelatin, hydroxylapatite, polylactic and polyglycolic acids, etc. |2).

Until now, various brands of biologically compatible hydrophilic polyacrylamide gel are widely used in experimental medicine, plastic, aesthetic and reconstructive surgery (SAqualift - Ukraine, Interfal - Ukraine, Farmacryl - Russia, Ata?ipde - China and others) .

There is a known method of stem cell cultivation, which consists in the cultivation of stem cells on a soft substrate (polyacrylamide gel of different consistency) in the presence of the appropriate complete nutrient medium ip miiko (ZI.

The authors managed to obtain long-term support in the culture of the proliferation of embryonic stem cells of the mouse and to prove that the reason for the long-term proliferation of cells lies in the degree of rigidity of the substrate on which the cells of the embryo are cultivated.

The determination of the Young's modulus (the degree of stiffness of the substrate in kilopascals (KPa) proved that the extinction of proliferation at a stiffness of 8 KPa (the stiffness of the gel corresponds to the stiffness of the plastic) occurs on the second day, and at 3.5 KPa - on the third day. When cultured on a substrate with a stiffness of 0.6 KPa fading of proliferation is not observed even with the removal of the factor that traditionally supports the proliferation of stem cells in culture (HIE-leukemia-inhibitory factor).

The presence of proliferation markers and the absence of differentiation markers on cells during five days of cultivation on a soft gel base indicate the preservation of proliferation in the culture of embryonic stem cells, that is, the expansion, accumulation of cellular elements.

There is also a known method of detecting stem cells by placing cellular material in a chamber filled with a nutrient medium with a semi-permeable filter, which is connected to a container containing a nutrient medium through a pump that ensures a uniform unidirectional movement of the medium through the filter at a speed of 2.1-2 ,4 mmz/min. The cultivation system is pre-filled with a suitable nutrient medium. Peripheral blood mononuclear cells with functional polypotency were selected for the study.

Peripheral blood mononuclear cells of healthy volunteer donors were isolated using a ficolverografin gradient.

Cells obtained by the fractionation method were brought to a final concentration of 3x105 mononuclear cells/ml with the nutrient medium.

Cellular material was injected into the chamber using a syringe through the valve opening in the side (4).

Thus, the cultivation takes place in conditions of directed movement of the nutrient medium at 37 "C for 24, 48 and 72 hours. After the end of the experiment, the filter is removed from the system, the cellular material is fixed and examined using cytological and cytochemical methods.

This method leads to the accumulation of a slightly larger cell mass. However, having significant advantages compared to the method of cultivation in Petri dishes, this method does not allow obtaining hematopoiesis in the culture, which would become a powerful producer of hematopoietic cells of all stages of differentiation necessary for transplantation.

In culture, according to this method, the first sprouts of hematopoietic cells appear during the first day in the form of macrophages and lymphocytes, and then there is the formation of fibroblast-like cells, which is proven by cytochemical research methods. The disadvantage of the method is cultivation on a rigid rigid substrate, which is glass and plastic (hardness 8KRa), which ultimately leads to the extinction of hematopoiesis and the maintenance of fibroblast-like structures.

The closest to the stated solution (its analogue) is the method of cell cultivation using diffusion chambers, into the cavity of which the cells under study are introduced (51.

Diffusion sealed chambers are made by polymerizing the working solution (a mixture of polyacrylamide gel components), which is poured into the space between two parallel Plexiglas plates, the upper of which has protrusions to form cavities in the lower plate.

The mixture is polymerized until a gel is formed and the top plate is removed.

A suspension of the studied cells is introduced into the formed cavities, and they are covered with round covers made of polyacrylamide gel of a slightly larger diameter than the diameter of the cavity.

The chambers are sealed by placing a plexiglass sheet on top with a gap between it and the lid. After that, the working solution is poured in and polymerized until a gel is formed, the sheet material is removed and the chambers are stamped, followed by their washing from the remains of unreacted gel components with a physiological solution of sodium chloride.

In the future, the cameras are implanted in the animal's body during the cultivation of ip mimo, or immersed in

The nutrient medium for the cultivation of ip myo. At the specified time, remove the cells from the appropriate nutrient medium and examine the cultured cells directly in the cells under an inverted microscope.

This method really ensures the accumulation of cellular elements in the chambers, thanks to cultivation on a soft gel basis.

At the same time, cameras made of polyacrylamide gel are characterized by optical transparency, which allows observation of the studied cells under light microscopy in the dynamics of their growth and reproduction, which greatly simplifies the research process.

But the main significant disadvantage of the analogue is that, despite all the listed advantages of the method of cultivating cells in gel chambers, which promotes their active growth and reproduction, stem cells and progenitor cells from them are not supported and, accordingly, long-term hematopoiesis is not achieved.

