The Effect of Rhodamine-Derived Superparamagnetic Maghemite Nanoparticles on the Motility of Human Mesenchymal Stem Cells and Mouse Embryonic Fibroblast Cells

molecules Article The Effect of Rhodamine-Derived Superparamagnetic Maghemite Nanoparticles on the Motility of Human Mesenchymal Stem Cells and Mouse Embryonic Fibroblast Cells Larisa Baiazitova 1, Josef Skopalik 1, Jiri Chmelik 1, Inna Zumberg 1, Vratislav Cmiel 1, Katerina Polakova 2and Ivo Provaznik 1,* 1Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, Technicka 3082/12, 61600 Brno, Czech Republic; [email protected].cz (L.B.); [email protected].cz (J.S.); [email protected].cz (J.C.); [email protected].cz (I.Z.); [email protected].cz (V.C.) 2Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacky University, 17 listopadu 12, 771 46 Olomouc, Czech Republic; dr[email protected] *Correspondence: [email protected].cz; Tel.: +420-54114-6666 Received: 20 February 2019; Accepted: 23 March 2019; Published: 27 March 2019   Abstract: Nanoparticles have become popular in life sciences in the last few years. They have been produced in many variants and have recently been used in both biological experiments and in clinical applications. Due to concerns over nanomaterial risks, there has been a dramatic increase in investigations focused on safety research. The aim of this paper is to present the advanced testing of rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R), which are used for their nontoxicity, biocompatibility, biodegradability, and magnetic properties. Recent results were expanded upon from the basic cytotoxic tests to evaluate cell proliferation and migration potential. Two cell types were used for the cell proliferation and tracking study: mouse embryonic fibroblast cells (3T3) and human mesenchymal stem cells (hMSCs). Advanced microscopic methods allowed for the precise quantification of the function of both cell types. This study has demonstrated that a dose of nanoparticles lower than 20 µ g · cm −2 per area of the dish does not negatively affect the cells’ morphology, migration, cytoskeletal function, proliferation, potential for wound healing, and single-cell migration in comparison to standard CellTracker ™ Green CMFDA (5-chloromethylfluorescein diacetate). A higher dose of nanoparticles could be a potential risk for cytoskeletal folding and detachment of the cells from the solid extracellular matrix. Keywords: magnetic nanoparticles; mesenchymal stem cells; fibroblast cells; cytotoxicity; wound healing assay; single-cell migration 1. Introduction Recently, nanoparticles have attracted the interest of scientists in various areas of biomedical research because of their unique properties. Nanoparticles of iron oxide, such as Fe 3 O 4 and γ -Fe 2 O 3, have been reported to be applicable as a material for use in drug delivery systems, magnetic resonance imaging [ 1 ], cancer therapy, and other fields. A potentially promising member of the family of iron oxides, maghemite (Fe(II)-deficient magnetite), allows the manufacture of nanoparticles that are biocompatible and non-toxic to living organisms. Maghemite nanoparticles can be introduced to living cells and their magnetic properties allow remote manipulation with external magnetic fields [ 2 , 3 ]; specific separation of cells from blood or mixed cell culture; inducing hyperthermia for selective Molecules 2019,24, 1192; doi:10.3390/molecules24071192 www.mdpi.com/journal/molecules Molecules 2019,24, 1192 2 of 17 destruction of cancer cells [ 4 ]; and delivery of pharmacoactive compounds or genes, and their selective activation by external stimuli [ 5 ]. Recently, cell labelling by superparamagnetic iron oxygen (SPIO) nanoparticles has also been used for cell tracking in vitro or in vivo . Multimodal probes based on SPIO nanoparticles may serve for cell detection and cell tracking via complementary contrast in light absorption or fluorescence. SPIO nanoparticles have also attracted much consideration for the possibility of replacing cytosolic or membrane probes, which are used in fluorescent confocal microscopy and microscopic time-lapse experiments due to their long-term chemical stability and photostability [6]. Rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R) display relatively high uptake by mammalian cells, very good stability in intracellular spaces, and long-term stability of their rhodamine fluorescence shell in intracellular space with a specific pH. SAMN-R have been previously tested on rat, rabbit and human stem cells, and are presented as a robust cell marker, a long-term stable probe for deposition in lysosomes, a probe that does not affect nuclei, and a probe with theranostic potential [7,8]. The main aim of this article is to expand on cell labelling experiments and refocus from the basic tests of acute toxicity and contrast properties to advanced evaluation of their interactions with mesenchymal stromal cells (MSCs) and fibroblasts, as well as evaluating their influence on the migration potential of cells. Fibroblasts and MSCs play a key role in regenerative medicine, especially in the regeneration of the dermis, chondral intercostal muscles, and other tissues for their motility and migration ability [ 9 – 11 ]. Thus, further investigation of SAMN-R properties will potentially bring new insight into the possibility of future biomedical applications. 