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      A novel chemically defined medium for the biotechnological and biomedical exploitation of the cell factory Leishmania tarentolae

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          Abstract

          The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

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          Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

          Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.
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            Serum-free cell culture: the serum-free media interactive online database.

            Fetal bovine serum (FBS) is a ubiquitously used essential supplement in cell culture media. However, there are serious scientific and ethical concerns about the use of FBS regarding its harvest and production. During the last three decades, FBS could be substituted by other supplements or by the use of defined chemical components in serum-free cell culture. A number of serum-free medium formulations have been described for mammalian and insect cell lines as well as for primary cultures. However, the switch to serum-free media still demands a time-consuming literature survey and a manufacturer search for appropriate medium formulations, respectively. Here we present the second collection of commercially available serum-free media in an updated, freely accessible interactive online database. Searches for serum-free media and continuous cell lines already adapted to serum-free culture can be performed according to various criteria. These include the degree of chemical definition, e.g. serum-free (SF), animal-derived component free (ADCF) or chemically defined (CD), and the type of medium, e.g. basal media, medium supplements, or full replacement media. In order to specify the cell lines that are adapted to serum-free media, search terms like species, organ, tissue, cell type and disease can be used. All commercially available serum-free media and adapted cell lines currently available from major distributors (e.g. ATCC, ECACC and DMSZ) are included in the database. Despite an extensive search for serum-free media and adapted cell lines, detailed information from certain companies and suppliers is still lacking and is specifically highlighted. It is intended to create a platform for the interactive exchange of information and experience by experts in the field in order to continuously improve and extend the serum-free online database. The database is accessible at http://www.goodcellculture.com/
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              Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications.

              Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs. Copyright 2005 Wiley-Liss, Inc.
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                Author and article information

                Contributors
                sara.epis@unimi.it
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                26 April 2024
                26 April 2024
                2024
                : 14
                : 9562
                Affiliations
                [1 ]Department of Biosciences, University of Milan, ( https://ror.org/00wjc7c48) 20133 Milan, Italy
                [2 ]Pediatric CRC ‘Fondazione Romeo ed Enrica Invernizzi’, University of Milan, ( https://ror.org/00wjc7c48) 20157 Milan, Italy
                [3 ]Department of Veterinary Medicine, University of Bari, ( https://ror.org/027ynra39) 70010 Valenzano, Italy
                [4 ]GRID grid.35030.35, ISNI 0000 0004 1792 6846, Department of Veterinary Clinical Sciences, , City University of Hong Kong, ; Hong Kong, Republic of China
                [5 ]Department of Molecular and Developmental Medicine, University of Siena, ( https://ror.org/01tevnk56) 53100 Siena, Italy
                [6 ]VisMederi, ( https://ror.org/05sv6xe54) 53100 Siena, Italy
                Article
                60383
                10.1038/s41598-024-60383-1
                11053126
                38671070
                78f79182-54f6-4693-b16f-44aa496cff5c
                © The Author(s) 2024

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 28 February 2024
                : 22 April 2024
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100012032, Fondazione Romeo ed Enrica Invernizzi;
                Award ID: LIB_VT22CBAND
                Award ID: LIB_VT21SEPIS
                Award Recipient :
                Funded by: Erogazione liberale per le attività di ricerca sul Coronavirus
                Award ID: LIB_VT20_COVID_19_SEPIS
                Award Recipient :
                Categories
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                © Springer Nature Limited 2024

                Uncategorized
                leishmania culture,leishmania tarentolae,chemically defined medium,protein expression,promastigotes,cell factory,microbiology,non-model organisms,assay systems

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