In the opinion of the Author of the stated solution, this is caused by the procedural shortcomings of the known method (analogue) of cultivation, which are directly related to the sequence of stages of preparation of the chambers and introduction of the suspension of the investigated cells specified in it.

Namely, the suspension of the studied cells is introduced into the semi-finished chambers (into the recesses of the gel plate) even before they are sealed with the working solution through its final polymerization to form a gel.

That is, living cells remain in the unwashed gel for a certain time until the chamber is stamped out and washed in physiological solution for a day. Having come into contact for a long time with toxic residues of unreacted components of the gel (and they always remain), and then instead of the CO" of the incubator and nutrient medium with additives, finding themselves in a physiological solution, they partially lose their viability.

And, as a result, their development can be observed, but their contact with toxic substances during the entire time of the formation of cells negatively affects the functioning of hematopoietic cells.

The purpose of the proposed useful model is to improve the method of cell cultivation in gel chambers by providing optimal physiological conditions for the cultivation of hematopoietic stem cells and their proliferation and differentiation during a long-term cultivation period due to the exclusion of contact of cultured cells with 60 toxic components of the gel.

The task is solved by the fact that the claimed method of long-term cultivation of hematopoietic stem cells using diffusion chambers, into the cavity of which the cells under study are introduced, includes the stages of manufacturing hermetic chambers by stamping them out of polyacrylamide gel due to its polymerization, introducing cell material into the chamber cavity, followed by cell cultivation by placing sealed chambers in the appropriate complete nutrient medium ip mito or ip mimo, extracting the chambers at a specified time from the appropriate nutrient medium and examining the cultured cells directly in the chambers under an inverted microscope, or extracting the cells from the chamber, fixing them on glass slides, staining and studying under a light microscope.

What is new, according to the claimed method, is that at the stage of camera manufacturing, a common two-layer gel plate for several cameras is created, formed by superimposing a flat gel plate on a gel plate with holes, and the formed internal cavities are sealed due to the final polymerization of the gel components.

Gel plates for the formation of a two-layer plate are created by superimposing two rigid covers, a flat cover and a cover with protrusions, respectively, on two layers of the required thickness of the mixture of gel components.

The obtained hermetic two-layer plate is washed from the toxic residues of the gel components in a physiological solution during the day with a 3-4-time change of the physiological solution.

After that, the cameras are stamped and disinfected, and in the future they are stored in a 40-95 solution of ethyl alcohol.

And a day before the start of cell cultivation, the chambers are moved from the alcohol solution to the physiological solution of 0.9 95 Mass.

After that, suspensions of the studied cells in the nutrient medium are injected into the inner cavity of the chamber with the help of a syringe through the puncture of its wall and the puncture site is sealed by introducing semi-liquid agar through the same needle while slowly withdrawing the syringe from the puncture site.

Sodium chloride solution or phosphate buffer solution (PB5) in a ratio of 1:10 is used as a physiological solution for washing the obtained gel chambers.

Zo As a nutrient medium, the organism of a laboratory animal is used, into which a hermetic gel chamber filled with a suspension of the studied cells is implanted.

A complete nutrient medium, the composition of which corresponds to the purpose of the experiment, is used as a nutrient medium for ip mice, into which a sealed gel chamber filled with a suspension of the cells under study is immersed.

Thus, according to the claimed method of cell cultivation, in comparison with the known method (similar), changes were made at the stage of formation of diffusion chambers, which made it possible to obtain integral sterile hermetic chambers, to ensure their long-term storage and use in the required quantities depending on the purpose of the study.

The proposed solution involves the introduction of a suspension of cells precisely into a fully formed sterile hermetic chamber in advance, which excludes their contact with the components of the future gel, which are toxic before polymerization. Due to which the cells do not lose viability for a long time.

The new approach leads to the emergence of new opportunities - a unique phenomenon is observed when outside the body, thanks to the optimal comfortable conditions created for the cells (mouse body, soft transparent non-toxic chambers, the stiffness of which is strictly regulated by following the recipe of the future gel), blood formation (hemopoiesis) is long-term supported for a long time (1.5...4.5 months), which ensures the accumulation of stem cells.

In addition, it was also found that according to the claimed method, thanks to the optimal comfortable conditions created for the cells (probably corresponding to the natural presence of cells in the body or at the stages of embryonic development), the growth of stem cells that cannot be seen (their morphology is unknown) is supported in the chambers ), but the result of their action, that is, their function, is that during long-term cultivation of the bone marrow (where the "sleeping" stem cells are initially located) blood formation (hemopoiesis) occurs in the chambers.

Examples of implementation of the claimed method.