2. Results 2.1. Cell Morphology and Flow Cytometry SAMN-R have been studied previously [ 7 , 8 , 12 ]. The stability of SAMN-R and their maghemite structure was confirmed by Mössbauer spectroscopy in the first days after preparation and also one year after preparation. The polydisperse index was quantified in all experiments using dynamic light scattering, its value being 0.22 ± 0.03 in dH 2 O and 0.47 ± 0.03 in culture medium. Their size varied from 20 to 50 nm [ 12 ]. It was found that nanoparticles are localized in lysosomes surrounding stem cell nuclei and are not localized in the cell nuclei themselves [8] (see Figure 1). Molecules 2018, 23, x 2 of 17 or genes, and their selective activation by external stimuli [5]. Recently, cell labelling by superparamagnetic iron oxygen (SPIO) nanoparticles has also been used for cell tracking in vitro or in vivo. Multimodal probes based on SPIO nanoparticles may serve for cell detection and cell tracking via complementary contrast in light absorption or fluorescence. SPIO nanoparticles have also attracted much consideration for the possibility of replacing cytosolic or membrane probes, which are used in fluorescent confocal microscopy and microscopic time-lapse experiments due to their long-term chemical stability and photostability [6]. Rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R) display relatively high uptake by mammalian cells, very good stability in intracellular spaces, and long-term stability of their rhodamine fluorescence shell in intracellular space with a specific pH. SAMN-R have been previously tested on rat, rabbit and human stem cells, and are presented as a robust cell marker, a long-term stable probe for deposition in lysosomes, a probe that does not affect nuclei, and a probe with theranostic potential [7,8]. The main aim of this article is to expand on cell labelling experiments and refocus from the basic tests of acute toxicity and contrast properties to advanced evaluation of their interactions with mesenchymal stromal cells (MSCs) and fibroblasts, as well as evaluating their influence on the migration potential of cells. Fibroblasts and MSCs play a key role in regenerative medicine, especially in the regeneration of the dermis, chondral intercostal muscles, and other tissues for their motility and migration ability [9–11]. Thus, further investigation of SAMN-R properties will potentially bring new insight into the possibility of future biomedical applications. 2. Results 2.1. Cell Morphology and Flow Cytometry SAMN-R have been studied previously [7,8,12]. The stability of SAMN-R and their maghemite structure was confirmed by Mössbauer spectroscopy in the first days after preparation and also one year after preparation. The polydisperse index was quantified in all experiments using dynamic light scattering, its value being 0.22 ± 0.03 in dH2O and 0.47 ± 0.03 in culture medium. Their size varied from 20 to 50 nm [12]. It was found that nanoparticles are localized in lysosomes surrounding stem cell nuclei and are not localized in the cell nuclei themselves [8] (see Figure 1). Figure 1. Human mesenchymal stem cells (hMSCs) treated with rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R). (A) Integrated modulation contrast in widefield microscopy, 40× magnification; (B) confocal microscopy, 63× magnification. Scale bar 50 µm. In this study, a 50 µg·mL −1 concentration of SAMN-R was used. Currently, there are many different types of dishes, well plates, or cultivation chambers, and labelling with SAMN-R has to be optimized for use in various types of cultivation chambers. However, SAMN-R are not uniformly distributed in the labelling suspension. Figure 1. Human mesenchymal stem cells (hMSCs) treated with rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R). ( A ) Integrated modulation contrast in wide-field microscopy, 40×magnification; (B) confocal microscopy, 63×magnification. Scale bar 50 µm. Molecules 2019,24, 1192 3 of 17 In this study, a 50 µ g · mL −1 concentration of SAMN-R was used. Currently, there are many different types of dishes, well plates, or cultivation chambers, and labelling with SAMN-R has to be optimized for use in various types of cultivation chambers. However, SAMN-R are not uniformly