Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions

Abstract

Background aims: Human mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations.

Methods: MSC aggregation and sphere formation was studied with the use of hanging drop cultures with medium containing fetal bovine serum or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with the use of gene expression and protein secretion measurements and with functional studies with the use of macrophages and cancer cells.

Results: MSCs did not condense into tight spheroids and express a full complement of therapeutic genes in α-minimum essential medium or several commercial stem-cell media. However, we identified a chemically defined xeno-free media, which, when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (prostaglandin E2, TSG-6 TNF-alpha induced gene/protein 6) and anti-cancer molecules (TRAIL TNF-related apoptosis-inducing ligand, interleukin-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage system and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death.

Conclusions: We demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed in the present study for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications.

Keywords: 3D; MSC; pre-activation; sphere; stem cell; xeno-free.

Figure 1. Effects of medium on sphere formation by MSCs in hanging drops

(A) Images of MSC aggregates/spheres formed in 3 day hanging drop cultures using various media formulations. Scale bar 200 µm. (B) Sphere-derived cell viability after enzymatic dissociation of the aggregates. Values are mean ± SD (n = 3). (C) Sphere-derived cell yield. Values are mean ± SD (n = 4). (D) Sphere-derived cell diameter measured by microscopy. Individual cell diameters are shown with mean (n = 40–120). (E) Sphere-derived cell diameter measured by flow cytometry. MSC sizes were estimated from forward scatter properties of the viable population (calceinAM⁺/7AAD⁻) relative to beads with known diameter (6, 10, and 15 µm). Values are mean ± SD (n = 4). ns, p ≥ .05; **, p < .01; ***, p < .001 compared to sphere-derived cell viability from CCM spheres in (B), compared to yield from CCM spheres in (C), and compared to cell diameter of adherent monolayer MSCs in (D). Abbreviations: Adh, adherent monolayer MSCs; CCM, complete culture medium; HSA, human serum albumin; MEMα, α-minimum essential medium; MesenC, Mesen Cult XF medium; MSCGM, MSC growth medium; Sph, sphere MSCs; StemP, StemPro XF medium.

Figure 2. Effects of medium on anti-inflammatory properties of MSC spheres

(A) Secretion of PGE2 by MSC spheres after 3 day culture in hanging drops in CCM or in chemically defined xeno-free conditions. (B,C) Anti-inflammatory effects of MSC sphere conditioned medium using various chemically defined xeno-free medium preparations measured as reduction in TNFα and increase in IL-10 secretion by LPS-stimulated macrophages. Values are mean ± SD (n = 4). ns, p ≥ .05; *, p < .05; **, p <.01; ***, p < .001 compared to conditioned medium from spheres cultured in CCM in (A) and compared to stimulated macrophage control in (B) and (C). Abbreviations: CCM, complete culture medium; CM, conditioned medium; HSA, human serum albumin; MEMα, α-minimum essential medium; MesenC, Mesen Cult XF medium; MSCGM, MSC growth medium; MΦ, macrophage; sMΦ, stimulated macrophage; Sph, sphere MSCs; StemP, StemPro XF medium.

Figure 3. Continuous secretion of anti-inflammatory factors PGE2 and TSG-6 by MSC spheres from chemically defined xeno-free cultures

(A,B) Secretion of PGE2 and TSG-6 by MSC spheres from chemically defined xeno-free conditions after 6 h transfer into low-protein medium. (C,D) Anti-inflammatory effects of low-protein medium conditioned by MSC spheres from chemically defined xeno-free hanging drop cultures measured as reduction in TNFα and increase in IL-10 secretion by LPS-stimulated macrophages. Values are mean ± SD (n = 4). ns, p ≥ .05; **, p < .01; ***, p < .001 compared to 6 h transfer conditioned medium from spheres cultured in CCM in (A) and (B) and compared to stimulated macrophage control in (C) and (D). Abbreviations: CCM, complete culture medium; CM, conditioned medium; Ctrl, macrophage only controls; HSA, human serum albumin; MEMα, α-minimum essential medium; MesenC, Mesen Cult XF medium; MSCGM, MSC growth medium; MΦ, macrophage; sMΦ, stimulated macrophage; Sph, sphere MSCs; StemP, StemPro XF medium.