According to the claimed method, at the stage of forming the diffusion chambers, the mixture of polyacrylamide gel components is immersed in pre-made Plexiglas cuvettes, which have two rectangular sectors of the same size. The mixture in each cuvette is covered with lids, one of which is flat, and the other has circular protrusions over the entire surface. because after polymerization of the gel, the lids are removed, and the gel plates remain in the cuvettes,

one of which has holes, and the other is flat.

The resulting plates are immediately placed on top of each other and kept connected until the gel is completely polymerized, and accordingly - until the cavities formed between them are completely sealed.

The two-layer gel plates obtained in this way are washed from the toxic residues of the gel components in a physiological solution of sodium chloride or a phosphate buffer solution (PB5) in a ratio of 1:10, during the day with 3-4 changes of the physiological solution.

Next, from the washed two-layer plates, all the obtained chambers are stamped with a metal cylinder of the appropriate size and immersed in 40 95 ethyl alcohol for disinfection.

It has been experimentally proven that the cameras can be stored in such a solution for up to five years without changes in the functional characteristics of cultured cells, which were determined by the Meisai method!

Mooge (1973) |bB)| (by the number of colonies that are formed during repeated cultivation in semi-liquid agar from cells that belong to the department of stem cells (hematopoietic progenitor cells).

Experimental data that confirm the long-term storage of cameras without losing their quality are presented in Table 1.

Table 1

Colonization efficiency of hematopoietic progenitor cells of the mouse bone marrow in the proposed diffusion chambers of different shelf life 22418011 11111111111111111збо111

That is, no statistically significant difference in the number of colonies growing in chambers of different storage periods was found (p20.05).

A day before the start of cultivation, the necessary number of cells (depending on the needs of the study) is moved from the alcohol solution to the physiological solution of 0.995 Mass for washing.

A suspension of the studied cells in a nutrient medium is introduced into the inner cavity of a sterile, washed from alcohol solution chamber with the help of a syringe through a puncture of its wall.

Seal the puncture site by injecting through the same needle semi-liquid agar (for example, Oiiso agar (0.33 95)) while slowly withdrawing the syringe from the puncture site until the hole in the chamber wall is completely closed.

The success of the procedure is checked under the control of an inverted microscope.

Absolutely sterile and reliably sealed chambers filled with a suspension of the cells under study are surgically implanted into the peritoneal cavity of a laboratory animal (recipient mice, etc.), which is a source of growth factors and nutrients for cells, during i.p. mimo cultivation.

Or in Petri dishes filled with a complete nutrient medium with growth factors, embryonic serum and additives for the cultivation of ip miigo.

In the case of culturing IP mimo, the cameras are removed from the abdominal cavity of the animals and studied under an inverted microscope.

The development of the cells cultivated in the migo chambers can be observed during the cultivation process.

After the end of the experiment, both ip mimo and ip mio cameras are dismantled. Cells are removed from their cavities, transferred to glass slides and prepared for cytological, cytochemical and histological study by separating cells with needles or using a cytocentrifuge.

Experimental studies have proven that according to the claimed method of cultivating ip mimo in a gel diffusion chamber filled with bone marrow cells in a liquid nutrient medium, that is, in a suspension culture, and implanted in the abdominal cavity of sexually mature linear mice, hematopoiesis is supported, which can last more than 1.5 month

The confirmation of this is the high viability of cells (suprivative staining with 0.1 95 trypan blue for survival shows up to 96 95 live cells), cytological and cytochemical characteristics of cells and the results of repeated cultivation of cells from chambers in semi-liquid agar, which leads to the formation of colonies of granulocytic-macrophage cells- precursors, which is evidence of the long-term culture of hematopoietic stem cells in the chamber.

Also, during cultivation according to the declared method, ip mygo in vials with medium

ERMI or OMEM (Zidta) from 20 95 embryonic calf serum in the presence of granulocyte-macrophage growth factor ZM-C5E, in which a diffusion chamber with bone marrow cells in semi-liquid agar is immersed, on the 14th day of cultivation, colonies of hematopoietic progenitor cells, the closest descendants are observed hematopoietic stem cells.

In order to compare the morphofunctional features of cells cultured according to the stated method and according to a known method (an analogue), an experiment was conducted, during which bone marrow cells of mice irradiated at a sublethal dose, in an equal amount (1x105 each), were introduced into a semi-finished chamber (until the fact of its sealing, according to the analogue method) and into a sterile and reliably sealed chamber (according to the declared method).

After 2 weeks of cultivation, the results were taken.

To do this, cells were washed from the inner cavity of each chamber, placed on a glass slide, and supravital staining was performed for viability.

It turned out that in the proposed (according to the claimed method) sample, all compared indicators (cell viability index, the number of cells in the cultures after their removal from the chamber cavity, colony formation in the culture with semi-liquid agar) were significantly higher than in the sample according to the known method- analogue

Cell counting was carried out in the Goryaev chamber.