distributed in the labelling suspension. The nanoparticles settle on the surface of cells and culture dishes due to agglomeration and gravity in the process of labelling. It should be noted that the optimal concentration may be close to the toxicity limit in samples with a small surface area. Therefore, we used dose per surface area rather than concentration as a volume in our study. In our research, we used fibroblast cell types—large size human mesenchymal stem cells (hMSCs) (about 250 µ m) and considerably smaller mouse embryonic fibroblast cells (3T3) (about 90 µ m). At the dose of SAMN-R under 20 µ g · cm −2 , visible changes in cell shape and membrane integrity were not observed (Figure 2A,B). When the dose increased to more than 20 µ g · cm −2 , we noticed a decline in cell proliferation ability and the appearance of necrotic and detached cells (Figure 2C,D). Molecules 2018, 23, x 3 of 17 The nanoparticles settle on the surface of cells and culture dishes due to agglomeration and gravity in the process of labelling. It should be noted that the optimal concentration may be close to the toxicity limit in samples with a small surface area. Therefore, we used dose per surface area rather than concentration as a volume in our study. In our research, we used fibroblast cell types—large size human mesenchymal stem cells (hMSCs) (about 250 µm) and considerably smaller mouse embryonic fibroblast cells (3T3) (about 90 µm). At the dose of SAMN-R under 20 µg·cm −2 , visible changes in cell shape and membrane integrity were not observed (Figure 2A,B). When the dose increased to more than 20 µg·cm −2 , we noticed a decline in cell proliferation ability and the appearance of necrotic and detached cells (Figure 2C,D). Figure 2. Influence of SAMN-R in different doses to mouse embryonic fibroblast cells (3T3) morphology. (A) Control; (B) dose of SAMN-R 20 µg·cm −2 ; (C) dose of SAMN-R 25 µg·cm −2 ; (D) dose of SAMN-R 35 µg·cm −2 . Bright-field microscopy, 10× magnification, scale bar 100 µm. We found out that even at the lowest tested doses, the SAMN-R emitted enough fluorescence to be detectable. According to the results of this experiment, it was decided to test the dose of SAMN-R of 20 µg·cm −2 , since it was determined as the limiting value. Flow cytometry evaluation gives a statistical comparison of cell size and cell granularity before and after nanoparticle incorporation. Granularity of both cell types was increased after nanoparticle deposition in intracellular space. However, size and distribution of cluster of differentiation (CD) molecules on the cell surface were not affected (Figure 3). Figure 2. Influence of SAMN-R in different doses to mouse embryonic fibroblast cells (3T3) morphology. ( A ) Control; ( B ) dose of SAMN-R 20 µ g · cm −2 ; ( C ) dose of SAMN-R 25 µ g · cm −2 ; ( D ) dose of SAMN-R 35 µg·cm−2. Bright-field microscopy, 10×magnification, scale bar 100 µm. We found out that even at the lowest tested doses, the SAMN-R emitted enough fluorescence to be detectable. According to the results of this experiment, it was decided to test the dose of SAMN-R of 20 µg·cm−2, since it was determined as the limiting value. Flow cytometry evaluation gives a statistical comparison of cell size and cell granularity before and after nanoparticle incorporation. Granularity of both cell types was increased after nanoparticle deposition in intracellular space. However, size and distribution of cluster of differentiation (CD) molecules on the cell surface were not affected (Figure 3). 2.2. Quantification of Reactive Oxygen Species (ROS) Generation after SAMN-R Labelling The value of ROS production is measured by CM-H2DCFDA after the exposure of cells to SAMN-R, which is an aspect that helps to quantify the very early changes in cell physiology. This method is very sensitive in many cases of cell stress, very often for several hours before the start of any apoptosis or cell malformation. The CM-H2DCFDA kit determines the rate of production of hydrogen peroxide (H 2 O 2 ), hydroxyl radicals ( • HO), and superoxides (O 2− ) in the cells. The final graphical overview of ROS value measured on our cells after SAMN-R labelling is shown in Figure 4. The results show that almost all used labelling concentrations do not have any negative effects on ROS value. 