Figure 4. HSA dose effect on activation of MSCs in chemically defined 3D xeno-free cultures

(A–C) Real-time PCR data of TRAIL, IL-24, and TSG-6 expression in MSC spheres cultured with various doses of HSA (1×, 3×, 6×, 9×) in chemically defined StemP xeno-free medium. Relative quantities were determined comparing to adherent monolayer MSCs cultured in CCM. (D) PGE2 secretion by MSC spheres formed in StemP medium supplemented with various HSA doses. (E,F) Anti-inflammatory effects of sphere conditioned medium from 3D cultures with StemP medium supplemented with various HSA doses measured as reduction in TNFα and increase in IL-10 secretion by LPS-stimulated macrophages. Values are mean ± SD (n = 4). ns, p ≥ .05; **, p < .01; ***, p < .001 compared to MSC spheres cultured in CCM in (A), (B), and (C), compared to conditioned medium from MSC spheres cultured in CCM in (D), and compared to stimulated macrophage control in (E) and (F). Abbreviations: Adh, adherent monolayer MSCs; CCM, complete culture medium; CM, conditioned medium; Ctrl, macrophage only controls; HSA, human serum albumin; MΦ, macrophage; RQ, relative quantity; sMΦ, stimulated macrophage; Sph, sphere MSCs; StemP, StemPro XF medium.

Figure 5. Comparison of different HSA preparations in activating MSCs in chemically defined 3D xeno-free cultures

(A–C) Real-time PCR data of TRAIL, IL-24, and TSG-6 expression with StemP medium supplemented with different HSA preparations. Relative quantities were determined comparing to adherent monolayer MSCs. (D) PGE2 secretion by MSC spheres formed in StemP medium supplemented with various HSA preparations. (E,F) Anti-inflammatory effects of sphere conditioned medium from 3D cultures with StemP medium supplemented with various HSA preparations measured as reduction in TNFα and increase in IL-10 secretion by LPS-stimulated macrophages. Values are mean ± SD (n = 3–4). ns, p ≥ .05; *, p < .05; ***, p < .001 compared to MSC spheres cultured in CCM in (A), (B), and (C), compared to conditioned medium from MSC spheres cultured in CCM in (D), and compared to stimulated macrophage control in (E) and (F). Abbreviations: Adh, adherent monolayer MSCs; CCM, complete culture medium; CM, conditioned medium; Ctrl, macrophage only controls; HSA (cli), clinical grade human serum albumin isolated from blood; HSA (res), research grade human serum albumin isolated from blood; rHSA (r), recombinant human serum albumin expressed in rice; rHSA (y), recombinant human serum albumin expressed in yeast; MΦ, macrophage; sMΦ, stimulated macrophage; Sph, sphere MSCs; StemP, StemPro XF medium.

Figure 6. Reduction of prostate cancer cell growth by MSC spheres prepared in chemically defined xeno-free conditions

(A) Images of LNCaP prostate cancer cells treated with various non-conditioned and conditioned medium preparations. Cells decreased in size and developed long and thin extensions in the conditioned medium from spheroids. Scale bar 200 µm (inset 100 µm). (B.C) MSC sphere conditioned medium reduces the growth of LNCaP cells measured with CyQUANT assay and manual cell counts after 3-day culture. Initial cell number is shown with a dashed line. (D) Sphere conditioned medium treatment of LNCaP cells results in cell cycle arrest with decrease in cells in S phase. (E) Sphere conditioned medium treatment causes LNCaP cell death with increase in both apoptotic and necrotic cells. Values are mean ± SD (n = 3–4). **, p < .01; ***, p < .001 compared to appropriate medium controls and to each other in (B,C, and D). Abbreviations: Anx, Annexin V; CCM, complete culture medium; CM, conditioned medium; Ctrl, non-conditioned medium controls; HSA, human serum albumin; PI, propidium iodide; RPMI, cancer cell medium control; Sph, sphere MSCs; StemP, StemPro XF medium.