RU2751603C2 - Method for production of labeled cell culture - Google Patents

Abstract

FIELD: biology.SUBSTANCE: invention relates to the field of cellular biology and biotechnology. Proposed is a method of producing a labeled cell culture, wherein said method includes processing a suspension of magnetite nanopowder with an arithmetic mean size of particles between 80 and 110 nm in the liquid phase with ultrasound with a frequency and duration sufficient to disperse large aggregates, followed by at least 10 minutes of incubation; a cell culture with a confluence of no more than 80% to a concentration of 50 to 600 μg/ml is then added and incubated for 2 to 14 hours at 37°C.EFFECT: invention provides increased viability and proliferative activity of labeled cells used in MRI diagnostics.5 cl, 6 dwg, 5 ex

Description

The invention relates to the field of cell biology and biotechnology, namely to cell technologies, in particular to methods of obtaining used in diagnostics, regenerative therapy of human cells containing components that allow them to be tracked in the human body and large animals.

The use of cellular technologies in the treatment of a wide range of diseases makes it possible to comprehensive and targeted treatment of serious disorders of various origins. However, there is a need to develop methods for in vivo labeling of cells in vitro to track cell migration and adhesion after their introduction into the human body. The previously developed methods of in vivo labeling of cells with phosphors and fluophores are suitable only for small organisms or for post mortem studies [Progatzky F., Dallman MJ, Lo Celso C. From seeing to believing: labeling strategies for in vivo cell-tracking experiments. Interface Focus, 2013, 3: 20130001]. Methods of radioactive labeling of cells pose a threat to the patient and affect the physiology of the transplanted cells, reducing the proliferation rate [Detante O., Moisan A., Dimastromatteo J., et al. Intravenous administration of 99mTc-HMPAO-labeled human mesenchymal stem cells after stroke: in vivo imaging and biodistribution // Cell Transplant. 2009; 18 (12): 1369-1379] and differentiation [Brenner W., Aicher A., Eckey T., et al. ¹¹¹ In-labeled CD34 + hematopoietic progenitor cells in a rat myocardial infarction model. J Nucl Med. 2004; 45: 512-518].

The use of the method of magnetic resonance imaging (MRI) for visualization of injected cells is considered promising. The use of MRI requires the development of a method for intravital cell labeling, in which cells capture particles of a contrast agent, retain them and can be visualized by MRI, while maintaining their viability, proliferative status and functional properties. However, many contrasting materials developed do not meet safety requirements when passing preclinical tests. So, contrasting preparations based on gadolinium were toxic to cells and patients [Hao D., Ai T., Goerner F., et al. MRI contrast agents: Basic chemistry and safety // J. Magn. Reson. Imaging, 2012, 36: 1060-1071]. The most popular and promising are preparations based on superparamagnetic iron oxide particles coated with various organic shells to protect particles from cells and cells from iron oxide particles. However, some of these drugs cause unwanted side effects, for example, most contrast agents based on dextran coated iron oxide particles, including the most famous of them - Ferridex, have been discontinued due to the high frequency of unwanted side effects [https: / /www.mr-tip.com/serv1.php?type=db1&gid=767, date of access: 15.11.2019; https://www.drugs.com/pro/feridex.html, date accessed: 15.11.2019].

The development of a method for in vivo labeling of cells, in which cells capture particles of a contrast agent, retain them and can be visualized by MRI, while maintaining their viability, proliferative status and functional properties, seems relevant.

In most cases, nanoparticles up to 100 nm in size or superparamagnetic particles (SPION, diameter up to 20 nm) of iron oxide (mixture of oxides, mixture formula Fe ₃ O ₄ ) coated with any organic substances (dextran, polyethyleneimine, etc.) are used as a contrast agent. etc.) to improve cell penetration and reduce cytotoxicity, etc. [Rakesh M. P., Nanasaheb DT, Prajkta BS, et al. Comprehensive cytotoxicity studies of superparamagnetic iron oxide nanoparticles // Biochemistry and Biophysics Reports, 2018, 13: 63-72]. However, already at a concentration of up to 80 μg / ml of such particles, the cytotoxicity of drugs begins to manifest [Rezaei M., Mafakheri N., Khoshgard K., et al. The Cytotoxicity of Dextran-coated Iron Oxide Nanoparticles on Hela and MCF-7 Cancerous Cell Lines // IJT. 2017; 11 (5): 31-36]. At this concentration, cytotoxicity is negligible: 10-15%. According to our own data, for successful visualization of SPION-labeled cells on clinical MRI tomographs, a particle concentration of more than 100 μg / ml is required, optimally 150-300 μg / ml. However, this concentration of magnetic particles can be expected to be highly toxic to cells.

