Cell-IQ Phase-contrast Microscopy for a Continuous Real-time Observation of Multipotent Mesenchymal Stromal Cell Culture

Continuous monitoring of multipotent mesenchymal stromal cells (MMSCs) is a promising tool that could be used in cellular biology, environmental research and biotechnology to study in vitro real-time morphology and behavioural response of stem cells alone, as well at contacts with other cells and for controlling a sustainable production of scaffolds for tissue engineering. The in vitro processes of human adipose-derived MMSCs (hAMMSCs) morphology, motility, cell division, and secretion were studied by means of Cell-IQ v2 MLF (CM  Technologies Oy, Finland) integrated phase-contrast microscopic platform for a continuous real-time surveillance imaging of living cells. 70 μL suspension (5×104 viable karyocytes) of the cells was applied into the center of the wells of 12-well plastic plates, and cells were allowed to adhere in a moist chamber for 120 min. Nonadherent cells were washed, and the wells were carefully filled with 1.5 mL of a nutrient medium DMEM/F12 (1 : 1) without osteogenic additions. Cells were cultured for 14 days at 100% humidity in a 5% carbondioxide atmosphere at 37°C until a monolayer formation. Digital images of cell culture growth were captured every 45 min. The cells were positively stained with alizarin red (osteoblasts), alcian blue (chondrocytes), or oil red (adipocytes). More than 95% of attached cells expressed CD73, CD90, and CD105 markers, mainly. Thus, the cells corresponded to the morphological criteria of MMSCs. The Cell-IQ system has allowed establishing 182 μm/h linear velocity of free (until the cell contacts) motility of spindle or fibroblast-like cells. Maximum number of cells achieved 136 cells per field of view; 13-24 % of cells divided each 1-3 h until a monolayer was formed. Chemokine cooperation between hAMMSCs and poor macrophages intermixture was proposed. Cell-IQ could be useful for in vitro real-time imaging of cell subpopulations and/or their response to biodegradable scaffolds and/or (micro)environmental factors.DOI: http://dx.doi.org/10.5755/j01.erem.74.3.20548


Introduction
New approaches to an investigation of adhesion, proliferation, migration, differentiation and maturation of stem cells in conditions of both two-dimensional (2D) and three-dimensional (3D) colonization of scaffolds are essential for effective tissue engineering.Various synthetic copolymers, such as PLGA (poly(lactic-co-glycolic acid) are environmentally and biologically compatible, and promising material for bone tissue engineering, but its degradation products can lower the pH around the implant and can induce inflammatory reactions (Kzhyshkowska et al., 2015).In turn, bone fracture triggers an inflammation that drives expansion/recruitment of endothelial, neuronal and mesenchymal stem/progenitor/precursor cells, leading to formation of a vascularized and innervated fibrous granulation tissue (Batoon et al., 2017).Bone and bone marrow contain multiple specialized resident macrophage subpopulations that contribute to bone biology and/or haematopoiesis (Kaur et al., 2017).
The interplay between macrophage inflammatory or regenerative subpopulations, stem and bone cells (Gu et al., 2017) and scaffolds is critical to physiologic and reparative bone tissue regeneration.Multipotent mesenchymal stromal cells (MMSCs) are able to migrate into inflammation and regeneration foci (Lepik et al., 2016).Thus, continuous monitoring of MMSCs is a promising tool that could be used in cellular biology, (micro)environmental research and biotechnology to study in vitro real-time morphology and behavioural response of stem cells alone, as well at contacts with other cells and for controlling a sustainable production of scaffolds for tissue engineering.However, few works have been devoted so far to modelling and determining the behaviour of MMSCs in real time (Moniri et al., 2015).
The aim of this work was to study human adipose-derived MMSCs (hAMMSCs) culture in vitro (cell morphology, motility, cell division, and secretory activity) using the Cell-IQ v2 MLF integrated phase-contrast microscopic platform for a continuous real-time surveillance imaging of living cells.

MMSCs isolation
MMSCs were isolated from lipoaspirate of 5 healthy subjects (permission no. 4 of October 23, 2013; Local Ethics Committee, Innovation Park, Immanuel Kant Baltic Federal University) as described previously (Zuk et al., 2001).To determine the stem nature of human adipose-derived MMSCs (hAMMSCs), they were cultivated in the medium with a StemPro® Differentiation Kit (Thermo Fisher Scientific, United States) for 21 days with medium exchange every 3-4 days.As we published previously (Litvinova et al., 2018), chondrogenic, osteogenic and adipogenic differentiations were detected by cell positive staining with alcian blue (Sigma-Aldrich, United States) and visualized proteoglycan synthesis by chondrocytes; alizarin red S (Sigma-Aldrich, United States) identified mineralization of intercellular substances in osteoblasts, and oil red (Sigma-Aldrich, United States) detected neutral triglycerides and lipids in adipocytes.This confirmed that obtained cells belonged to the pool of stem cells according to the minimal morphological criteria of the International Society for Cellular Therapy (Dominici et al., 2006).

