Programmable microencapsulation for enhanced mesenchymal stem cell persistence and immunomodulation

Abstract

Mesenchymal stem cell (MSC) therapies demonstrate particular promise in ameliorating diseases of immune dysregulation but are hampered by short in vivo cell persistence and inconsistencies in phenotype. Here, we demonstrate that biomaterial encapsulation into alginate using a microfluidic device could substantially increase in vivo MSC persistence after intravenous (i.v.) injection. A combination of cell cluster formation and subsequent cross-linking with polylysine led to an increase in injected MSC half-life by more than an order of magnitude. These modifications extended persistence even in the presence of innate and adaptive immunity-mediated clearance. Licensing of encapsulated MSCs with inflammatory cytokine pretransplantation increased expression of immunomodulatory-associated genes, and licensed encapsulates promoted repopulation of recipient blood and bone marrow with allogeneic donor cells after sublethal irradiation by a ∼2-fold increase. The ability of microgel encapsulation to sustain MSC survival and increase overall immunomodulatory capacity may be applicable for improving MSC therapies in general.

Keywords: MSC; biomaterials; immune modulation; microfluidics; regenerative medicine.

Fig. 1.

Encapsulation of mMSCs into alginate and alginate–PDL–alginate (APA) microgels. (A) Schematic of encapsulation and subsequent treatments. Diameters (B) and viability (C) of mMSC-encapsulating microgels before and after cryopreservation (2-tailed Student’s t test or χ² test). (D) Encapsulated mMSCs after 2 d of culture. Green, alginate; blue, DAPI. (E) Distribution of cells per microgel at day 0 and day 2. (F) Fluorescent and bright-field overlay of encapsulated cell before and after PDL exposure. Green, alginate. (G) Microgel diameter after exposure to 5-kDa PDL as a function of PDL concentration in serum-free buffer, except where noted. (H) Half-life of fluorescent recovery of dextrans after photobleaching. (I) Encapsulated mMSCs viability with and without PDL treatment (χ² test). (J) Fraction of mMSCs that remained inside, produced extensions out of, or egressed from calcium-treated and APA encapsulates. Error bars show SD from 2 independent experiments (χ² test). *P < 0.05; **P < 0.01. N.S., not significant.

Fig. 2.

In vivo persistence of infused mMSCs with different encapsulation parameters. (A) Luminescent signal of bare and MAPA encapsulates in B6 recipient mice postinjection. Total radiance over time normalized to initial 2-h signal of (B) bare mMSCs (Bare cells), mMSCs encapsulated in alginate (Single cell enc), or mMSCs encapsulated in alginate followed by proliferation (Multicell enc); and (C) mMSCs encapsulated followed by APA (Single cell, APA), by proliferation and calcium (Multicell enc., Ca), and by proliferation and APA (MAPA). Normalized radiance of bare mMSCs included for comparison (faded gray). The colors in B and C match x-axis labels in D. (D) Half-life of signal from infused mMSCs. See SI Appendix, Methods. Bare cells, or microgels with single or multiple cells, were subject to a 1-way ANOVA followed by multiple comparisons (Inset). Other significant testing was conducted using a 2-way ANOVA with multiple comparisons on ranked observation or 2-way Mann–Whitney tests. (E) Half-life of primary bare cells and MAPA encapsulates. *P < 0.05; **P < 0.01. N.S., not significant.

Fig. 3.

Interaction of host immunity and infused mMSCs. (A) H&E (Left) and Masson’s trichrome (Right) stains of lungs explanted from recipient mice 2 wk after injection of mMSCs encapsulated in APA microgels. Fluorescent (Left) and transmitted light images (Right) of mMSC-encapsulating APA microgels explanted and stained for C3 protein (B) or CD68 (C) and DAPI. Green, alginate; red, C3; yellow, CD68. (D) Evans blue extravasation in pulmonary vasculature permeability. One-way ANOVA, Sidak’s multiple-comparison test. Half-life of signal from mice that had received infused mMSCs in Hepes buffer (E) or complete DMEM (F), with or without clondronate or CVF administration. (G) Half-life of mMSC in primed B6. mMSC were injected either bare, in multicellular encapsulates (multicell enc.), or as MAPA encapsulates. Two-tailed Student’s t test in E, Mann–Whitney test in F, and Kruskal–Wallis with multiple comparisons in G. *P < 0.05; **P < 0.01. N.S., not significant.

Fig. 4.

Effects of encapsulation and licensing on mMSC gene expression. Gene expression in MAPA mMSCs, normalized to mMSCs on tissue culture plastic (A) and in encapsulates without APA treatment (B), all without cytokine licensing. (C) Gene expression of licensed bare mMSCs versus licensed MAPA-encapsulated mMSCs, both normalized to unlicensed mMSCs grown on tissue culture plastic. (D) Gene expression of licensed multicellular encapsulates, normalized to unlicensed, compared with licensed MAPA encapsulates, normalized to unlicensed. (E) Gene expression of MAPA mMSCs licensed immediately after fabrication, normalized to unlicensed, compared with MAPA mMSCs licensed after cryopreservation, normalized to unlicensed. r² in C–E calculated from Pearson’s correlation of log transforms. Normality assessed using the D’Agostino–Pearson test. Gray line shows x = y for reference. *P < 0.05.

Fig. 5.

Effects of MAPA-mMSC on allogeneic engraftment. (A) Schema of competitive allogeneic transplant. All animals received a 1:1 mixture of congenic and allogeneic donor BM cells. (B) Representative flow cytometry plots from BM at 9 d. (C) Percent allogeneic cells in blood (C) and femur (D) in mice that had received donor BM cells with buffer and various MSC conditions. Student’s t test with Welch’s correction. Correlation between luminescent signal from licensed bare or MAPA mMSCs at 24 h and percent allogeneic cells in blood (E) and femur (F) at 9 d using the nonparametric Spearman test. The dashed line shows best-fit linear regression and was not used for statistical testing. (G) Percent allogeneic cells in femur in mice that had received donor BM cells with buffer; empty APA microgels; and licensed primary mMSC, bare or MAPA. One-way ANOVA with Sidak’s multiple comparisons. (H) Percent allogeneic cells in femur in mice that had received donor BM cells with buffer, licensed bare primary hMSCs, and licensed MAPA primary hMSCs. One-way ANOVA with Fisher’s least significance difference. *P < 0.05; **P < 0.01. N.S., not significant.