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      Adaptation of the HEp-2 cell line to totally animal-free culture systems and real-time analysis of cell growth

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          Abstract

          Routine cell culture demands the use of animal-derived products, mainly fetal bovine serum and swine or bovine trypsin. According to the 3Rs principle and to the European Centre for the Validation of Alternative Methods, animal-free substitutes are strongly recommended for in vitro methods. In this study, the HEp-2 cell line was adapted to different totally animal-free culture systems, such as a serum-free complete medium (VP-SFM), human platelet lysate and a synthetic trypsin (TrypLE™ Express); afterward, cell growth was assessed with the xCELLigence instrument. Animal-free products provided promising results, with performances similar or preferable to the common reagents; therefore their use could be encouraged for both ethical and technical advantages.

          METHOD SUMMARY

          HEp-2 cells were sequentially adapted to the serum-free complete medium VP-SFM, performing subcultures into 25, 50, 75, 90, 95, 98, 99 and 100% VP-SFM. A direct adaptation to the medium was achieved with 5% platelet lysate. TrypLE™ Express was employed as a direct replacement of animal trypsin for the detachment of HEp-2 cells, without the use of an inhibitor. Real-time analysis of cell growth and proliferation and the comparison of different culture conditions were determined with the xCELLigence method.

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          Most cited references 22

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          Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods.

          Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media. Copyright 2010 Elsevier Ltd. All rights reserved.
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            The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture.

            Fetal bovine serum (FBS) is a common supplement to in vitro culture media. A workshop was organized to discuss whether or not fetuses might suffer when blood is withdrawn, and to discuss serum replacement methods. When bovine fetuses are exposed after slaughter of the dam, they can suffer only if they inflate their lungs with air and increase their blood oxygen to levels compatible with awareness. Preventing fetuses from breathing air or killing them by an efficient method, according to clearly defined safeguards, ensures that fetal blood collection is humane. Since serum is a supplement of unknown composition, which could be contaminated with unwanted factors, there are scientific and safety reasons for omitting FBS from culture media. Several media have been developed in which minimal or no animal derived components are present. Also, different cell types have been adapted to serum-free media. As yet, no standard serum free media are present, and each cell type requires its own medium composition. Among other recommendations, the establishment of a public database with information on cell types and their serum-free medium composition is proposed.
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              Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future

              The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing–thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2. PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.
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                Author and article information

                Journal
                BTN
                BioTechniques
                Future Science Ltd (London, UK )
                0736-6205
                1940-9818
                24 May 2021
                April 2021
                : 70
                : 6
                : 319-326
                Affiliations
                1Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, via Bianchi 9, 25124, Brescia, Italy
                Author notes
                [* ]Author for correspondence: Tel.: +39 030 229 0620; marafusi707@ 123456gmail.com
                Article
                10.2144/btn-2020-0162
                © 2021 Mara Fusi

                This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

                Page count
                Pages: 8
                Product
                Self URI (journal page): https://www.biotechniques.com/
                Funding
                Funded by: Italian Ministry of Health
                Award ID: Project MINSAL_XCELLIGENCE
                Funded by: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna
                Award ID: Writing assistance
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