hAMMSCs behaviour
The morphology of the attached cells and monolayer formation were monitored in vitro with a Cell-IQR v2 MLF device for continuous phase-contrast visualization of living cells in real time (CM Technologies Oy, Finland).A 70-μL cell suspension (5 × 10 4 viable cells, fifth passage) was placed in the well centre of sterile 12-well plastic plates (Orange Scientific, Belgium).The cells were allowed to adhere for 120 min in a moist chamber.Unattached cells were washed out with a phosphate buffer.Then, 1.5 mL of the medium (90% DMEM/ F12 (1 : 1) (Gibco Life Technologies, United States), 10% fetal bovine serum (Sigma-Aldrich, United States), 50 mg/mL gentamicin (Invitrogen, United Kingdom), and 280 mg/mL L-glutamine (Sigma-Aldrich, United States) were carefully added.The cells were cultivated in the Cell-IQ system at 37°C, 100% humidity, and 5% СО 2 for 14 days (until monolayer formation).Microscopic identification of cell morphology and cell motility is difficult in multilayer cultures.
In each well, four points were chosen for Cell-IQ phase-contrast microscopy around the initial drop of cell suspension.The points were located as follows: on the cell layer boundary (01), distantly from the cell layer and the same points on the opposite side of cell suspension.Cell culture images were captured every 45 min, and 1308 images were obtained for each of the four points.Every tenth image was used for automatic analysis with the Cell-IQ Imagen software.Several electronic libraries of digital images were created for effective cell identification.During manual processing of videos, we determined the linear velocity of free movement of cells before they formed cell-cell contacts.The distance travelled by the cell was evaluated by the shift of the nucleus due to the variation in the shape and different sizes of migrating AMMSCs.

hAMMSCs immunophenotype
Before and after 14-day culturing, hAMMSCs cells were reattached with 0.05% trypsin (PanEco, Russia) in 0.53 mM EDTA (Sigma-Aldrich, United States) and washed twice with phosphate buffer.The analysis of the surface markers of the viable AMMSCs was performed with human MSC Phenotyping Miltenyi Biotec,Germany), which allowed evaluating the expression of antigenic clusters CD14, CD20,CD34, CD45, CD73, CD90, and CD105 using a MACS Quant flow cytometry (Miltenyi Biotec, Germany) according to the protocol of the manufacturer and the KALUZA Analysis Software (Beckman Coulter, United States).The concentration and viability of cells were determined with a CountessTM Automated Cell Counter (Invitrogen, United Kingdom) using 0.4% trypan blue solution (Invitrogen, United Kingdom).

Cytokines and chemokines secretion in hAMMSCs culture
Concentrations of cytokines and chemokines in the supernatants was assayed with monoclonal antibodies (Bio-Plex Pro TM Human Cytokine 21-plex Assay, Bio-Rad, United States) and the flow cytometry according to the manufacturer's instructions using automatic analyzer (Bio-Plex Protein Assay System, Bio-Rad, United States).The concentration was expressed in pg/mL.

Statistics
The results were treated with the STATISTICA software for Windows 10.0.Testing for normality was performed using the Kolmogorov-Smirnov criterion.Median (Me), 25% (Q1), and 75% (Q3) quartiles were calculated.The non-parametric Mann-Whitney U test was used to determine the statistical significance of differences.The differences were statistically significant at p < 0.05.
The Cell-IQ system allowed us to establish 182 μm/h linear velocity of free (until the cell contacts) motility of spindle or fibroblast-like human AMMSCs (Table 1).Maximum number of cells achieved 136 cells per field of view; 13-24% of cells divided each 1-3 h until a monolayer was obtained.
Low expression of CD14 determinant and secretion of pro-inflammatory (van den Broek et al., 2014) monokines into supernatants (Table 2) favoured to the poor macrophage intermixture of the hAMMSCs parametric Mann-Whitney U test was used to determine the statistical significance of differences.Th 118 were statistically significant at p < 0.05.

131
The Cell-IQ system allowed us to establish 182 μm/h linear velocity of free (until the cell contacts 132 spindle or fibroblast-like human AMMSCs (Table 1).Maximum number of cells achieved 136 cells per f 133 13-24% of cells divided each 1-3 h until a monolayer was obtained.
134 135  2) favoured to the poor macrophage intermixture of the hAMMSCs cult 141 chemokines influenced macrophages and expressed by them were revealed (Table 2).

150
Thus, humoral interplay between hAMMSCs and macrophages may control a migration and morp 151 reactions of cell subpopulations in vitro.

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Table 1 Motility and cell division of hAMMSCs before monolayer formation Manual processing of Cell-IQ video images Software processing of Cell-IQ video images Table 2 Certain chemokines (pg/mL) detected in the supernatants of hAMMSCs culture in 14 days of cultivation
Thus, humoral interplay between hAMMSCs and macrophages may control a migration and morphofunctional reactions of cell subpopulations in vitro.

Conclusions
The Cell-IQ phase-contrast microscopic platform allowed obtaining the multiple data of the hAMMSCs culture morphofunctional state and behaviour.It could be a useful tool for in vitro real-time imaging of an interplay between cell subpopulations and/or their morphofunctional response to biodegradable scaffolds and/or (micro)environmental factors.

Table 1
Motility and cell division of hAMMSCs before monolayer formation136 137

Manual processing of Cell-IQ video images Software processing of Cell-IQ video images
n = number of cells studied.Data are represented as median (Q1-Q3).138 139 Low expression of CD14 determinant and secretion of pro-inflammatory (van den Broek et al., 2014 140 into supernatants (Table