2.3. Cell Proliferation A fluorescence probe designed for live cell imaging can be toxic for cells [ 13 , 14 ]. Negative effects on vital functions could otherwise affect the positive properties and application potential of the dye. As an initial study, we used a cell growth curve to identify the effect on cell proliferation (Figure 5). For comparison with SAMN-R, we tested CellTracker ™ Green CMFDA (5-chloromethylfluorescein diacetate) because it is a low toxic reagent. The result of the statistical t-test showed that both dyes do Molecules 2019,24, 1192 4 of 17 not have a significant effect on cell proliferation. This means that SAMN-R can be successfully used in long-term experiments such as a cell tracking dye similar to CMFDA. Molecules 2018, 23, x 4 of 17 Figure 3. Flow cytometry analysis of labelled cells. Site scatter and forward scatter: (A) 3T3 before labelling; (B) mesenchymal stromal cell (MSC) before labelling; (C) 3T3 after 24 h of labelling; (D)MSC after 24 h of labelling; (E) quantification of CD90 and CD45 positive 3T3; (F) quantification of CD90, CD73, and CD45 positive MSC. 2.2. Quantification of Reactive Oxygen Species (ROS) Generation after SAMN-R Labelling The value of ROS production is measured by CM-H2DCFDA after the exposure of cells to SAMN-R, which is an aspect that helps to quantify the very early changes in cell physiology. This method is very sensitive in many cases of cell stress, very often for several hours before the start of any apoptosis or cell malformation. The CM-H2DCFDA kit determines the rate of production of hydrogen peroxide (H2O2), hydroxyl radicals (•HO), and superoxides (O2−) in the cells. The final graphical overview of ROS value measured on our cells after SAMN-R labelling is shown in Figure Figure 3. Flow cytometry analysis of labelled cells. Site scatter and forward scatter: ( A ) 3T3 before labelling; ( B ) mesenchymal stromal cell (MSC) before labelling; ( C ) 3T3 after 24 h of labelling; ( D )MSC after 24 h of labelling; ( E ) quantification of CD90 and CD45 positive 3T3; ( F ) quantification of CD90, CD73, and CD45 positive MSC. Molecules 2019,24, 1192 5 of 17 Molecules 2018, 23, x 5 of 17 4. The results show that almost all used labelling concentrations do not have any negative effects on ROS value. Figure 4. Measurement of reactive oxygen species (ROS) after cell labelling. The intensity of indicator CM-H2DCFDA was measured for four different labelling concentrations. The horizontal segment (whiskers) marks the standard deviation of fluorescence intensity. 2.3. Cell Proliferation A fluorescence probe designed for live cell imaging can be toxic for cells [13,14]. Negative effects on vital functions could otherwise affect the positive properties and application potential of the dye. As an initial study, we used a cell growth curve to identify the effect on cell proliferation (Figure 5). Figure 5. 3T3 cell growth. Each box represents the number of cells in an area of 1 mm 2 from 10 experiments. Each box plots the 25th and 75th percentiles. The circle inside the box is the mean, the band inside the box is the median. Whiskers (the most extreme values not considered as outliers) and outliers (red squares) are outside the box. For comparison with SAMN-R, we tested CellTracker™ Green CMFDA (5chloromethylfluorescein diacetate) because it is a low toxic reagent. The result of the statistical t-test showed that both dyes do not have a significant effect on cell proliferation. This means that SAMNR can be successfully used in long-term experiments such as a cell tracking dye similar to CMFDA. Figure 4. Measurement of reactive oxygen species (ROS) after cell labelling. The intensity of indicator CM-H2DCFDA was measured for four different labelling concentrations. The horizontal segment (whiskers) marks the standard deviation of fluorescence intensity. Molecules 2018, 23, x 5 of 17 4. The results show that almost all used labelling concentrations do not have any negative effects on ROS value. Figure 4. Measurement of reactive oxygen species (ROS) after cell labelling. The intensity of indicator CM-H2DCFDA was measured for four different labelling concentrations. The horizontal segment (whiskers) marks the standard deviation of fluorescence intensity. 2.3. Cell Proliferation A fluorescence probe designed for live cell imaging can be toxic for cells [13,14]. Negative effects on vital functions could otherwise affect the positive properties and application potential of the dye. As an initial study, we used a cell growth curve to identify the effect on cell proliferation (Figure 5). Figure 5. 3T3 cell growth. Each box represents the number of cells in an area of 1 mm 2 from 10 experiments. Each box plots the 25th and 75th percentiles. The circle inside the box is the mean, the band inside the box is the median. Whiskers (the most extreme values not considered as outliers) and outliers (red squares) are outside the box. For comparison with SAMN-R, we tested CellTracker™ Green CMFDA (5chloromethylfluorescein diacetate) because it is a low toxic reagent. The result of the statistical t-test showed that both dyes do not have a significant effect on cell proliferation. This means that SAMNR can be successfully used in long-term experiments such as a cell tracking dye similar to CMFDA. Figure 5. 