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9Cellular Therapy and Transplantation (CTT). Vol. 12, No. 1, 2023 doi: 10.18620/ctt-1866-8836-2023-12-1-60-67 Submitted: 05 October 2022, accepted: 03 March 2023
Application of fluorescent potential-sensitive probes for the study of living blood and marrow cells
Tatyana V. Parkhomenko, Oleg V. Galibin, Vladimir V. Tomson
RM Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantology, Pavlov University, St. Petersburg, Russia
Tatyana V. Parkhomenko, Senior Research Associate, Phone/fax: +7 (812) 499-71-54
Laboratory of Pathomorphology, Research Center, Pavlov E-mail: [email protected]
University, St. Petersburg, Russia
Citation: Parkhomenko TV, Galibin OV, Tomson VV. Application of fluorescent potential-sensitive probes for the study of living cells. Cell Ther Transplant 2023; 12(1): 60-67.
Summary
Usage of membrane potential-sensitive probes is of great importance for assessing both the viability and functional integrity of the cells and their structural components (mitochondria, nuclei, cytoplasmic membranes, ion channels). Potential advantage of this approach includes studies of native viable cells in order to assess functional state of donor hematopoietic cells before transplantation as well as upon their storage and cultivation. These staining tools allow to assess the state of cellular bioenerget-ics, i.e., the balance between production and consumption of energy in living cells. The production of energy in
mitochondrial structures ensures the cell viability, whereas its impairment leads to the development of different disorders and aging. The purpose of this literature review is to demonstrate the opportunities of using potential-sensitive probes to assess the viability of blood system cells and to study the state of their energy potential.
Keywords
Mitochondria, hematopoietic cells, membrane potential, electron transport, ion channels, energy status, cell viability, potential-sensitive probes.
Introduction
Mitochondria (MX) are the main source of energy in different cell populations, including blood cells, thus ensuring their vital activity, homeostasis and differentiation [1,2]. Mi-tochondrial transmembrane potential is one of the important indices of the functional state of mitochondria, which provides effective coupling, between the substrate oxidation and energy accumulation in ATP pool [3, 4]. The transmembrane potential is detectable not only in mitochondrial membranes but also in the membranes of chloroplasts, pho-tosynthetic bacteria and aerobic heterotrophic bacteria, in erythrocyte membranes and the sarcoplasmic reticulum of muscles. Hence, the membrane potential is a universal index of energy conjugation systems [5]. Disturbance of the functional state of MX leads to metabolic disorders in cells [6, 7]. Thus, the maintenance of membrane potential serves as an index of the energy balance in MX and the level of metabolic activity of cells [8, 9].
Electron transport and ion channels
Among different membrane structures in the cell, the outer plasma membranes separating the cell cytoplasm from the outer space, and the membranes of intracellular organelles (nucleus, endoplasmic reticulum - EPR, Golgi apparatus, MX, vesicles, etc.) should be treated separately [10,11]. Transmembrane electron transport in biological systems has been studied for many years [12]. Transfer of electrons through the inner membrane of mitochondria occurs during the formation of the proton-motive force required for the ATP synthesis. Along with studies of the mitochondrial potential in nucleated cells, the mechanisms of transmembrane electron transport through the surface plasma membrane of red blood cells have been actively studied in recent years [13]. The electron transport activity of plasma membranes has been shown for a number of cell types [14]. Currently,
electron transport chains of the plasma membrane are also described which transfer the electrons from cytoplasmic NADH to molecular oxygen or disulfides [15].
Several membrane enzymes are involved in the transport of electrons across the plasma membrane, stimulation of the activity of the electron transport chain in cells is observed under the action of hormones and certain growth factors [16]. This is probably due to participation of the electron transport chain in regulation of cellular growth. Increased activity of this system is also observed in cells with damaged mitochondria. E.g., the processes of oxidative phosphoryla-tion do not occur in the cells of the lymphoblastic Namal-wa rho 0 line, and the needs for energy are here provided by glycolysis chain producing NADH. NAD+ in these cells is formed mainly via electron transport system of the plasma membrane, using NADH in order to reduce molecular oxygen [17].
All biological membranes contain the molecular electric pumps, which function as is the molecular proton and ion channels [18,19]. Membrane-spanning transmembrane proteins form structures that ensure the movement of ions through the membrane, i.e., ion carriers and ion channels. To date, the ion channels are known to present a large family of proteins (>400 peptide species) encoding 1-2% of the human genome. The cell ion channels are complex protein structures with molecular systems of opening, closing, selectivity, inac-tivation, and regulatory functions. Ion channels are of special importance in excitable cells of nervous system [20]. It is known that ion channels provide their main properties, e.g., of excitable cells: formation of the resting membrane potential (MPP), action potential (AP), and others [21].
