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Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

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Abstract

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 109 BHK-21 cells from 4 × 106 cells in 13 day with 1,051 mL culture medium.

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Acknowledgments

The authors would like to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for post-doctoral fellowship (2010/52521-6), Fundação para o Desenvolvimento Tecnológico da Engenharia (FDTE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: 483009/2010-5) for scientific grants. First author gratefully acknowledges his wife and daughter, Relma and Giovanna for the inspiration to write this paper.

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Authors and Affiliations

  1. Laboratório de Células Animais, Departamento de Engenharia Química, Escola Politécnica, Universidade de São Paulo, Av. Prof. Luciano Gualberto, trav. 3, 380, Butantã, São Paulo, SP, 05508-900, Brazil

    Eutimio Gustavo Fernández Núñez, Letícia de Almeida Parizotto, Carlos Augusto Pereira & Aldo Tonso

  2. Departamento de Ciências Biológicas, Universidade Estadual “Júlio de Mesquita Filho”, Campus-Assis, Avenida Dom Antonio 2100, Bairro Parque Universitário, 19806-900, Assis, SP, Brazil

    Eutimio Gustavo Fernández Núñez

  3. Laboratório Especial de Pesquisa e Desenvolvimento em Imunológicos Veterinários, Instituto Butantan, Av. Vital Brasil, 1500, Butantã, 05503-900, São Paulo, SP, Brazil

    Jaci Leme & Celso Pereira Caricati

  4. Laboratório de Imunologia Viral, Instituto Butantan, Av. Vital Brasil, 1500, Butantã, São Paulo, SP, 05503-900, Brazil

    Alexandre Gonçalves de Rezende, Vera Lucia Lopes Boldorini, Soraia Attie Calil Jorge, Renato Mancini Astray & Carlos Augusto Pereira

  5. Laboratório de Biotecnologia Industrial, Núcleo de Bionanomanufatura, Instituto de Pesquisas Tecnológicas do Estado de São Paulo, Av. Prof. Almeida Prado 532 Cid. Universitária - Butantã, São Paulo, SP, 05508-901, Brazil

    Bruno Labate Vale da Costa

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  1. Eutimio Gustavo Fernández Núñez
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  2. Jaci Leme
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  3. Letícia de Almeida Parizotto
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  4. Alexandre Gonçalves de Rezende
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  5. Bruno Labate Vale da Costa
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  6. Vera Lucia Lopes Boldorini
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  7. Soraia Attie Calil Jorge
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  8. Renato Mancini Astray
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  9. Carlos Augusto Pereira
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  10. Celso Pereira Caricati
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  11. Aldo Tonso
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Correspondence to Eutimio Gustavo Fernández Núñez.

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Fernández Núñez, E.G., Leme, J., de Almeida Parizotto, L. et al. Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture. Cytotechnology 68, 95–104 (2016). https://doi.org/10.1007/s10616-014-9756-6

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  • DOI: https://doi.org/10.1007/s10616-014-9756-6

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