Encapsulating non-human primate multipotent stromal cells in alginate via high voltage for cell-based therapies and cryopreservation

Abstract

Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15-30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application.

Figure 1. Schematic presentation of alginate high voltage encapsulation.

(A) Application of encapsulation of cells in alginate using high voltage (B) in cell-based therapy for immunoisolation, controllable drug release through semi-permeable membrane (SPM) and long-term storage of cells. Scale bar is 100 µm.

Figure 2. Characterization of amnion derived multipotent stromal cells.

(A) FACS analysis with characteristic markers CD105 and 73 (mean±SD, n = 4). (B–E) Immunohistochemistry stainings with MSC markers CD105, CD90, Bra and Snail1. (F) RT-PCR analysis displays presence of characteristic MSC markers CD90, ITGA6, GFRa1, CD73, CD105, ALCAMm CD44, whereas CD34 as marker for hematopoietic progenitors is absent (housekeeper RPS29). Scale bars are 50 µm.

Figure 3. Optimization of cell concentration and cell number.

(A) Effect of applied voltage, initial cell concentration and alginate-cell mixing procedure on diameter of alginate beads (mean±SD). (B) Shrinking of alginate in presence of 100 mM CaCl₂ depending on gelling time for different initial surface-to-volume ratio (S/V) and (C) calculation of encapsulation efficiency of MSCs in alginate beads. Shrinking rate was analyzed based on decrease in volume during gelling caused by water release for: MD – manual dropping of alginate solution, AF – using air-flow method and ES – using electro-spraying; dotted lines show the tendency of shrinking outside the studied cross-linking time. (C) Effect of initial cell concentration on number of encapsulated cells and respective photographs of alginate encapsulated MSCs at initial concentration of 1×10⁶ (C1), 5×10⁶ (C2) and 10×10⁶ (C3) cells/ml. The next process parameters were kept constant: both for AF and ES – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration from 1.2% to 1.6% (w/v); for AF – air flow 150 l/h; for ES – applied voltage 20 kV. The data are presented as a mean±SD (n = 3, N = 10). Scale bars are 200 µm.

Figure 4. Viability of encapsulated MSCs vs. applied voltage and incubation time.

Live-dead staining with CalceinAM (2 µM) and Ethidium Homodymer (4 µM). Note: beads obtained under 15 kV and using air flow (AF) had slightly bigger diameter as compared to the others. Viable cells stained in green, cells with massive membrane damages – in red. No visible difference in viability of encapsulated cells depending on applied voltage can be observed. The next process parameters were kept constant: both for AF and electro-spraying – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration 1.6% (w/v); for AF – air flow 150 l/h. Scale bar is 100 µm.

Figure 5. Effect of applied encapsulation methods and incubation on proliferation of MSCs post-encapsulation and cryopreservation.

Proliferation of MSCs: (A1) after encapsulation in alginate; (A2) after recovery post-encapsulation; (B1) cryopreserved in alginate; (B2) after recovery post-thawing. “Recovery” – incubation of cells in culture (37°C, 5% CO₂ in a humidified incubator) for 5 days after encapsulation and/or cryopreservation; “Immediate” – immediately after encapsulation; “Incubated” – after 24 h of incubation inside alginate beads in culture; AF – air flow; CF – frozen not encapsulated cells; CN – not encapsulated and not frozen MSCs. The next process parameters were kept constant during encapsulation: both for AF and electro-spraying – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration 1.6% (w/v); for AF – air flow 150 l/h. In B1–B2 freezing parameters included cooling rate 1 K/min down to −80°C and freezing medium with 10% DMSO and 10% FBS. The data are shown as a mean±SD (n = 3, N = 5). One-way ANOVA: NS – not significant (p = 0.05); *, **, *** - significantly different with p<0.05, p<0.01, p<0.001, respectively.

Figure 6. Effect of encapsulation method and period of incubation of MSCs inside beads on proliferation of cells.

MSCs were encapsulated in alginate beads using air flow (AF) and high voltage (15, 20, 25 kV) and incubated for 0, 1, 3, 5 and 7 days inside beads in culture (37°C, 5% CO₂ in a humidified incubator). Culture medium was replaced each second day to a fresh one. The next process parameters were kept constant: both for AF and electro-spraying – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration 1.6% (w/v); for AF – air flow 150 l/h. The data are shown as a mean±SD (n = 2, N = 5). One-way ANOVA: NS – not significant (p = 0.05); *** – significantly different (p<0.001).

Figure 7. Differentiation capacity of encapsulated MSCs.

MSCs were differentiated into adipogenic (Oil Red O) and osteogenic direction (Von Kossa) after encapsulation using air flow (AF) and high voltage (20 kV) followed by dilution of alginate with 55 mM sodium citrate for 5 min, or as not encapsulated MSCs. Cells expanded in regular culture medium represent the negative control (37°C, 5% CO₂ in a humidified incubator) and stained the same as positive cells of respective lineage. The next process parameters were kept constant: both for AF and electro-spraying (ES) – spraying distance 10 cm, concentration of gelling solution 100 mM CaCl₂, alginate flow rate 10 ml/h, and alginate concentration 1.6% (w/v); for AF – air flow 150 l/h; for ES – applied voltage 20 kV. Scale bar is 100 µm.