Using the methods of molecular genetics and electrophysi-ology, it has been shown that many nervous and muscular disorders, including myopathies and periodic paralysis, are caused by dysfunctions of ion channels in the membrane. A study published in 2022 demonstrated the important role of the channel (So Cs) associated with several physiological roles in endothelial dysfunction and vascular smooth muscle proliferation, which contribute to the progression of cardiovascular diseases [22], diseases of the blood system [23]. The authors revealed a disturbed regulation of the main mech-anosensory ion channel Piezo 1 in several blood lineages in the patients with type 2 diabetes mellitus (DM2). The increased activity of Piezo1 in platelets, erythrocytes and neu-trophils in DM2 triggered discrete antithrombotic cellular reactions. Inhibition of Piezo1 protected against thrombosis. Based on the results of this work, the authors propose a potential screening method for predicting patient-specific risk (thrombosis). A large number of scientific papers have been devoted to the study of the properties of the Piezo1 channel.
When the functioning of the ion channel is disturbed, not only dysfunction of excitable cells is observed, but also a violation of ion transport in non-excitable tissues [24, 25].
Investigation of ion channel functions
Various methods are used to study ion channels: electrophys-iological, biochemical, pharmacological, etc. When using
electrophysiological methods, the potentials and currents of ions are recorded using metal electrodes or glass micropipettes, mostly, in neural cells. This method makes it possible to check the function of ion channels in an indirect way, by changing the shape of the action potential (AP) [26]. It is not always possible to use the microelectrode registration method, since serious damage to the cell and its death may occur.
Changes in the magnetic field and, consequently, the activity of ion channels can be recorded using voltage-sensitive dyes which exhibit shifts in their spectral characteristics with changing levels of electric potentials. The most widely studied fluorescent probes dyes are as follows: di-4 ANEPPS [27], di-8-ANEPPS [28], created on the basis of amino naphthyl ethenyl pyridinium (ANEP), RH237 [29], di-8-ANEPPS, [30].
According to the current concept, the functional state of living cells is closely related to the number of active mitochondria in the cytoplasm, the total transmembrane potential on plasmatic and mitochondrial membranes whose changes can be monitored using voltage-sensitive fluorescent probes [31, 32]. However, when assessing mitochondrial energy metabolism in isolated MXs the isolation procedures can lead to irreversible clumping and damage to MX [33]. When working with viable cells, despite the fact that the MX inside the cells remain intact, the cell membranes may be impermeable to a number of substances necessary for evaluation of mito-chondrial functions, Membrane potential of MX can be assessed using membrane-penetrating cationic fluorescent or phosphorescent dyes, which are distributed in MX according to the Nernst equation, thus enabling detection of MX membrane depolarization [34]. The following fluorescent probes are used: safranin O [35], rhodamine [36], cyanines [37], DiSC2 (3) [38].
All the potential-sensitive dyes mentioned above are toxic and themselves affect the energy state of living cells, which limits their use as vital dyes.
Usage of fluorescent probes that interact non-covalently with membrane systems makes it possible to study the state of biological membranes in any living cells, including mitochondria [39].
Choosing fluorescent probes to assess the energy status of cells
The choice of a voltage-sensitive fluorescent probe for evaluating the energy status of cells is very important. The probe must be a cationic molecule able to penetrate into living cells. It should accumulate in the energized structures, showing fluorescent signal when binding them, and having no pronounced toxic effects. If possible, such probe should be also polychromatic, i.e., create fluorescence of different colors for distinct cell components. An example of such a probe is 2-[p-(dimethiamino)styryl]-1-methylpyridinium iodide (2-Di-1-ASP, or DASPMI). For the first time, this probe was used by Bereiter-Hahn (1976) in order to study the state of mitochondria in a culture of pigeon heart cells [39]. It was later proposed as a probe for assessing mitochondrial potential in other cellular models.
Further, it was found that the absorption and intensity of DASPMI fluorescence in mitochondria is a dynamic indicator of the membrane potential and the influence of the local electric field on the transient dipole moment of the probe provide information about changes in the energy status of mitochondria in living cells.
