Expansion of Human Mesenchymal Stromal Cells from Fresh Bone Marrow in a 3D Scaffold-Based System under Direct Perfusion

<div><p>Mesenchymal stromal/stem cell (MSC) expansion in conventional monolayer culture on plastic dishes (2D) leads to progressive loss of functionality and thus challenges fundamental studies on the physiology of skeletal progenitors, as well as translational applications for cellular therapy and molecular medicine. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor system. The 3D-perfusion system generated a stromal tissue that could be enzymatically treated to yield CD45- MSC. As compared to 2D-expanded MSC (control), those derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7–8 doublings) better maintained their progenitor properties, as assessed by a 4.3-fold higher clonogenicity and the superior differentiation capacity towards all typical mesenchymal lineages. Transcriptomic analysis of MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability and a significant upregulation of multipotency-related gene clusters following 3D-perfusion- as compared to 2D-expansion. Interestingly, the differences in functionality and transcriptomics between MSC expanded in 2D or under 3D-perfusion were only partially captured by cytofluorimetric analysis using conventional surface markers. The described system offers a multidisciplinary approach to study how factors of a 3D engineered niche regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems.</p></div

Schematic overview of the experimental setup.

<p>Bone marrow aspirates were seeded into the 3D perfusion system and in conventional Petri dishes. After culture, cells from both systems were enzymatically retrieved and CD45- sorted cells using magnetic beads were analyzed as described.</p

Analysis of the expression of surface markers in 2D and 3D cultured MSC.

<p>Colored lines display the frequency of positive cells compared to isotype (gray lines). Most markers were similarly expressed in the two experimental groups. CD90, CD105, CD166, and ALP positive populations were more represented in monolayer culture, while CD146 and SSEA-1 were more represented in 3D-perfusion culture.</p

Phenotypical and growth characteristics for 2D and 3D perfused MSC.

<p>(a) Scanning electron microscopy imaging of cells within the scaffold display a complex network of branched fibroblastic-like adherent cells and the presence of rounded cells possibly of hematopoietic origin. (b) 2D cultured MSC display a typical flat fibroblastic morphology. (c) Flow cytometry of cultured cells shows a higher frequency of CD45+ cells in the perfusion system. (d) Proliferation rates indicate higher proliferation in 2D as compared to 3D perfusion cultured MSC. Statistically significant differences (P<0.05) are indicated with an asterisk (*; n = 5).</p

Functional differences between 2D and 3D perfused MSC.

<p>Higher (a) frequency of clonogenic cells and (b) differentiation capacity for osteogenic, adipogenic, and chondrogenic lineages with the associated quantifications of 3D perfusion- as compared to 2D-expanded cells. Scale bar: 50 um. Statistically significant differences (P<0.05) are indicated with an asterisk (*; n = 3).</p

Skeletal muscle Beclin and ATG5 protein levels in patients with or without >10% weight loss (Group II).

<p>Western blot analysis with indicated antibodies, α-tubulin was used as a loading control. Graph shows the mean (± SEM) protein level represented in arbitrary units (A.U). (*, P<0.05 and **, P<0.01, by Student's t test).</p

Inflammatory pathways in patients with (CRP >10 mg/L) and without (CRP ≤10 mg/L) systemic inflammation.

<p>(A) Western blot analysis in the presence or absence of systemic inflammation with indicated antibodies, α-tubulin was used as a loading control. (B) Graph shows the mean (± SEM) protein level of phospho-NF-κB, represented in arbitrary units (A.U). (C) Representative immunohistochemistry and nuclei count of phospho-STAT3 (area shown is representative of field) of a patient with or without systemic inflammation. (D) Graph shows the staining density of phospho-STAT3 nuclei (A.U.) (± SEM) in the presence or absence of systemic inflammation.</p

Variations in protein and nucleic acid content according to the different definitions of cancer cachexia.

<p>A comparison is made between patients with the proposed cachexia definition absent (dark grey) and those with the proposed cachexia definition present (light grey) for the four definitions set out in the methods (I–IV). (A) Mean (± SEM) wet weight protein content. (B) Mean (± SEM) RNA content. (C) Mean (± SEM) DNA content. (C) Mean (± SEM) RNA/DNA ratio. (*, P<0.05 by Student's t test).</p

Demographic data of the patients involved in the study.

<p>Data (except gender split) are presented as mean (SEM).</p><p> = cachexia group significantly different from the non-cachexia group, (p<0.05 by Student's t test).</p><p>Abbreviations – CRP = C - reactive protein, BMI = Body Mass Index.</p

Total SMAD3, phospho-SMAD3 and ratio of phospho-SMAD3/SMAD3 in patients with or without >5% WL (Group I) levels.

<p>Western blot analysis with indicated antibodies, α-tubulin was used as a loading control. Graph shows the mean (± SEM) protein level represented in arbitrary units (A.U). (*, P<0.05 by Student's t test).</p