GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC

PLOS ONE, Dec 2019

Background Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. Methods and Findings We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. Conclusions The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043255&type=printable

GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC

et al. (2012) GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC. PLoS ONE 7(8): e43255. doi:10.1371/journal.pone.0043255 GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC Natalie Fekete 0 Markus T. Rojewski 0 Daniel Fu rst 0 Ludwika Kreja 0 Anita Ignatius 0 Julia Dausend 0 Hubert Schrezenmeier 0 Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, United States of America 0 1 Institut f u r Transfusionsmedizin, Universita tsklinikum Ulm , Ulm, Germany , 2 Institut f u r klinische Transfusionsmedizin und Immungenetik , Ulm, Ulm, Germany , 3 Institut fu r Unfallchirurgische Forschung und Biomechanik , Ulm , Germany Background: Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. Methods and Findings: We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. Conclusions: The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion. - Funding: This work was supported by grants from the 7th Framework Program of the European Commission: CASCADE (Cultivated Adult Stem Cells as Alternative for Damaged Tissue) (number 223236; HEALTH-F5-2009-223236) and REBORNE (Regenerating BOne defects using New biomedical Engineering approaches) (number HEALTH- 2009-1.4.2-241879), and the University Centre of Musculoskelettal Research, Ulm, Germany). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interest: NF, MTR, DF, MW, and HS are working for the German Red Cross Blood Donor Service, Institute for Clinical Transfusion Medicine and Immunogenetics, which is a non-profit organization producing and marketing platelet lysate. There are no patents or products in development to declare. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. . These authors contributed equally to this work. MSC present an exciting prospect for fulfilling previously unmet needs in cell therapy, tissue repair, tissue engineering and gene therapy. Translational research involving MSC aims at developing an off-the-shelf cellular therapeutic product tailored to a myriad of clinical scenarios. As per the definition of the ISCT [1], MSC have to be plasticadherent when maintained in vitro and must be able to differentiate into osteoblasts, adipocytes and chondroblasts following standard cell culture differentiating conditions. In addition, $95% the MSC population has to express CD73, CD90 and CD105 and must lack expression of hematopoietic markers such as CD14, CD34, CD45 and HLA-DR [1]. Due to the low content of primary MSC in the bone marrow (0.0010.01% of total nucleated cells [2]), significant ex vivo cell amplification prior to clinical application is strictly necessary to obtain therapeutic applicable cell doses of 15 millions cells/kg body weight [3]. While more than 100 MSC-related clinical trials are currently registered at www.clinicaltrials.gov, a considerable variation can be observed in the mode and stringency regarding the pro (...truncated)


This is a preview of a remote PDF: https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0043255&type=printable

Natalie Fekete, Markus T. Rojewski, Daniel Fürst, Ludwika Kreja, Anita Ignatius, Julia Dausend, Hubert Schrezenmeier. GMP-Compliant Isolation and Large-Scale Expansion of Bone Marrow-Derived MSC, PLOS ONE, 2012, 8, DOI: 10.1371/journal.pone.0043255