It was also found that the absorption and intensity of DASPMI fluorescence in mitochondria is a dynamic indicator of the membrane potential. The influence of local electric field upon the transient dipole moment of the probe was shown to provide information about changing energy status of mitochondria in living cells [40 ]. In 1981, DASPMI was proposed as a probe for evaluating mitochondrial potential [41]. When observing changes in HeLa cells treated by apopto-sis inducers, three fluorescent dyes (2-[4-(dimethylamino)) were tested styryl ]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide). The obtained results reveal the advantage of cationic dyes for studying the membrane transport [42].
In 1981, a related 2-Di-1-ASP compound, 4-(p-dimethyl-aminostyryl) was synthesized at the Institute of Organic Synthesis, Latvian Academy of Sciences, Riga (1-methylpyrid-inium, DSM), being also tested as a fluorescent probe [43]. With DSM, the values of free energy accumulation in the MX of lymphocytes and the levels of transmembrane and mitochondrial potentials were determined in intact lymphocytes without preliminary isolation of MX from the cells, and a technique was described for determining the ranges magnitude of transmembrane and mitochondrial potentials in intact lymphocytes using the Nernst equation [44]. A method for assessing the energy state of lymphocytes and neutrophils using a DSM probe was developed with donor blood samples [45]. The special importance of these studies is that they clearly demonstrate the opportunity of studying living blood cells and their MX in native environment [46].
Compounds of this family have also been investigated for binding to double-stranded DNA structures [47]. Recent studies show that a decrease in the membrane potential of mitochondria of T-lymphocytes leads to their death and, possibly, to the disturbances of antitumor immunity with an increased risk of tumor development [48].
Effects of different chemical factors on various cell types
Usage of fluorescent probes makes it possible to study primary reactions of intact cells (lymphocytes and neutrophils) and after exposure to various chemical compounds and drugs [49, 50, 51]. Fluorescent rhodamine derivatives allow us to assess the state of mitochondrial functions and redox potential of cells [52]. Experimental studies using fluorescent probes have provided information on the functional activity of various cells, including erythrocytes [53], myo-cardiocytes [54], cells of the immune system [55], smooth muscle cells [56].
Effect of erythropoietin on T-lymphocytes assessed with DSM probe
The DSM probe (4-(p-dimethylaminostyryl)-1-meth-ylpyridinium) was tested with T-lymphocytes isolated from the thymus of white rats and placed in a standard Hanks' solution. The suspensions (2-3x106 cells/ml) in Eppendorf test tubes were incubated with DSM probe (final concentration of 0.15 and 0.5 ^m) for 30 minutes at 37°C. The fluorescence was excited at the wavelength of 405-436 nm. To register fluorescence, a photometric nozzle FMEL-1 and an interference filter with transmission of 585 nm were used. The fluorescence intensity of each individual cell was recorded, with photometry field being equal to the cell diameter. The number of luminous mitochondria per cell (n m/c) was counted, being recognized by granular yellow fluorescence of the DSM probe. The fluorescence of 50-70 cells was measured per sample, and the fluorescence intensity per cell (F arb.units) was calculated. The image capture and analysis of DSM-stained cells were performed with a Lumam-P8 microscope, TCA-5.0 camera; and Micro-Analysis View software package (LOMO-Microsystems LLC, Russia). The excitation and absorption maximum were 485 nm, and 450 nm, respectively, with fluorescence maximum of 590nm [50].
Using the DSM fluorescent probe, we studied in vitro effects of erythropoietin (EPO) on some energy-coupled characteristics of lymphoid cells from the rat thymus, directly related to the level of their functional activity, i.e., number of active mitochondria in the cytoplasm, total transmembrane potential of the plasma and mitochondrial membranes. In control samples, the levels of fluorescence from the cells showed direct correlation with the number of energized mitochondria. Activation of T-lymphocytic cells (TLC) was observed at EPO concentrations of 2-20 u/ml, whereas reduction of their activity was shown at 20-200 u/ml EPO, thus reflecting a phasic modulatory effect of EPO on the energy activity of these cells. The study of lymphoid cells from 27 rat thymuses with mitochondrial fluorescence probe made it possible to divide the entire population of thymocytes into 3 groups that differently react to EPO, which may be due to the varying proportion of mature (differentiated) cells in these samples, which, would reflect different physiological state of the thy-muses [50]. The first group included cells with initially low potential and limited response to EPO. The second group included cells with initially high potential which, however, lose their potential when exposed to EPO. The maximal activating effect of EPO was achieved at the average initial level of thymic cell fluorescence (AF). Apparently, the effect of EPO on thymocytes depends on the state of the outer cell surface, and initial activity of cell membrane erythropoietin receptors. It was found that EPO has an activating effect on thymocytes, accompanied by an increased number of cells with energized MX, and higher contents of luminescent MX in the cells (n m/c) having a proton potential Aq>m).