The cell labeling preparations known in the art typically use SPIONs prepared by chemical reactions. Zhu H.M., Wang Y.X., Leung K.C., et al. Enhanced cellular uptake of aminosilane-coated superparamagnetic iron oxide nanoparticles in mammalian cell lines // Int J Nanomedicine. 2012; 7: 953-964 describe methods for the synthesis of nanoparticles in which an iron oxide core is synthesized by alkaline coprecipitation of two parts of ferric chloride and one part of ferrous sulfate in NaOH solution under nitrogen atmosphere with stirring. The precipitate is separated with a magnet and washed with deoxygenated water. SPIO @ SiO2 and SPIO @ SiO2-NH2 particles are obtained by hydrolysis of tetraethoxysilane or (3-aminopropyl) triethoxysilane on the surface of SPIO nanoparticles. To obtain SPIO @ dextran nanoparticles, dextran with a molecular weight of 70 kDa is preliminarily dissolved in a NaOH solution and boiled under reflux for 2 hours, then this solution is added dropwise to the ultrasonically dispersed SPIO nanoparticles in water with pH 3. The mixture is stirred and boiled with reflux for 30 minutes. The products are separated with a magnet and washed with deoxygenated water.

Such methods of obtaining superparamagnetic nanoparticles are laborious and expensive; in addition, the preparations obtained by these methods contain residual traces of the cytotoxic substances used.

Superparamagnetic nanoparticles of magnetite, or iron oxide Fe ₃ O ₄ , obtained by the method of electric explosion of a conductor in an air atmosphere, do not contain by-products of chemical reactions, are readily available and cheaper than their coated counterparts. Such particles have been used as a contrast agent, presumably meeting the safety requirements for cells. In work: Levchuk K.A., Shumeev A.N., Zolina T.L., Kotova A.V., Aleksandrova L.V., Bagaeva V.V., Aizenshtadt A.A., Kotkas I.E. , Maslennikova I.I., Enukashvili N.I. The method of intravital labeling of human mesenchymal stromal cells with superparamagnetic particles of magnetite / Translational medicine: from theory to practice: Proceedings of the 5th scientific-practical conference of young scientists and specialists // Ed. d.m.s. A.V. Silina. - SPb .: Publishing house of the North-Western State Medical University named after I.I. Mechnikov, 2017 .-- p. 54-55 describe a method for obtaining a culture of labeled mesenchymal stromal cells (MMSC) from the umbilical cord and human adipose tissue. The method consists in incubating MMSC in a culture medium containing 200 ng / ml magnetite nanopowder with a particle size of 5-20 nm for 24 hours. The suspension of nanoparticles is preliminarily treated with ultrasound at a frequency of 37 kHz in order to break up the aggregates of nanoparticles. After 24-hour incubation with magnetite particles, MMSC cell cultures retained viability at the control level, that is, 95% of the total number of cells, and contained captured nanoparticles. However, according to our own data, this method does not allow obtaining a consistently reproducible result. In addition, it requires too long incubation time of the cell culture with magnetite nanoparticles. Particles 5-20 nm in size require coating with organic components (dextran, etc.) to prevent aggregation, which is laborious and expensive. At the same time, the actual size of the particles increases, which means that their number per cell decreases, which leads to a deterioration in the degree of visualization during scanning. In addition, magnetite nanopowder with such a particle size is not produced in Russia, so their use is inconvenient, impractical and depends on expensive and long-term supplied raw materials.

The objective of the present invention is to provide a method for obtaining a culture of labeled cells that provides stable and reproducible labeling of cells while maintaining their viability and proliferative status, with the ability to visualize cells by MRI.