3T3 cell growth. Each box represents the number of cells in an area of 1 mm 2 from 10 experiments. Each box plots the 25th and 75th percentiles. The circle inside the box is the mean, the band inside the box is the median. Whiskers (the most extreme values not considered as outliers) and outliers (red squares) are outside the box. 2.4. Cell Migration Study Currently, researchers use many methods to study cell motility such as wound healing assays, trans-well cell migration, cell exclusion zone assays, spheroid migration assays, single-cell motility assays, and others [ 15 ]. Wound healing assays, a widely used method for the analysis of cell migration, are treated with a specific compound [ 16 – 18 ]. This method is technically non-demanding, and it is possible to observe changes in cell motility in a short time [ 19 ]. To understand the effect of changes in the environment on the mechanical responses of cells, single-cell migration was usually used in other studies [20,21]. Molecules 2019,24, 1192 6 of 17 2.4.1. In Vitro Wound Healing Assay In each experiment, three groups of 3T3 cells were investigated: control (non-labelled), treated SAMN-R, and those labelled with CMFDA dye. We studied the effect of these treatments on the ability of the collective migration of 3T3 cells by wound healing assays. In 10 experiments, we obtained 60 images from each group: 30 with a narrow scratch and 30 with a wide scratch, which were compared separately (Figure 6). Molecules 2018, 23, x 6 of 17 2.4. Cell Migration Study Currently, researchers use many methods to study cell motility such as wound healing assays, trans-well cell migration, cell exclusion zone assays, spheroid migration assays, single-cell motility assays, and others [15]. Wound healing assays, a widely used method for the analysis of cell migration, are treated with a specific compound [16–18]. This method is technically nondemanding, and it is possible to observe changes in cell motility in a short time [19]. To understand the effect of changes in the environment on the mechanical responses of cells, single-cell migration was usually used in other studies [20,21]. 2.4.1. In Vitro Wound Healing Assay In each experiment, three groups of 3T3 cells were investigated: control (non-labelled), treated SAMN-R, and those labelled with CMFDA dye. We studied the effect of these treatments on the ability of the collective migration of 3T3 cells by wound healing assays. In 10 experiments, we obtained 60 images from each group: 30 with a narrow scratch and 30 with a wide scratch, which were compared separately (Figure 6). Figure 6. Wound healing assay applied to 3T3 cells: control, SAMN-R, and CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate) labelled cells. Images were detected at the beginning and after 24 and 48 hours. The white line marks the initial scratch border. Confocal microscopy, 10× magnification, scale bar 150 µm. Since the cells migrate as a loosely connected population, the analysis was based on calculating the clear area sites present at the wound. Quantitative analysis was provided using a custom-made algorithm in MATLAB software (R2018a 64-bit, MathWorks®, Natick, MA, USA). As can be seen from the data in Tables 1 and 2, the areas with a narrow scratch were overgrown within approximately 24 h, and the areas with a wide scratch were overgrown within 48 hours. Timedependent changes in the open area due to 3T3 cell migration are shown in Figure 7. Figure 6. Wound healing assay applied to 3T3 cells: control, SAMN-R, and CellTracker ™ Green CMFDA (5-chloromethylfluorescein diacetate) labelled cells. Images were detected at the beginning and after 24 and 48 h. The white line marks the initial scratch border. Confocal microscopy, 10 × magnification, scale bar 150 µm. Since the cells migrate as a loosely connected population, the analysis was based on calculating the clear area sites present at the wound. Quantitative analysis was provided using a custom-made algorithm in MATLAB software (R2018a 64-bit, MathWorks ® , Natick, MA, USA). As can be seen from the data in Tables 1and 2, the areas with a narrow scratch were overgrown within approximately 24 h, and the areas with a wide scratch were overgrown within 48 h. Time-dependent changes in the open area due to 3T3 cell migration are shown in Figure 7. Table 1. Size values of the open area of narrow scratch in the wound healing assay experiment. Item Time Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Scratch width (µm) 311.54 493.62 408.01 325.04 477.73 416.44 329.51 491.50 427.76 Scratch area (mm2)0 h 0.36 0.58 0.48 0.38 0.56 0.49 0.38 0.57 0.50 24 h 0.00 0.02 0.00 0.00 0.02 0.00 0.00 0.02 0.00 Molecules 2019,24, 1192 7 of 17 Table 2. Size values of the open area of wide scratch in the wound healing assay experiment. Item