Cryopreserved cells from a biobank were thawed and characterization was performed in a previous research that evaluated the feasibility of isolation of mule MSCs. Cell culture was performed in culture flasks maintained in an incubator at 37°C and 5% CO₂, according to Yamada et al. (2013) and Carvalho et al. (2013), using Knockout DMEM^®, 10% Fetal Bovine Serum, 1% L-glutamine, 1% antibiotic-antimycotic solution (penicillin, streptomycin and amphotericin B), 1% essential amino acids and 0.5% non-essential amino acids. All the medium components were obtained from Thermo Fisher Scientific, Grand Island, New York, USA.

Cell encapsulation was performed following the technique previously described by Ma et al. (2003). For that, 5 × 10⁴ cells were homogenized in sterile 1.2% low viscosity alginate solution (Aldrich Materials Science Sigma-Aldrich, Milwaukee, USA) with pH adjusted to 7.4. Alginate concentration was chosen based on the study of Ewa-Choy et al. (2017).

The solution was dripped through a 30 G needle (0.30 mm × 13 mm) in Petri dishes containing a 102 mM calcium chloride aqueous solution, at a controlled rate of 40 ml/h and 10 cm of height. Previous tests were performed to determine optimal rate and height, defined when dripping without drop overlap and coalescence occurred. The drops were maintained for 20 min in the CaCl₂ solution for the ionic cross-linking occurrence. Each well of a 24-well plate received 10 capsules that were maintained for 24 h in culture medium for clearance of the calcium excess and for cell adaptation to the new tridimensional structure.

At each time point the respective capsules were transferred to 15 ml conical tubes, dissolved with sodium citrate and centrifuged for cell count and cell viability assessment using Trypan Blue technique in a hemocytometer, following the technique described by Tennant (1964). Briefly, capsules were dissolved for 10 min at 37°C in 3.2% sodium citrate solution. After centrifugation and resuspension in PBS, cell concentration and viability were calculated in a hemocytometer filled with a 1:1 solution of cells and trypan blue. The remaining cells attached to the well were counted in a microscope (Zeiss Axiovert40CFL, Axiovision, Carl Zeiss, Oberkochen, Germany) to assess cell release from the capsules.

Analysis of the swelling behavior (SB) was conducted gravimetrically based on Hanna et al. (2013) and Bajpai and Kirar (2016) to evaluate the potential of nutrient transportation across the capsule membrane. Two groups were evaluated: capsules alone (n = 10) and encapsulated cells (n = 10). An overnight post-hydration triplicate was also additionally evaluated in order to verify the capsule behavior and its absorption capability over an extended period.

Briefly, previously weighed capsules were immersed in culture medium at 37°C. Capsules were removed at regular intervals (hourly for up to 7 h), wiped to remove the excess of liquid content, and weighed in a semi-analytical scale (AUY220 scale 0.1 mg, Shimadzu Scientific Instruments Incorporated, Kyoto, Japan). After the measurement, capsules were placed again in the medium. The percentage of absorption was determined using the expression below, where M0 and Mt are the initial mass and the mass at time “t,” respectively. The measurements were performed in triplicate and mean values were obtained. S w e l l i n g   b e h a v i o r = ( M T − M 0 ) ( 100 × M 0 ) (1)

Moisture content (Xp%) was determined according to the Association of Official Analytical Chemists (Association of Official Analytical Chemists (AOAC), 2016), with adaptation to the capsule size. A portion of Xg = 5 capsules was maintained at 50°C for 3 h. The capsules were weighed and the procedure of dehydration was repeated till a constant weight was obtained. Since cells would not survive to these steps, Xp% was determined using only naïve capsules. The Xp% was obtained using the equation below, where “cw” means the constant weight obtained after dehydration. The tests were performed in triplicate. After dehydration, the capsules were re-hydrated and their final mass was assessed post-overnight. X p ( % ) = [ 5 − c w 5 ] x 100 (2)

Cell integrity was observed in a Confocal Laser Scanning Microscope (CLSM - TCS SP5, Leica), using the HeNe 633 nm laser wavelength to excite the Qtracker and the 405 nm laser for the 4′,6-diamidino-2-phenylindole (DAPI), to excite and detect the fluorescence of the encapsulated cell sample, according to the technique described by Santos et al. (2019). The whole capsules were placed in excavated slide blanks and observed by CLSM in 2 µm cuts in a 20x objective.

Cell immunophenotype of CD44 and MHC class II was observed by immunohistochemistry. In brief, 4 µm sections were obtained and stretched on histologically charged glass slides (Immunoslide, EasyPath, São Paulo, Brazil) and antigen retrieval was performed using citrate buffer (pH 6) in a pressure cooker (Pascal, Dako, Carpinteria CA, USA). Primary antibodies against CD44 (AbD Serotec, Kidlington, Oxford, UK) and MHC class II mouse anti-horse (AbD Serotec, Kidlington, Oxford, UK) were used. The primary concentration was 1:50 for both antibodies. The polymer detection system (Envision, Dako, USA) was used as the secondary antibody and the sample was incubated at 27°C for 1 h. The molecule 3,3′-diaminobenzidine (DAB) was used as a chromogen for 5 min and then the slides were immersed in Harris-Hematoxylin for 2 min for nuclei staining. Slides were observed in the Confocal Zeiss LSM 510 microscope. The entire section was evaluated to determine CD44 and MHC-II expression, considering either positive or negative labelling. All images were obtained by cameras attached to the microscope.

