Phenotypic and functional characterization of human bone marrow stromal cells in hollow-fibre bioreactors

Abstract

The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fibre bioreactor devices in which the cells, separated by a semipermeable polyethersulphone (PES) membrane, can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibres. The cells also preserved their immunophenotype under physiological fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per-cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow-fibre bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells.

Figure 1. BMSC adhere to polyethersulfone hollow fibers and remain viable after long-term culture

Fluorescent micrographs of BMSC adherence and morphology on PES hollow fibers after (A) 24 hours, (B) 48 hours, and (C) 6 days. Cells were stained with Calcein AM for viability. Scanning electron microscopic images show (D) BMSCs with typical fibroblastoid morphology within the constructs of a (E) complex three-dimensional environment.

Figure 2. BMSCs are metabolically active during device operation

(A) Schematic of a recycling flow circuit with an intracapillary flow rate of 200 mL/min. and extracapillary flow rate of 10 mL/min. BMSCs are seeded on the extraluminal space. (B) Photograph of multi-device operation in vitro. (C) Glucose consumption and lactate production over time of perfusion show cells maintain viability throughout a 48-hour time course in a bioreactor under flow. N=3.

Figure 3. BMSCs retain their identity and differentiation potential after bioreactor operation

(A) Schematic of methods used to isolate and analyze BMSCs after device perfusion. After two days of operation, cells were harvested by trypsin and were analyzed by flow cytometry or differentiation assays thereafter. (B) Histograms of classical immunomarkers used to identify BMSCs. A phenotype of CD44⁺, CD45⁻, CD11b⁻, CD73⁺ surface expression is preserved after a 48-hour bioreactor perfusion. Analysis was performed on a device seeded with 200 × 10⁶ cells exposed to an ultrafiltrate flow rate of 10 mL/min. Black histogram is a stained sample and grey histogram is isotype control. N=2 (C) Microscopic images of Alizarin Red staining of BMSCs directly after perfusion. Negative staining confirms that cells taken directly from the device showed no signs of osteogenic differentiation. (D) Alizarin Red stain of BMSCs isolated from the device and differentiated for three weeks in osteoinductive medium demonstrating BMSCs still retain the potential to become an osteoblastic lineage after device operation. N=2.

Figure 4. BMSCs secrete two well-known factors in a bioreactor

ELISA results of (A) Interleukin-6 (IL-6) and (B) Vascular Endothelial Growth Factor (VEGF) during a 48-hour bioreactor perfusion. Secreted factors were measured in the reservoir of the bioreactor circuit from 200 × 10⁶ cells exposed to an extraluminal flow rate of 10 mL/min. The results are compared to BMSCs seeded in 2D tissue culture plates; *p < 0.05. All data is normalized to cell mass. N=3.

Figure 5. BMSC bioreactor effluents have enhanced anti-inflammatory properties

Upregulation of IL-10 in peripheral blood mononuclear cells (PBMCs) after exposure to unconcentrated medium harvested from BMSCs cultured in 2D flasks or 3D bioreactors. PBMCs were incubated in the medium for 18 hours, after which, cells were stimulate with LPS for 5 hours. Supernatant was collected and measured for IL-10 through ELISA; *p < 0.05, **p < 0.001. Baseline results represent PBMCs incubated with unconditioned medium. N=3.