However, the positive effect of EPO can be caused not only by an increase in A^m, but also by an increase in the external membrane potential (A^p). To determine, which type
of potential activates EPO in TLC, Aq>m or (and) A^p, the reaction of TLC to EPO after exposure to specific inhibitors of phosphorylation reactions in the respiratory chain was studied.
Dinitrophenol (DNF), a known inhibitor of oxidative phos-phorylation, had a maximal effect on TLC. EPO did not restore energy balance after exposure to DNF. Meanwhile, the maximal restoring effect of EPO on thymocytes was observed after exposure to an inhibitor of the membrane-bound part of ATP-ase - dicyclohexylcarbodiimide (DCCD). EPO partially restored the polarization of the MX membranes after exposure to DCCD, while up to 42% of the luminescent MX and 38% of the total luminescence from the controls are detectable. These data may indicate that EPO affects the metabolism of thymocytes associated with the energy activity of mitochondria [57].
We have also investigated the effect of EPO in vitro in rat native blood preparations suspensions on transmembrane potentials of granulocytes, which are a sufficient component of innate immunity. In blood samples stained with a DSM probe, the intensity of probe-specific fluorescence was measured before and after incubation with EPO. These data suggest that EPO, activating the physiological activity of granulocytes, may indirectly affect the cells of the immune response [58].
Examination of bone marrow cells with 2-Di-1-ASP probe
In 2013, we developed a method using a 2-Di-1-ASP supravital fluorescent probe to assess the condition of bone marrow cells (BMC). We have found optimal working conditions with the probe, i.e., incubation terms and temperature, probe concentration. It has been experimentally confirmed that the tests with 2-Di-1-ASP are register changes in the energy potential of BMC when exposed to various factors: cryopreser-vation with dimethyl sulfoxide, thawing procedure, suspension in saline solution, Hanks medium, standard stabilizer medium, storage duration [59].
The state of BMC transplants was assessed by their energy activity, in order to determine the quality of the graft and its safety. The study was performed with bone marrow of 20 healthy donors taken for allogeneic transplantation. The preparation of BMC suspensions was carried out according to the standard procedure [60]. A total of 9130 cells were examined in 500 preparations. It was found that, in all BMC samples, after 3-3.5 hours of storage at room temperature, an increased intensity of probe-specific fluorescence (F) was recorded [61].
Over last years, several methods were proposed for BMC viability detection. E.g., in a German publication [62], the viability issue of BMC transplants and stem cells from peripheral blood (PSCs) under various temperature conditions was discussed. The cell viability was assessed by the activity of the ADH (aldehyde dehydrogenase) marker enzyme. According to this study, the number of cells able to divide in these transplants remained unchanged for 72 hours at room temperature, thus being consistent with our data.
In the studies from last decade, some data on the energy activation of cells at the initial stage of mitosis were presented for the suspension cell cultures of T-lymphoblastoid lines MT 4 and SEM. The associated effects included enhanced 2-Di-1-ASP fluorescence of BMC (after 3-3.5 hours) and DSM probe on ferret brain lymphoblastoid cells (after 6-12 hours) they may indicate similar processes occurring in the nuclei of these cells, namely, their metabolic activation and proliferation [63].
Manual and automated techniques for mitochondrial staining
DSM probe (4-(p-dimethylaminostyryl)-1-methylpyridini-um) was applied for studies of lymphocytes and neutrophils in whole native blood using microfluorometric method. As simplest approach, an aqueous solution of DSM was added to heparinized donor blood. Non-toxic working concentrations of DSM were determined (0.5-20.0 ^mol/L). Optimal staining conditions were as follows: for neutrophils, 1 ^mol/L of DSM; for lymphocytes, 10 ^mol/L of probe followed by incubation for 30-40 min at 37°C. To study the blood samples, a solution of DSM was used at the concentration range of 0.2 to 20.0 ^m/L. The incubated sample was placed on a glass slide, covered with a cover glass and subjected to UV microscopy (Lumam-P8, St. Petersburg, Russia). The native properties of lymphocyte and neutrophil membranes and the high energy status of MX in them are preserved under adequate blood plasma condition, at a weak background dye fluorescence [64].