The problem is solved by the fact that in the method of obtaining a culture of labeled cells, magnetite nanopowder with an arithmetic mean particle size of 80-110 nm is combined with a liquid phase, the resulting suspension is treated with ultrasound with a frequency and duration sufficient to disperse large aggregates. The resulting suspension of nanoparticles is kept for at least 10 minutes to obtain microaggregates with a size of no more than 5 microns, in the optimal version, the average size of macroaggregates is about 1 micron. The resulting suspension of microaggregates is introduced into a cell culture grown to no more than 80% confluence, to a concentration of 50-600 μg / ml, and then the cell culture is maintained in a culture medium with the mentioned microaggregates for 2-14 hours at a temperature in the range of 37 ° C ...

Unlike the prototype, the inventive method uses an affordable and relatively inexpensive magnetite powder with an arithmetic mean particle size of 80-110 nm, while the concentration of particles in the suspension is 250-3000 times higher, which provides the number of particles required for effective labeling of all cells in culture ... In addition, keeping the cell culture in the presence of magnetite microaggregates is carried out for a shorter time, which ensures better preservation and proliferative properties of cells. Numerous repetitions of the method showed that it is reproducible, the cells are viable, capable of proliferation, and they can also be visualized using MRI [Kotkas I.E., Enukashvili N.I., Asadulaev Sh.M., et al. Visualization of the distribution of autologous mesenchymal cells introduced into the body of a patient suffering from cirrhosis of the liver of alimentary etiology // Bulletin of the North-Western State Medical University named after I. I.I. Mechnikova, 2017, 9 (4): 76-78; Kotkas I.E., Asadulaev Sh.M., Marchenko N.V., Enukashvili N.I. Evaluation of changes in blood biochemical parameters in a patient with a diagnosis of liver cirrhosis against the background of the use of autologous mesenchymal stem cells. Experimental and Clinical Gastroenterology 2018; 150 (2): 174-176; Kotkas I.E., Asadulaev Sh.M., Enukashvili N.I., et al. The possibility of visualizing the distribution in the human body of autologous mesenchymal cells introduced into the arterial bed of the liver // Medical imaging, 2017, 6: 72-75.] ... A suspension of magnetite microaggregates is added by any available method, both in the culture liquid and in a balanced salt solution.

In one embodiment of the method, magnetite nanopowder obtained by the method of electric explosion of conductors is used. This method makes it possible to obtain a polydisperse powder of particles without impurities of by-products, which occurs during the synthesis of nanoparticles by the method of chemical reactions.

In one embodiment of the method, a culture of mesenchymal stem cells is used as a cell culture, in another embodiment of the method, a culture of fibroblasts is used. For labeling, a culture of any cells suitable for introduction into a human or animal organism is used, for example, a culture of mesenchymal stem cells or a culture of fibroblasts. The choice of specific cells depends on the type of treatment and is limited only by medical indications.

In one embodiment of the method, after keeping the magnetite microaggregates in suspension, the cells are washed and sieved in a ratio not lower than the permissible concentration limit. This provides an increase in the number of labeled cells and additionally removes microaggregates of magnetite, which did not penetrate into the cells, but were attached to them from the outside.

The problem is also solved by the fact that the implementation of the claimed method produces a culture of labeled cells for introduction into the human body.

The invention is illustrated by the following drawings.

FIG. 1 shows the curves of particle size distribution immediately after sonication (line 1), 3 and 4 hours after turning off the stirrer (lines 2 and 3). Particle size was determined by laser diffraction.

FIG. 2. presents the data of the analysis of the immunophenotype and viability of cells containing microaggregates of iron oxide. Four histograms show examples of flow cytometry of labeled cells for positive (CD73, CD90) and negative (CD34, CD45) markers. The average immunophenotype is also shown. Viability was determined by the 7-AAD staining method. The percentage of unstained cells (99.49) corresponds to the percentage of living cells.

FIG. 3. Appearance of the culture of cells containing microaggregates of iron oxide after cryopreservation (0 h) and subsequent cultivation for 2 days (48 h).

FIG. 4. shows an electron micrograph of the fine ultrastructure of cells containing microaggregates of iron oxide.

FIG. 5. presented MRI of the abdominal organs (from: Kotkas I.E., Enukashvili N.I., Asadulaev Sh.M., et al. Visualization of the distribution of autologous mesenchymal cells injected into the body of a patient suffering from liver cirrhosis of alimentary etiology // Bulletin Mechnikov North-West State Medical University, 2017, 9 (4): 76-78).

