Efficacy of 3D Culture Priming is Maintained in Human Mesenchymal Stem Cells after Extensive Expansion of the Cells

Abstract

The use of non-optimal preparations of mesenchymal stem cells (MSCs), such as extensively expanded cells, might be necessary to obtain the large numbers of cells needed for many clinical applications. We previously demonstrated that minimally expanded (early passage) MSCs can be pre-activated as spheroids to produce potentially therapeutic factors in 3D cultures. Here, we used extensively expanded (late passage) MSCs and studied their 3D-culture activation potential. MSCs were culture-expanded as 2D monolayers, and cells from various passages were activated by 3D culture in hanging drops with either fetal bovine serum (FBS)-containing media or a more clinically-applicable animal product-free (xeno-free) media. Gene expression analyses demonstrated that MSC spheroids prepared from passage 3, 5, and 7 cells were similar to each other but different from 2D MSCs. Furthermore, the expression of notable anti-inflammatory/immune-modulatory factors cyclooxygenase-2 (PTGS2), TNF alpha induced protein 6 (TNFAIP6), and stanniocalcin 1 (STC-1) were up-regulated in all spheroid preparations. This was confirmed by the detection of secreted prostaglandin E2 (PGE-2), tumor necrosis factor-stimulated gene 6 (TSG-6, and STC-1. This study demonstrated that extensively expanded MSCs can be activated in 3D culture through spheroid formation in both FBS-containing and xeno-free media. This work highlights the possibility of activating otherwise less useable MSC preparations through 3D culture generating large numbers of potentially therapeutic MSCs.

Keywords: 3D; MSC; PGE2; TSG-6; anti-inflammatory; culture-expansion; immunomodulatory; passage; spheroid; xeno-free.

Figure 1

Mesenchymal stem cells (MSCs) produce up to 10²⁵ cells from a small bone marrow aspirate. MSCs from three different donors were cultured in low density and passaged every seven days. Cells were counted and seeded in a low density for the next passage. This was repeated until the cell number did not double during the seven days growth. (A) Cell growth as cumulative population doublings. (B) Cell growth as cumulative population expansion.

Figure 2

Gene expression is similar in spheroids generated from both extensively expanded and early passage MSCs. Spheroids were generated from passage 3, 5, and 7 MSCs and employed for gene expression microarrays. (A) Principal component analysis of the microarray data. Cube characters are spheroid MSCs from different passages and ball character is adherent monolayer MSCs. (B) Hierarchical clustering of the differentially expressed genes between adherent monolayer MSCs and spheroid MSCs from different passages. Red color indicates a high gene expression, and blue color indicates a low gene expression. Scale demonstrates log₂ gene expression value. Abbreviations: Adh, adherent monolayer MSCs; PCA1, principal component 1; P3, passage 3; Sph, spheroid MSCs.

Figure 3

Spheroids generated from extensively expanded MSCs maintain high expression of potentially therapeutic molecules. Gene expression data were searched for differentially expressed genes between adherent monolayer MSCs and spheroids from various passages. Furthermore, the expression of potentially therapeutic genes and IL-1 signaling molecules were queried from the microarray data. (A) Venn diagram of differentially expressed genes. (B) Expression of potentially therapeutic genes in spheroid MSCs. (C) Expression of IL-1 signaling related genes in spheroid MSCs. Abbreviations: Adh, adherent monolayer MSCs; P3, passage 3; Sph, spheroid MSCs.

Figure 4

Spheroids generated from extensively expanded MSCs secrete high amounts of prostaglandin E2 (PGE2). MSCs from various passages (3, 5, and 7) were cultured as spheroids and as very high density monolayers in fetal bovine serum (FBS)-containing and xeno-free media. The conditioned medium was harvested for PGE2 ELISA. Spheroids were also dissociated and the resulting cells were tested for their ability to maintain PGE2 secretion. (A) PGE2 secretion by spheroids and very high density monolayer MSCs in FBS-containing media and xeno-free media at different passages. (B) PGE2 secretion by spheroid-derived cells from FBS-containing media and xeno-free media at different passages. Abbreviations: Adh VH, adherent very high density monolayer MSCs; CCM, complete culture medium; P3, passage 3; Sph, spheroid MSCs; Sph DC, spheroid-derived MSCs; StemP HSA, StemPro xeno-free media with human serum albumin.

Figure 5

Spheroids generated from extensively expanded MSCs secrete high amounts of tumor necrosis factor-stimulated gene 6 (TSG-6). MSCs from various passages (3, 5, and 7) were cultured as spheroids and as very high density monolayers in FBS-containing and xeno-free media. The conditioned medium was harvested for TSG-6 ELISA. Spheroids were also dissociated and the resulting cells were tested for their ability to maintain TSG-6 secretion. (A) TSG-6 secretion by spheroids and very high density monolayer MSCs in FBS-containing media and xeno-free media at different passages. (B) TSG-6 secretion by spheroid-derived cells from FBS-containing media and xeno-free media at different passages. Abbreviations: Adh VH, adherent very high density monolayer MSCs; CCM, complete culture medium; P3, passage 3; Sph, spheroid MSCs; Sph DC, spheroid-derived MSCs; StemP HSA, StemPro xeno-free media with human serum albumin.

Figure 6

Spheroids generated from extensively expanded MSCs secrete high amounts of stanniocalcin 1 (STC-1). MSCs from various passages (3, 5, and 7) were cultured as spheroids and as very high density monolayers in FBS-containing and xeno-free media. The conditioned medium was harvested for STC-1 ELISA. Spheroids were also dissociated and the resulting cells were tested for their ability to maintain STC-1 secretion. (A) STC-1 secretion by spheroids and very high density monolayer MSCs in FBS-containing media and xeno-free media at different passages. (B) STC-1 secretion by spheroid-derived cells from FBS-containing media and xeno-free media at different passages. Abbreviations: Adh VH, adherent very high density monolayer MSCs; CCM, complete culture medium; P3, passage 3; Sph, spheroid MSCs; Sph DC, spheroid-derived MSCs; StemP HSA, StemPro xeno-free media with human serum albumin.

Figure 7

Spheroids generated from extensively expanded MSCs exhibit anti-inflammatory properties. MSCs from various passages (3, 5, and 7) were cultured as spheroids and as very high density monolayers in FBS-containing and xeno-free media. Conditioned media from these cultures were applied on lipopolysaccharide (LPS)-stimulated macrophages and anti-inflammatory effects were studied. (A) Conditioned media effects on TNFα secretion by LPS-stimulated macrophages. (B) Conditioned media effects on IL-10 secretion by LPS-stimulated macrophages. Compared to the appropriate media control (i.e., CCM or StemP HSA). Statistical significance was defined as ns, p ≥ 0.05; and ***, p < 0.001, Abbreviations: Adh VH, adherent very high density monolayer MSCs; CCM, complete culture medium; Ctrl, control unstimulated macrophages; P3, passage 3; Sph, spheroid MSCs; StemP HSA, StemPro xeno-free media with human serum albumin.