Cell migration and viability were assessed in triplicate at time points 0, 24, 48, 72 h and 7 days. Cell migration from the capsules to the plastic was determined by direct counting of attached cells, while concentration and viability of encapsulated cells per well were obtained after capsule dissolution in sodium citrate. In order to evaluate the expression of surface markers of AdMSCs after encapsulation, cells were submitted to immunophenotyping according to Carvalho et al. (2014) and Santos et al. (2018). Flow cytometry was performed 24 h and 7 days after encapsulation to assess the behavior of AdMSCs and possible phenotypic changes due to encapsulation. For that, capsules were dissolved using sodium citrate before incubation with the antibodies, and then analyzed by FACS Calibur cytometer (BD, San Jose, CA, USA), as described above.

Metabolic activity was assessed by colorimetric cytotoxicity assay using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) salt, adapted from Uludag and Sefton (1993). Evaluations were made at 0, 24, 72 h and 7 days. Analysis of MTT in adherent cells cultured in monolayers was used as control. In brief, on the day of analysis the conventional medium was replaced with 300 µl of MTT solution (5 mg/ml) and cells were incubated for 3 h at 37°C in the dark. After that, MTT was replaced with isopropyl alcohol to dissolve the crystals formed and the 96-well plate was read. Absorbance was measured at 570 nm using a BioTeK PowerWave XS2 microplate scanning spectrophotometer (Highland Park, Winooski, VT, USA), Gen5 software.

Experimental data are reported as mean ± SD. The SD was also used as an error bar in the figures. After the descriptive analysis of the data, they underwent a normality test and Dunn’s test was applied for multiple comparisons. The t-test or Mann-Whitney was used for double comparisons. Differences were considered statistically significant when p-value was <0.05. The software used for all analyses was GraphPad Prism v.7 (GraphPad, San Diego, CA, USA).

Encapsulation using a continuous infusion pump generated approximately 120 capsules/ml of solution dripped in calcium chloride.

Capsule observation through the magnifier showed higher cell concentration in the core of the capsule and lower concentration close to the hydrogel margins (Figure 1). Measurement of capsules was performed before and after immersion in toluidine blue solution. Mean initial perpendicular diameters of capsules (D1 and D2) were 1,459 and 1,596 μm, respectively. After immersion in 1:20 toluidine blue solution, the following perpendicular diameters were obtained: D1 = 2,610 ± 40 µm and D2: 2,660 ± 5 µm.

The structure of the microcapsules observed by SEM is shown in Figures 2A,B, 3. Different pore morphologies and measurements were observed, from circular morphology (100–165 µm) to irregular (20–45 µm).

Weight variation (%) of mass measurements from the swelling behavior test are shown in Figure 4. Although mass oscillation was more evident in the group with capsules and MSCs, there was a significant increase in mass of the group of capsules at 21 h compared to the initial time point (p < 0.05).

Reduction in moisture content was evident after the drying process (p < 0.0001) (90.75 ± 6.26%). Only the group of capsules without cells could be evaluated, since the temperature to which they were subjected aimed the evaluation of the biomaterial per se and not the cells, as observed in Figure 5. Capsules showed a good absorption potential after being immersed in culture medium overnight. However, capsules showed a structural modification under direct visualization, acquiring a softer aspect between 72 h and 7 days of culture.

The sample marked with the Qtracker and DAPI was observed by Confocal Laser Scanning Microscopy, showing cell membrane and nuclei integrity, confirming cell viability after encapsulation (Figure 6).

Encapsulated cells maintained mean viability of 93.57 ± 3.53% (Figure 7A). Cell migration from the scaffolds to the plastic bottom started after 48 h of evaluation, becoming more evident in 7 days with a significant increase of almost 10x compared to the two initial moments (p = 0.0434) (Figure 7B).

Despite the absence of statistical difference, MTT assay showed a decline of metabolic activity of encapsulated cells 24 h after encapsulation. Although there was an initial decrease in MTT, subsequent evaluations showed a recovery through time (Figure 8).

Immunophenotypic profile revealed negativity for CD11b, CD45 and MHC class II and positivity for CD90 at initial time point and after 7 days (Figure 9).

Immunohistochemistry labeling of the encapsulated cells did not show marking in the negative control (Figure 10A). Cells showed strong labeling for CD44 (Figure 10B) and no expression of MHC II (Figure 10C). Thus, cells were classified into (CD44 +++)/(MHC class II-).