FIG. 6 shows the type of MSCs containing microaggregates of iron oxide on the day of transplantation (A) and cells isolated 1 year later from a liver biopsy in the area of transplantation (B).

The method was carried out as follows.

Example 1. Obtaining a culture of labeled mesenchymal stem cells.

In a penicillin vial containing 30 mg of magnetite nanopowder with a particle size of 80-110 nm (OOO "Advanced Powder Technologies", Tomsk), 10 ml of sterile saline was added, mixed and transferred into 15 ml test tubes. Then the tubes were sonicated at a frequency of 37 kHz for 15 minutes, shaking the tube every 3 minutes. The resulting suspension of particles with a particle concentration of 3 mg / ml is stable during the day. Measurement of the particle size (Fig. 1) showed that the average particle size after ultrasonic treatment is 80-100 nm, however, microaggregates 0.5-1.5 μm (80% 0.9-1.1 μm) are formed within 10 minutes and then the size of the micro-aggregates does not change for 12 hours. Before use, 1 ml of the resulting suspension of microaggregates was transferred into 4 ml or 9 ml of saline or culture medium, thus obtaining a finished preparation of microaggregates with a concentration of 600 μg / ml or 300 μg / ml, respectively. A suspension of microaggregates with a concentration of 300 μg / ml, if necessary, was used to obtain suspensions with a lower concentration of microaggregates.

The primary material of a biopsy of the liver or adipose tissue was obtained by surgery in a volume of 100 μl. Liver tissues were kept in a 0.1% solution of type I and IV collagenase for 20 minutes in a thermal shaker at 37 ° C with constant stirring. The resulting mixture was centrifuged, the supernatant was removed, the pellet was resuspended in 4 ml of culture medium, for example, Advanced Stem Cell Media (HyClone, Germany) supplemented with 20% fetal bovine serum for stem cells (SC) or Advanced Supplement for Stem Cells ( HyClone, Germany) and 10 μg / ml penicillin-streptomycin solution (HyClone, Germany). The cell suspension was placed in a culture flask with an area of 25 cm ² . The vials were placed in a CO ₂ incubator, for example, BBD 6220 (Thermo, Germany) at a temperature of 37 ° C, the exposure time is 3-5 days, depending on the state of the cells. Then the SCs were cultivated by the known method of cultivating mesenchymal SCs.

Upon reaching 80% confluence, the medium was changed by adding a suspension of microaggregates with a concentration of 3, 30, 50, 100, 200, 300, or 600 μg / ml. For this, the old medium was removed and 27 ml of the prepared new medium and 3 ml of the prepared prepared solution with microaggregates were added. Before adding a suspension of microaggregates, it must be well resuspended. Then the cells were incubated in a medium with microaggregates for 2 to 14 hours, after which the medium was removed and replaced with a new one without microaggregates. The cells included microaggregates over the entire range of microaggregates concentration and incubation time. Further experiments to identify labeled cells on MRI showed that labeled cells incubated in a suspension of microaggregates with a concentration of at least 50 μg / ml are available for visualization.

The immunophenotype and viability of cells containing microaggregates of iron oxide were determined by flow cytometry of labeled cells using positive (CD73, CD90) and negative (CD34, CD45) markers. FIG. 2 shows the results of the analysis, and also shows the average immunophenotype. Cell viability was determined by the 7-AAD staining method. The percentage of unstained cells (99.49) corresponds to the percentage of living cells (Fig. 2).

Example 2. Obtaining a culture of labeled fibroblasts.

Primary skin biopsy material with an area of 0.8-1 cm ² was obtained by surgery. The remains of adipose tissue and epithelium were removed. The dermal layer was dissected and incubated in a 0.1% solution of collagenase I and IV types for 45 minutes in a thermal shaker at 37 ° C with constant stirring. The resulting mixture was centrifuged, the supernatant was removed, the precipitate was resuspended in a 0.1% solution of collagenase I and IV types and trypsin and incubated for another 30 min. The dissociated biopsy sample was washed from enzymes and resuspended in 4 ml of culture medium, for example, Advanced Stem Cell Media (HyClone, Germany) with the addition of 20% fetal bovine serum for SC or Serum Replacement Advanced Supplement for Stem Cells (HyClone, Germany) and 10 μg / ml penicillin-streptomycin solution (HyClone, Germany). The cell suspension was placed in a culture flask with an area of 25 cm ² .

