Article
Effects of cell culture conditions and passaging on the phenotype and proliferation rate of primary human osteoblasts
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Authors
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Nicole Dychus
- IfADo - Leibniz Research Centre for Working Environment and Human Factors, Department of Immunology, Neuroimmunology research group, Dortmund
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Elena Schwendich
- IfADo - Leibniz Research Centre for Working Environment and Human Factors, Department of Immunology, Neuroimmunology research group, Dortmund
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Anke Flegel
- IfADo - Leibniz Research Centre for Working Environment and Human Factors, Department of Immunology, Neuroimmunology research group, Dortmund
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Noureddine Bouchkhachakh
- Orthopädische Klinik Volmarstein, Abteilung für primäre Knie- und Hüftgelenksendoprothetik, Wetter (Ruhr)
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Helge Bast
- Orthopädische Klinik Volmarstein, Abteilung für primäre Knie- und Hüftgelenksendoprothetik, Wetter (Ruhr)
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Silvia Capellino
- IfADo - Leibniz Research Centre for Working Environment and Human Factors, Department of Immunology, Neuroimmunology research group, Dortmund
| Published: | September 9, 2020 |
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Outline
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Introduction: Isolation and cell culture of primary human osteoblasts is a technical challenge. Indeed, it is extremely difficult to cut the hard bone and isolate a sufficient amount of osteoblasts. Moreover, cell culture conditions of primary human osteoblasts are not yet well standardized. Due to these technical issues, many studies are based on osteoblasts differentiated from mesenchimal stem cells, but a well-established method for cell culture of primary human osteoblasts would be preferable for in vitro studies of diseases affecting bone matrix, such as rheumatoid arthritis and ankylosing spondylitis.
First aim of this study was to develop an efficient method for isolation of primary human osteoblasts from bone tissue. Furthermore, we aim to compare the phenotype of isolated cells at different passages and under different cell culture conditions, in order to find out the best protocol for in vitro culture of primary human osteoblasts.
Methods: Osteoblasts were isolated from the bone spongiosa of osteoarthritis patients undergoing knee replacement surgery. The study was approved by the ethical committee and all patients gave written consent. A “bone cutter” was designed and constructed at the workshop of the IfADo. Isolated cells were cultured for four passages and with three different culture media. Proliferation rate and mineralization assay were performed at each passage and culture condition. Real-time PCR for osteoblast marker expression are ongoing.
Results: Thank to the new developed “bone cutter”, we were able to isolate a big amount of cells and to create a cell bank for the study (n= 60). Our first results (n= 4) show no significant changes in cell proliferation during passaging, but a morphological change of the cells at passage 4 in some patients. The composition of cell culture medium strongly influences mineralization. Also, the amount of osteoblasts within the mixed cell population isolated from the bone can strongly vary within patients and should be taken into account.
Conclusion: In this study, we present a new protocol for isolation and standardized cell culture of human osteoblast. This protocol could be useful for in vitro study on many diseases, such as rheumatoid arthritis and ankylosing spondylitis.
Disclosures: We declare that there are no competing interests in relation to this work.