Time Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Scratch width (µm) 502.18 689.57 554.24 438.18 711.15 548.73 447.23 621.20 541.37 Scratch area (mm2) 0 h 0.59 0.80 0.65 0.51 0.83 0.64 0.52 0.73 0.63 24 h 0.17 0.53 0.31 0.14 0.57 0.32 0.16 0.56 0.36 48 h 0.00 0.09 0.03 0.00 0.05 0.02 0.00 0.23 0.03 Molecules 2018, 23, x 7 of 17 Table 1. Size values of the open area of narrow scratch in the wound healing assay experiment. Item Time Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Scratch width (µm) 311.54 493.62 408.01 325.04 477.73 416.44 329.51 491.50 427.76 Scratch area (mm2) 0 h 0.36 0.58 0.48 0.38 0.56 0.49 0.38 0.57 0.50 24 h 0.00 0.02 0.00 0.00 0.02 0.00 0.00 0.02 0.00 Table 2. Size values of the open area of wide scratch in the wound healing assay experiment. Item Time Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Scratch width (µm) 502.18 689.57 554.24 438.18 711.15 548.73 447.23 621.20 541.37 Scratch area (mm2) 0 h 0.59 0.80 0.65 0.51 0.83 0.64 0.52 0.73 0.63 24 h 0.17 0.53 0.31 0.14 0.57 0.32 0.16 0.56 0.36 48 h 0.00 0.09 0.03 0.00 0.05 0.02 0.00 0.23 0.03 Figure 7. Time-dependent changes in the size of the open area due to 3T3 cell migration (left) in narrow scratch and (right) in wide scratch. Each box represents scratch areas in 30 images in each group. Each box plots the 25th and 75th percentiles. The circle inside the box is the mean, the band inside the box is the median. Whiskers (the most extreme values not considered as outliers) and outliers (red squares) are outside the box. A statistical t-test shows that the effect of SAMN-R and CMFDA dye to collective cell migration is not statistically significant. 2.4.2. Single-Cell Migration To investigate single-cell migration, we used a time-lapse microscopy scan to observe hMSCs. The selected scan field was scanned over six hours at intervals of five minutes. In the primary experiments (Patient 1), there were scans only in one field of view. The following experiments were made with a choice of two to four fields (Patients 2–4) with the help of multi-view mode (mark and find function). The calculated value of velocity and accumulated and Euclidean distance hMSCs are listed in Tables 3 to 6. Figure 7. Time-dependent changes in the size of the open area due to 3T3 cell migration ( left ) in narrow scratch and ( right ) in wide scratch. Each box represents scratch areas in 30 images in each group. Each box plots the 25th and 75th percentiles. The circle inside the box is the mean, the band inside the box is the median. Whiskers (the most extreme values not considered as outliers) and outliers (red squares) are outside the box. A statistical t-test shows that the effect of SAMN-R and CMFDA dye to collective cell migration is not statistically significant. 2.4.2. Single-Cell Migration To investigate single-cell migration, we used a time-lapse microscopy scan to observe hMSCs. The selected scan field was scanned over six hours at intervals of five minutes. In the primary experiments (Patient 1), there were scans only in one field of view. The following experiments were made with a choice of two to four fields (Patients 2–4) with the help of multi-view mode (mark and find function). The calculated value of velocity and accumulated and Euclidean distance hMSCs are listed in Tables 3–6. In our research, the most important parameter during cell migration of single cells is velocity. Figure 8presents the complete set of single-cell velocities from four patients. The number of cells in groups from one patient was the same. Using a statistical t-test, it was shown that the effect of SAMN-R and CMFDA dye on individual hMSCs’ velocity was not statistically significant. The Rayleigh test confirms that cell distribution is homogeneous. Cell tracking examples are shown in Figure 9. Table 3. Migration of hMSCs in Patient 1 from 18 cells of control, CellTracker ™ Green CMFDA (5-chloromethylfluorescein diacetate) labelled, and rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R) treatment groups. The velocity, accumulated, and Euclidean distance values for a six-hour period are shown. Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Accumulated distance (µm) 62.76 271.85 153.99 86.36 241.65 148.85 73.92 200.72 136.70 Euclidean distance (µm) 1..23 217.19 81.90 8.83 123.17 55.36 7.91 97.74 41.92 Velocity (µm/min) 0.17 0.76 0.43 0.24 0.67 0.41 0.21 0.56 0.38 Molecules 2019,24, 1192 8 of 17 Table 4. Migration of hMSCs in Patient 2 from 30 cells of control, CMFDA-labelled, and SAMN-R treatment groups. The velocity, accumulated, and Euclidean distance values for a six-hour period are shown. Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Accumulated distance (µm) 32.82 294.80 104.90 44.64 362.05 118.83 51.73 221.71 114.01 Euclidean distance (µm) 7.24 248.21 69.32 6.87 316.28 80.86 12.22 194.51 82.98 Velocity (µm/min) 0.09 0.82 0.29 0.12 1.01 0.33 0.14 0.62 0.32 Table 5. Migration of hMSCs in Patient 3 from 50 cells of control, CMFDA-labelled, and SAMN-R treatment groups. The velocity, accumulated, and Euclidean distance values for a six-hour period are shown. Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Accumulated distance (µm) 48.06 288.48 156.00 45.15 270.70 143.62 43.02 282.62 154.66 Euclidean distance (µm) 9.72 190.26 65.91 15.74 226.10 99.33 2.26 193.26 68.11 Velocity (µm/min) 0.13 0.80 0.43 0.13 0.75 0.40 0.12 0.79 0.43 Table 6. Migration of hMSCs in Patient 4 from 50 cells of control, CMFDA-labelled, and SAMN-R treatment groups. The velocity, accumulated, and Euclidean distance values for a six-hour period are shown. Control SAMN-R CMFDA Min Max Mean Min Max Mean Min Max Mean Accumulated distance (µm) 33.29 179.74 90.96 30.09 181.34 84.78 37.08 263.26 79.96 Euclidean distance (µm) 9.71 147.88 61.31 8.03 102.06 40.66 3.41 243.35 42.51 Velocity (µm/min) 0.09 0.50 0.25 0.08 0.50 0.24 0.10 0.73 0.22 Molecules 2018, 23, x 9 of 17 groups from one patient was the same. Using a statistical t-test, it was shown that the effect of SAMNR and CMFDA dye on individual hMSCs’ velocity was not statistically significant. The Rayleigh test confirms that cell distribution is homogeneous. Cell tracking examples are shown in Figure 9. Figure 8. Calculated velocity from single hMSCs. Each box represents the complete set of single-cell velocities from four patients. There are 18 cells in each group from Patient 1, 30 cells from Patient 2, and 50 cells from Patients 3 and 4. Boxes are composed of main box edges (25th and 75th percentiles), blue dots (mean), central red horizontal line (median), whiskers (the most extreme values not considered outliers), and red squares (outliers). Figure 9. Tracks of hMSCs. (A) Non-labelled cells (control); (B) SAMN-R treated cells; (C) CMFDAlabelled cells. The blue circle is a detected cell, the blue line is accumulated distance, and the yellow line is Euclidean distance. Confocal microscopy, 10× magnification, scale bar 250 µm. 3. Discussion Fibroblasts and mesenchymal stem cells have prominent status in the hierarchy of mammal cells. Fibroblasts are one of the most populous and important cells of connective tissue, and play the crucial role in normal preand postnatal organ development. They represent key components for wound healing and the occurrence of tumors. MSCs are relatively more latent cells in normal development; however, their effective migration, proliferation, and interaction with other cell types are essential in the critical time after tissue scarring or ischemic events, when wound healing and correction of abnormal immune reactions begins. Unfortunately, fibroblasts and MSCs could be negatively affected by modern cell markers, cell labels, or labelling nanoparticles [22,23]. The negative effect of markers and nanoparticles may occur a short time after their application. Such markers and nanoparticles with acute toxicity cause visible rapid morphological changes and cells rapidly die. Markers and nanoparticles, that can be seemingly non-toxic, may have a hidden long-term negative effect that can manifest after several days. Moreover, the effect need not necessarily lead to apoptosis of the cell, but rather to the abnormal production of certain factors in the cell (e.g., reactive oxygen) or to altered metabolism or cell motility. Figure 8. Calculated velocity from single hMSCs. Each box represents the complete set of single-cell velocities from four patients. There are 18 cells in each group from Patient 1, 30 cells from Patient 2, and 50 cells from Patients 3 and 4. Boxes are composed of main box edges (25th and 75th percentiles), blue dots (mean), central red horizontal line (median), whiskers (the most extreme values not considered outliers), and red squares (outliers). Molecules 2019,24, 1192 9 of 17 Molecules 2018, 23, x 9 of 17 groups from one patient was the same. Using a statistical t-test, it was shown that the effect of SAMNR and CMFDA dye on individual hMSCs’ velocity was not statistically significant. The Rayleigh test confirms that cell distribution is homogeneous. Cell tracking examples are shown in Figure 9. Figure 8. Calculated velocity from single hMSCs. Each box represents the complete set of single-cell velocities from four patients. There are 18 cells in each group from Patient 1, 30 cells from Patient 2, and 50 cells from Patients 3 and 4. Boxes are composed of main box edges (25th and 75th percentiles), blue dots (mean), central red horizontal line (median), whiskers (the most extreme values not considered outliers), and red squares (outliers). Figure 9. Tracks of hMSCs. (A) Non-labelled cells (control); (B) SAMN-R treated cells; (C) CMFDAlabelled cells. The blue circle is a detected