The numerical indicators of the obtained results, shown in Table 2, eloquently testify to the significant advantages of the claimed method of cultivating hematopoietic cells.

Table 2

Comparative characteristics of morphofunctional features of cultured cells

Compared indicators - - - - - (Colony formation//-/-:/ | 77777770 36b535 ССС

The proposed method makes it possible to cultivate cells in optimal physiological conditions and not only to identify stem cells at an early stage, but also to reproduce hematopoiesis in the chamber,

Which ensures the expansion of hematopoietic cells, that is, their proliferation and differentiation during a long-term cultivation period.

The achieved results are stable and convincing.

What will allow to experimentally investigate the process of accumulation of hematopoietic cells, and, in the future, to apply the principles and understanding of the process as a basis for creating ways to restore hematopoiesis in patients, that is, for regenerative medicine.

Sources of information: 1. Adams R. Cell culture methods for biochemists/Ler. with English MA. Popova // Edited by V. Yu. Polyakov - M.: Mir, 1983.- 2646. 2. Gupsn 5.5iet sei! todei!v5 az ap ip mygo toavai! Fog rgedisiime iochisoiodu. / Gupsy 5., Rgiddeop b. 5., biskKzhmop S.A., Z5Nagta R., Rak V.K., / Viosnet U. -2019. - 476(7): 1149-4158.

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Rom/pgediiaypd SeiI-Mayih Tgasiyop5. E. Spomzhapigu, M. Gi, M.-S. Ron, T. Tataki, M. Mapa, T.5. Tapaka,

IBA/Oes 2010, RiIo5 OME mmlyu.riozope.oga). 4. RF Patent No. 95663 01. Device for cell cultivation / Zapuskalov I.V. (CI),

Kryvosheyna O.I. (KO), Khlusov I.A. (CI), Martusevich Ya.A. (KO), Kochmala O.B. (KU) //from 29.07.2009 - Official bulletin "Inventions. Useful models" - 2010.- Мо 19. 5. Patent of Ukraine for the invention Мо 2692. Method of manufacturing a chamber for cell cultivation / N. M. Bil'ko (OA); Bilko I. P. (USA); V.V. Hashynskyi (USA); V. G. Voytsekhovskyi (USA)//from 04/15/1994.- Official Bulletin "Industrial Property".- 1994. - Mo 5. b. Mooge M.A.5 Netaiyoroiiviiis 5iet Seiv Riipsirieeee oi Tivvce Epdipeegipud (Bosch Eayop), 2014.

Claims (5)

USEFUL MODEL FORMULA 1. The method of long-term cultivation of hematopoietic stem cells using diffusion chambers, into the cavity of which the cells under study are introduced, which includes the stages of manufacturing sealed chambers by stamping them out of polyacrylamide gel due to its polymerization, introducing cell material into the cavity of the chambers, subsequent cultivation of cells by placing sealed chambers in the appropriate complete nutrient medium ip my or ip mimo, removal of cells in a specified time from the appropriate nutrient medium and examination of cultured cells directly in the cells under an inverted microscope, or removal of cells from the chamber, their fixation on glass slides, staining and study under a light microscope, which differs in that, at the stage of camera manufacturing, a common two-layer gel plate is created for several cameras, formed by superimposing a flat gel plate on a gel plate with holes, and the formed internal cavities are sealed thanks to the accurate polymerization of the gel components, the obtained hermetic two-layer plate is washed from the toxic residues of the gel components in a physiological solution during the day, with a 3-4-fold change of the physiological solution, chambers are stamped from it, which are then stored in a 40 95 solution of ethyl alcohol, and a day before at the beginning of cell cultivation, the chamber is moved from an alcohol solution to a physiological solution of 0.99% by weight, after which a suspension of the studied cells in a nutrient medium is introduced into the inner cavity of the chamber with the help of a syringe through a puncture in its wall and the puncture site is sealed by introducing semi-liquid agar through the same needle under the time of slow removal of the syringe from the puncture site. 2. The method according to claim 1, which is characterized by the fact that gel plates for forming a two-layer plate are created by superimposing two rigid covers, a flat cover and a cover with protrusions, respectively, on two layers of the required thickness of the mixture of gel components. C. The method according to any of claims 1, 2, which differs in that sodium chloride solution or phosphate buffer solution (PB5) in a ratio of 1:10 is used as a physiological solution for washing the obtained gel chambers. 4. The method according to any of claims 1-3, which is characterized by the fact that the body of a laboratory animal is used as a nutrient medium, into which a sealed gel chamber filled with a suspension of the investigated cells is implanted. 5. The method according to any of claims 1-3, which is characterized by the fact that a complete nutrient medium, the composition of which corresponds to the purpose of the experiment, is used as a nutrient medium for ip mio, in which a sealed gel chamber filled with a suspension of the investigated cells is immersed.