University of Natural Resources and Life Sciences, Vienna Department of Biotechnology

Universität für Bodenkultur Wien Department für Biotechnologie

INTRODUCTION

MSC were isolated from umbilical cord by explant method approach under xeno-free conditions.1

Anne Neumann1,2, Antonina Lavrentieva2, Dominik Egger2, Tim Hatlapatka1, Cornelia Kasper1 1 Department for Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria, anne.neumann@boku.ac.at 2 Institute for Technical Chemistry, Leibniz University of Hanover, Callinstraße 5, 30167 Hanover, Germany

Refenences Hatlapatka and Moretti et al., Adv. Biomedical Engin/Biotechol (2010), 123, 29-45 Lavrentieva, Dissertation, Institute for Technical Chemistry, Leibniz University of Hanover, 2012 Acknowledgement: The authors would like to thank the mechanical workshop of the Institute for Technical Chemistry for the excellent technical support.

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Normoxia (21% O2) Hypoxia (5% O2)

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A main challenge in cell therapies and other tissue regeneration approaches is to produce a therapeutically significant cell number. For expansion of mesenchymal stem cells (MSC) the cultivation on 2D plastic surfaces is still the conventional procedure, even though the culture conditions differ significantly from the 3D environment in vivo. Additionally, static amplification of MSC is a labour-intensive procedure. We therefore used a specialized rotating bed bioreactor in order to maximize ex vivo expansion of MSC. Furthermore the bioreactor system was designed to enable integration of sensors for online monitoring of various parameters (e.g. pH, pO2, pCO2) and hence, allow ensured cultivation under well controlled and reproducible conditions. Beside cell expansion, directed differentiation of MSC was also achieved in bioreactors. MSC lack the ability to grow in 3D direction and build functional tissue in vitro. Thus, it is necessary to seed and culture MSC on 3D matrices to obtain functional implants. For guided differentiation towards the osteogenic lineage, MSC were cultivated on ceramic porous matrices under dynamic conditions. Custom-made miniaturized perfusion bioreactors for parallel testing were designed, characterized and optimized for that purpose.

CONCLUSION

CELL ISOLATION CELL EXPANSION

MSC were thereafter expanded under dynamic conditions in a novel rotating bed bioreactor, and subsequently examined regarding their proliferations capacity, senescence and differentiation potential.2

MSC were seeded onto 3D ceramic matrices and cultivated under dynamic conditions using miniaturized perfusion bioreactors.

Approaches for Automized Expansion and Differentiation of human MSC in specialized bioreactors

MSC could be successfully isolated from human umbilical cord tissue. MSC expansion in the rotation bed bioreactor provides a high number of cells, maintaining their stem cell properties such as specific surface markers, proliferation capacity and differentiation potential (data not shown). Cultivation of MSC in perfusion bioreactors have been shown to support and improve osteogenic differentiation as mechanical plays an important role in directing MSC fate. The influence of various osteo-supportive parameters will be part of further studies, using the mini-perfusion bioreactor systems, allowing to achieve a statistically relevant number of experiments at the same time und der well controled and reproducible conditions. Our results support the argument that the application of tailor-made bioreactors are an essential step toward the production of stem cell based therapeutics and tissue engineering products.

Marker Expression, %

CD31 0.0 %

CD34 0.3 %

CD44 98.3 %

CD45 0.3 %

CD73 99.9 %

CD90 98.1 %

CD105 99.8 %

Fig. 1: Isolation process of MSC from umbilical cord tissue A) umbilical cord in buffer B) cut in tissue pieces (blood removed) C) filled in T-flascs D) MSC grow out of tissue in 14 days of culture in αMEM supplied with 15% human serum.

Fig. 2: Rotation bed bioreactor A) set up in breeder B) reactor inlay containing cell culture plastic slides C) MSC after 5 days of dynamic cultivation D) no expression of senescence-associated β-galactosidase of MSC after dynamic cultivation.

Fig. 4: A) Incubator for parallel bioreactor setup and monitoring B) mini-perfusion bioreactors C/D) MSC on macroporous ceramic scaffold after dyn. cultivation in perfusion bioreactor (C) DAPI and D) Alizarine Red and Von Kossa staining.

DIRECTED DIFFERENTIATION

CONTROL

PERFUSION

A B C D

Fig. 3: A) Glucose consumption of MSC in rotation bed bioreactor B) key parameters and results form rotation bed bioreactor cultivation (n=3) C) MSC surface markers after 5 days dynamic cultivation.

Seeding 3 x 106 cells

Yield 24.6 ± 2.4 x 106 cells

Amplification 8.2 ± 0.8

Population 39.6 ± 1.8 h doubling time A B C

A B C D