cell, the blue line is accumulated distance, and the yellow line is Euclidean distance. Confocal microscopy, 10× magnification, scale bar 250 µm. 3. Discussion Fibroblasts and mesenchymal stem cells have prominent status in the hierarchy of mammal cells. Fibroblasts are one of the most populous and important cells of connective tissue, and play the crucial role in normal preand postnatal organ development. They represent key components for wound healing and the occurrence of tumors. MSCs are relatively more latent cells in normal development; however, their effective migration, proliferation, and interaction with other cell types are essential in the critical time after tissue scarring or ischemic events, when wound healing and correction of abnormal immune reactions begins. Unfortunately, fibroblasts and MSCs could be negatively affected by modern cell markers, cell labels, or labelling nanoparticles [22,23]. The negative effect of markers and nanoparticles may occur a short time after their application. Such markers and nanoparticles with acute toxicity cause visible rapid morphological changes and cells rapidly die. Markers and nanoparticles, that can be seemingly non-toxic, may have a hidden long-term negative effect that can manifest after several days. Moreover, the effect need not necessarily lead to apoptosis of the cell, but rather to the abnormal production of certain factors in the cell (e.g., reactive oxygen) or to altered metabolism or cell motility. Figure 9. Tracks of hMSCs. ( A ) Non-labelled cells (control); ( B ) SAMN-R treated cells; ( C ) CMFDAlabelled cells. The blue circle is a detected cell, the blue line is accumulated distance, and the yellow line is Euclidean distance. Confocal microscopy, 10×magnification, scale bar 250 µm. 3. Discussion Fibroblasts and mesenchymal stem cells have prominent status in the hierarchy of mammal cells. Fibroblasts are one of the most populous and important cells of connective tissue, and play the crucial role in normal preand postnatal organ development. They represent key components for wound healing and the occurrence of tumors. MSCs are relatively more latent cells in normal development; however, their effective migration, proliferation, and interaction with other cell types are essential in the critical time after tissue scarring or ischemic events, when wound healing and correction of abnormal immune reactions begins. Unfortunately, fibroblasts and MSCs could be negatively affected by modern cell markers, cell labels, or labelling nanoparticles [22,23]. The negative effect of markers and nanoparticles may occur a short time after their application. Such markers and nanoparticles with acute toxicity cause visible rapid morphological changes and cells rapidly die. Markers and nanoparticles, that can be seemingly non-toxic, may have a hidden long-term negative effect that can manifest after several days. Moreover, the effect need not necessarily lead to apoptosis of the cell, but rather to the abnormal production of certain factors in the cell (e.g., reactive oxygen) or to altered metabolism or cell motility. We have focused on the evaluation of the abovementioned hidden long-term effects after the application of SAMN-R to cells. Acute toxicity and critical concentration/dose for immediate effective marking of cells were described in our previous studies [7,8]. In the majority of publications dealing with the toxicity of nanoparticles, the authors take as a standard measure the absolute concentration of the nanoparticles in a given cultivation medium presuming colloidal stability of nanoparticle suspension. However, there is a certain agglomeration rate and sedimentation rate dependent on particles’ concentration even in case of almost ideally colloidal particle suspensions [ 24 ]. With regard to increasing concerns about the health risks of nanoparticles, we propose to define concentrations of each used solution in µ g · mL −1 with a value of a dose in µ g · cm −2 per area of the dish with medium, although an increase of particles’ concentration in the microenvironment of cells does not exceed 10% (data not shown). The first set of experiments included cultivation of fibroblasts at the bottom of standard in vitro dishes treated with SAMN-R. The used SAMN-R treatment solutions were prepared in a gradient series. Nanoparticles were diluted in a cultivation medium, and a colloid profile of solution was prepared by 20 min of ultrasound exposure immediately before cell incubation. The used absolute concentrations were 0, 10, 20, 30, 40, 50, 60, and 70 µ g · mL −1 , which corresponded to doses of 0, 5, 10, 15, 20, 25, 30, and 35 µ g · cm −2 per area of the dish. The effects of different doses of SAMN-R were evaluated after 24 h of incubation. 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