Expansion of human mesenchymal stromal cells from fresh bone marrow in a 3D scaffold-based system under direct perfusion.

Adam Papadimitropoulos,David Wendt,Andrea Barbero,Sophie Brachat,Elia Piccinini,Alessandra Braccini,Ivan Martin,Carsten Jacobi,Zoran Ivanovic

doi:10.1371/journal.pone.0102359

Adam Papadimitropoulos, David Wendt + Show 7 more

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https://doi.org/10.1371/journal.pone.0102359

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Abstract

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.

Highlights

Mesenchymal stromal/stem cell (MSC) are receiving an increasing experimental and clinical interest, owing to the large degree of plasticity and the capacity to modulate the immune system or the phenotype of cancer cells [1]
3D-perfusion expansion of freshly isolated MSC Using a bioreactor system as described in [26] and graphically illustrated in Figure 1, total BM cells were perfused through the scaffold pores for 5 days, followed by perfusion of culture medium for further 14 days
Based on the retrospectively calculated density of CFU-f from the five donors (0.08%60.02%) and assuming that all CFU-f attached to the ceramic scaffolds, an estimated average of 5.46103MSC were perfused through each scaffold, corresponding to 1.6 MSC per cm2 of ceramic surface area

Summary

Introduction

MSC are receiving an increasing experimental and clinical interest, owing to the large degree of plasticity and the capacity to modulate the immune system or the phenotype of cancer cells [1] Their use is advocated for treatment of various genetic, haematologic or immunologic pathologies and in the emerging field of regenerative medicine [2,3,4]. For most of these potential applications, given the low frequency among bone marrow nucleated cells (around 0.01%), MSC are typically expanded by sequential passages in monolayer (2D) cultures. Non-mesenchymal bone marrow cells were proposed to be involved in regulating MSC function [21] and have been demonstrated to enhance growth of MSC with clonogenic properties [22,23]

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