Efficiency of fluorescent 2-Di-1-ASP probe (iodide 2-[p-(di-methylamino)styryl]-1-ethylpyridinium) was tested with bone marrow cell samples (BMC) being stored in standard stabilizing solution at 2--3x106 cells/mL at room temperature (20-22°C). The cell aliquots (30 ^l) were supplied with 2-Di-1-ASP to the concentration of 40 ^mol, and incubated at 37°C for 60 min. Fluorescence was measured with a Lum-am-P8 luminescence (St. Petersburg, Russia) at a 900x magnification. A photometric device was used with interference filter at maximal wavelength of 585 nm, with excitation and emission values at, respectively, 470 and 560 nm. Fluorescence intensity signals were recorded from single cells. Seventy to hundred cells per sample were tested manually, and the mean fluorescence intensity of single cells (F, arbitrary units) was calculated for each specimen. Photographic pictures of luminescent objects were performed with TCA-5.0 camera, using "Micro-Analysis View" software ("LOMO LLC Microsystems", Russia) [61].
RH237 probe was used in experiments with murine pluripo-tent stem cells (iPSCs) induced for differentiation transplanted into the mice with experimental myocardial infarction. In preliminary tests, electrophysiological activity of clustered iPS-derived beating cells was recorded by an optical mapping system using an RH237 voltage-sensitive dye [29]. The cell clusters from 3-4 week-cultures were incubated with the voltage-sensitive dye RH237 (1 ^mol/l) for 5 min. The optical signal was excited with custom-made LED and recorded with a high frequency CCD camera (710 frames/s). The data was processed with an algorithm on the Matlab platform,
and phase maps were constructed. These results were compared to the records of whole-cell action potentials using single-cell technique with micropipette-assisted current-clamp mode of Axon multiclamp 700 A amplifier and patchclamp 10.0 software from Molecular Devices, CA, USA.
Rodamine-123 (Rh-123), another well-known dye was used by Indian workers in order to determine toxic effects of di-chlorophene on the rat blood leukocytes [36]. The leukocytes isolated from blood were tested with propidium iodide stain to assess cell cycle distribution. The based assessment of mitochondrial membrane potential (Jm) was made in order to evaluate cellular energy state by flow cytometry of Jm in single cells as a result of mitochondrial changes after 60-min incubation of cells with rodamine-123 dye (Rh-123) using FITC channel on FACS ARIA II flow cytometer (Bec-ton Dickinson, USA). Leukocytes were identified with the characteristics of forward scatter and side scatter.
Conclusion
Laboratory and clinical studies with potential-sensitive probes capable of penetrating into the cells, have shown that it is possible to obtain objective information about the functional state of the cell, its energy status without disturbing the integrity of the cell. According to a number of studies, currently such information can be currently obtained using fluorescent probes. This may be especially relevant when examining functional state of donor cells during its storage and cultivation before transplantation. It is necessary to develop specialized techniques and standards of flow fluorimetry for quantitative assessments of the membrane potential in the cellular populations of the blood system.
Conflict of interest
The authors have no conflicts of interests to declare.
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Применение флуоресцентных потенциал-чувствительных зондов для изучения живых клеток крови и костного мозга
Татьяна В. Пархоменко, Олег В. Галибин, Владимир В. Томсон
НИИ детской онкологии, гематологии и трансплантологии им. Р. М. Горбачевой, Первый Санкт-Петербургский государственный медицинский университет им. акад. И. П. Павлова, Санкт-Петербург, Россия
Резюме
Применение синтетических зондов, чувствительных к мембранному потенциалу, имеет большое значение для оценки жизнеспособности и функциональной целостности клеток и их структурных компонентов (митохондрий, ядер, цитоплазматических мембран, ионных каналов). Потенциальное преимущество такого подхода состоит в исследовании нативных жизнеспособных клеток для оценки функционального состояния донорских гемопоэтических клеток до трансплантации, а также в ходе их хранения и при культивировании. Эти красители позволяют оценивать состояние клеточной биоэнергетики, т.е. - баланс между продукцией и потреблением энергии в живых клетках. Продукция энергии в структурах митохондрий обеспечивает жизнеспособность клеток, а ее нарушения ведут к развитию
различных заболеваний и старению. Целью данного обзора литературы является демонстрация возможностей применения потенциал-чувствительных зондов для определения жизнеспособности клеток системы крови и изучения состояния их энергетического потенциала.
Ключевые слова
Митохондрии, гемопоэтические клетки, мембранный потенциал, транспорт электронов, ионные каналы, энергетический статус, жизнеспособность клеток, потенциал-чувствительные зонды.
