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5 Publications visible to you, out of a total of 5
Evaluation of GMP-compliant culture media for in vitro expansion of human bone marrow mesenchymal stromal cells.

Abstract (Expand)

Mesenchymal stromal cells (MSCs) from human bone marrow serve as a resource for cell-based therapies in regenerative medicine. Clinical applications require standardized protocols according to good … manufacturing practice (GMP) guidelines. Donor variability as well as the intrinsic heterogeneity of MSC populations must be taken into consideration. The composition of the culture medium is a key factor in successful MSC expansion. The aim of this study was to comparatively assess the efficiency of xeno-free human platelet lysate (HPL)-based cell expansion with two commercially available media-StemPro MSC SFM CTS (for human ex vivo tissue and cell culture processing applications) and MSCGM (non-GMP-compliant, for research only)-in an academic setting as the first optimization step toward GMP-compliant manufacturing. We report the feasibility of MSC expansion up to the yielded cell number with all three media. MSCs exhibited the typical fibroblastoid morphology, with distinct differences in cell size depending on the medium. The differentiation capacity and characteristic immunophenotype were confirmed for all MSC populations. Proliferation was highest using StemPro MSC SFM CTS, whereas HPL medium was more cost-effective and its composition could be adjusted individually according to the respective needs. In summary, we present a comprehensive evaluation of GMP-compatible culture media for MSC expansion. Both StemPro and HPL medium proved to be suitable for clinical application and allowed sufficient cell proliferation. Specific differences were observed and should be considered according to the intended use. This study provides a detailed cost analysis and tools that may be helpful for the establishment of GMP-compliant MSC expansion.

Authors: P. Wuchter, M. Vetter, R. Saffrich, A. Diehlmann, K. Bieback, A. D. Ho, P. Horn

Date Published: 26th Feb 2016

Publication Type: Journal

Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells.

Abstract (Expand)

In previous studies human mesenchymal stromal cells (MSCs) maintained the "stemness" of human hematopoietic progenitor cells (HPCs) through direct cell-cell contact in two-dimensional co-culture … systems. We establish a three-dimensional (3D) co-culture system based on a custom-made chip, the 3(D)-KITChip, as an in vitro model system of the human hematopoietic stem cell niche. This array of up to 625 microcavities, with 300 mum size in each orientation, was inserted into a microfluidic bioreactor. The microcavities of the 3(D)-KITChip were inoculated with human bone marrow MSCs together with umbilical cord blood HPCs. MSCs used the microcavities as a scaffold to build a complex 3D mesh. HPCs were distributed three-dimensionally inside this MSC network and formed ss-catenin- and N-cadherin-based intercellular junctions to the surrounding MSCs. Using RT(2)-PCR and western blots, we demonstrate that a proportion of HPCs maintained the expression of CD34 throughout a culture period of 14 days. In colony-forming unit assays, the hematopoietic stem cell plasticity remained similar after 14 days of bioreactor co-culture, whereas monolayer co-cultures showed increasing signs of HPC differentiation and loss of stemness. These data support the notion that the 3D microenvironment created within the microcavity array preserves vital stem cell functions of HPCs more efficiently than conventional co-culture systems.

Authors: P. Wuchter, R. Saffrich, S. Giselbrecht, C. Nies, H. Lorig, S. Kolb, A. D. Ho, E. Gottwald

Date Published: 3rd Feb 2016

Publication Type: Journal

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

Abstract (Expand)

BACKGROUND AIMS: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different … MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Authors: P. Wuchter, K. Bieback, H. Schrezenmeier, M. Bornhauser, L. P. Muller, H. Bonig, W. Wagner, R. Meisel, P. Pavel, T. Tonn, P. Lang, I. Muller, M. Renner, G. Malcherek, R. Saffrich, E. C. Buss, P. Horn, M. Rojewski, A. Schmitt, A. D. Ho, R. Sanzenbacher, M. Schmitt

Date Published: 27th May 2014

Publication Type: Journal

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells.

Abstract (Expand)

The interaction between the stromal cell-derived factor-1 alpha (SDF-1alpha, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The … CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1alpha axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1alpha levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1alpha significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1alpha interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1alpha gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1alpha axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.

Authors: P. Wuchter, C. Leinweber, R. Saffrich, M. Hanke, V. Eckstein, A. D. Ho, M. Grunze, A. Rosenhahn

Date Published: 17th Dec 2013

Publication Type: Journal

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor.

Abstract (Expand)

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C … chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1alpha (SDF-1alpha) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro. METHODS: We monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor. RESULTS: Co-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34(+) cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations-and conversely their hematopoiesis supportive functions-may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor. CONCLUSIONS: These results support the notion that the CXCR4/SDF-1alpha axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.

Authors: A. Ludwig, R. Saffrich, V. Eckstein, T. Bruckner, W. Wagner, A. D. Ho, P. Wuchter

Date Published: 15th Oct 2013

Publication Type: Journal

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