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<p>A scalable and controlled process for expanding human liver stem cells (HLSCs) under chemically defined conditions is crucial for their translation into clinical applications. To this end, we screened several customized media formulations, alongside commercially available recombinant collagen&#xa0;1, fibronectin, and Cellbind surfaces as well as a surface matrix in a 96-well plate. We also investigated different adaptation methods and cell detachment using TrypLE and Accutase at various concentrations and incubation times. Defining a suitable basal medium, using bone sialoprotein as a growth surface matrix, applying a gentle adaptation protocol and Accutase detachment, enabled HLSC expansion at 1.2% fetal calf serum (FCS) with 22.1 cumulative population doublings while increasing resistance to toxic metabolites. We then transferred this process to a fully controlled 2-L stirred tank bioreactor, achieving growth rates comparable to standard αMEM medium with 10% FCS, while maintaining cell phenotype and functionality as assessed by an islet formation assay. Finally, we reduced FCS to 0.5% using a design of experiments approach, testing the addition of human serum albumin, fibroblast growth factors (FGF-2, FGF-1), epidermal growth factor (EGF) and an insulin-transferrin-selenium (ITS) premix in an attempt to achieve chemically defined conditions. While fully chemically defined conditions were not achieved, medium supplementation with ITS, EGF and FGF-2 doubled HLSC growth in serum-reduced culture. This study outlines the critical steps in developing a serum-free process that complies with good manufacturing practice and can be scaled up for the expansion of HLSCs, paving the way for future clinical applications.</p>

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Adaptation and expansion of human liver stem cells in serum-reduced culture conditions

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月16日

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  • Jan Thorbow,
  • Viktoria Pfening,
  • Andrea Strauch,
  • Jan-Philip Klee,
  • Patricia Brücher,
  • Björn Boshof,
  • Florian Petry,
  • Peter Czermak,
  • Maria Beatriz Herrera Sanchez,
  • Denise Salzig

摘要

A scalable and controlled process for expanding human liver stem cells (HLSCs) under chemically defined conditions is crucial for their translation into clinical applications. To this end, we screened several customized media formulations, alongside commercially available recombinant collagen 1, fibronectin, and Cellbind surfaces as well as a surface matrix in a 96-well plate. We also investigated different adaptation methods and cell detachment using TrypLE and Accutase at various concentrations and incubation times. Defining a suitable basal medium, using bone sialoprotein as a growth surface matrix, applying a gentle adaptation protocol and Accutase detachment, enabled HLSC expansion at 1.2% fetal calf serum (FCS) with 22.1 cumulative population doublings while increasing resistance to toxic metabolites. We then transferred this process to a fully controlled 2-L stirred tank bioreactor, achieving growth rates comparable to standard αMEM medium with 10% FCS, while maintaining cell phenotype and functionality as assessed by an islet formation assay. Finally, we reduced FCS to 0.5% using a design of experiments approach, testing the addition of human serum albumin, fibroblast growth factors (FGF-2, FGF-1), epidermal growth factor (EGF) and an insulin-transferrin-selenium (ITS) premix in an attempt to achieve chemically defined conditions. While fully chemically defined conditions were not achieved, medium supplementation with ITS, EGF and FGF-2 doubled HLSC growth in serum-reduced culture. This study outlines the critical steps in developing a serum-free process that complies with good manufacturing practice and can be scaled up for the expansion of HLSCs, paving the way for future clinical applications.