When 80% confluence was reached, the cells were incubated with a suspension of microaggregates as described in Example 1.

Example 3. Cell viability after cryopreservation

MSC cells were grown and labeled as described in Example 1. Upon reaching 80% confluence, the cells were removed from the support with trypsin: Versene solution, washed from this solution by centrifugation in culture medium and resuspended in cryo-media containing 50% fetal bovine serum, 8% dimethyl sulfoxide and 42% low glucose DMEM. Cells were frozen using a CryoMed programmable cell freezer (Thermofisher Sci). Frozen cells were stored in liquid nitrogen for 2-5 months. The cells were thawed by immersing the tubes in a water bath at 37 ° C, after which the cells were washed from the cryo medium and plated under standard cultivation conditions described in Example 1. The presence of iron oxide nanoparticles in the cells, the viability of the cells, and the possibility of their passaging (reseeding) were assessed. FIG. 3 shows the data confirming the presence of nanoparticles in cells after cryopreservation, as well as the ability of labeled cells to grow in culture after thawing.

Example 4. Study of the fine ultrastructure of cells containing microaggregates by electron microscopy.

To assess the fine ultrastructure of cells, preparations for electron microscopy were prepared using the standard method after fixation with glutaraldehyde. Electron micrographs show that magnetite microaggregates are surrounded by a membrane that separates them from the rest of the cell. (Fig. 4).

Example 5. Identification of labeled cells after transplantation into the human body.

Patient K., 59 years old, diagnosed with cirrhosis of the liver of alimentary etiology, was sampled of adipose tissue on the inner surface of the thigh, followed by isolation of mesenchymal cells from it. Subsequently, 4 culturing passages of the isolated mesenchymal cells were performed. The cells obtained as a result of culturing were labeled with microparticles of iron oxide, as described in example 1. Subsequently, selective angiography was performed at the level of the own hepatic artery, followed by the introduction of labeled autologous mesenchymal cells in the amount of 25 million. One day after the cells were injected into his own hepatic artery, the patient underwent magnetic resonance therapy of the abdominal organs. Based on the results of this study, it was possible to visually assess the distribution of the introduced cellular structures in the liver tissue. All the cellular structures introduced into the own hepatic artery were fixed in this patient mainly in the left lobe of the liver, in the region of II and III segments (Fig. 5, from: Kotkas I.E., Enukashvili N.I., Asadulaev Sh.M., and etc. Visualization of the distribution of autologous mesenchymal cells injected into the body of a patient suffering from liver cirrhosis of alimentary etiology // Bulletin of the Mechnikov North-Western State Medical University, 2017, 9 (4): 76-78). After 1 year, when carrying out a standard diagnostic biopsy for medical indications, 1/5 of the biopsy was inoculated into the culture medium as described in example 1. In cultured cells, the presence of iron nanoparticles was assessed both by morphological analysis of cultures and by staining with Prussian blue. Microscopic analysis of cultures showed the presence of microaggregates of iron oxide both immediately before transplantation (Fig. 6A) and one year after transplantation (Fig. 6B).

The applicant asks to consider the submitted materials of the application "Method for obtaining a culture of labeled cells" for the issue of a RF patent for an invention.

Claims (5)

1. A method of obtaining a culture of labeled cells, which consists in the fact that magnetite nanopowder with an arithmetic average particle size of 80-110 nm is combined with a liquid phase, the resulting suspension is treated with ultrasound with a frequency and duration sufficient to disperse large aggregates, and not earlier than after 10 minutes after that, add to a concentration of 50-600 μg / ml into a cell culture preliminarily grown to no more than 80% confluence, incubate for 2-14 hours at a temperature of 37 ° C, while any liquid can be used as a liquid phase, compatible with the maintenance and / or culturing of cells. 2. The method according to claim 1, wherein magnetite nanopowder obtained by the method of electric explosion of conductors is used. 3. The method according to claim 1 or 2, wherein a culture of mesenchymal stem cells is used as the cell culture. 4. A method according to claim 1 or 2, wherein a fibroblast culture is used as the cell culture. 5. The method according to any one of claims. 1-4, in which, after keeping in a suspension of magnetite, the cells are washed and sieved in a ratio not lower than the permissible concentration limit.

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