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Porcine Mesenchymal Stem Cell Derivation by Plating Bone Marrow Cells Directly and After Erythrocyte Lysis

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Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

Porcine mesenchymal stem cells (pMSC) have high demand for research, transgenic animal production and as alternate cells for human regenerative therapy. Bone marrow is the most popular source of MSC; however, it contains huge number of erythrocytes. Erythrocytes from bone marrow cells are selectively removed using the plastic adherence property of MSC either by plating directly or after enrichment by differential density centrifugation and erythrocyte lysis. Enrichment of MSC by differential density centrifugation has disadvantages. However, the relative advantage of direct plating vs plating after erythrocyte lysis is not known. Therefore the present experiment was designed to understand the derivation efficiency of pMSC by direct plating and plating after erythrocyte lysis. As a proof-of-concept, pig bone marrow cells were processed for derivation by direct plating and after erythrocyte lysis under uniform culture condition and media formulations. The time taken for confluence in the first passage, cell morphology, presence of pMSC marker genes and the lineage specific differentiation potential at passage 5 were tested in both the groups. The results indicated that the cells adhered similarly in both the methods, reached 80% confluence in about 10 days, appeared with same morphology, with no difference in quality and expression of pMSC markers at passage 5, maintaining similar differentiation potential to lineage specific osteogenic, adipogenic and chondrogenic cells. In conclusion, erythrocyte lysis did not give advantages over non lysis, it is therefore recommended to seed the bone marrow cells directly for pMSC derivation which is easy, fast and inexpensive.

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Acknowledgements

Funding of DST-WOSA to the first author, facilities extended by the Director ICAR-NIANP and the support and help of the colleagues and contractual workers of other projects is thankfully acknowledged.

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Authors and Affiliations

  1. Molecular Biology Laboratory, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore 560 030, Karnataka, India

    Sangeetha Kannan & Jyotirmoy Ghosh

  2. Stem Cell Laboratory, Division of Animal Biotechnology, ICAR-Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, 243 122, India

    Sujoy K. Dhara

Authors
  1. Sangeetha Kannan
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  2. Sujoy K. Dhara
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Correspondence to Jyotirmoy Ghosh.

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Significance statement Study proved that plating bone marrow cells directly to plastic surface is best for MSC derivation in porcine being easy, straightforward, fast and inexpensive method compared to the plating after RBC lysis. Method is also suitable for other species.

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Kannan, S., Dhara, S.K. & Ghosh, J. Porcine Mesenchymal Stem Cell Derivation by Plating Bone Marrow Cells Directly and After Erythrocyte Lysis. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 89, 565–573 (2019). https://doi.org/10.1007/s40011-018-0966-0

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