Presentation Information
[P01-145]Thermo-Responsive Microcarriers for Efficient Cell Expansion in Upstream Bioprocessing
○YOUNGJIN KIM1, Shoya Hiratoko1, Sora Ikeya1, Nezu Yusuke1, Misaki Kato1, Itsuki Miyaguni1, Toshifumi Mogami1 (1. Life Science Research Laboratory, Tosoh Corporation (Japan))
Keywords:
Temperature-Responsive Microcarrier,Upstream Bioprocessing,Cell Expansion Culture,Seed Train Strategy,Bead-to-Bead Cell Transfer,Cooled Bead-to-Bead Cell Transfer
[Purpose]
In upstream bioprocessing, microcarrier (MC)-based seed train strategies typically involve bead-to-bead transfer or enzymatic subculture. These approaches can lead to non-uniform cell distribution, increased operational complexity, process variability, and elevated cost-of-goods (CoGs). To overcome these limitations, we developed thermo-responsive microcarriers (trMCs) and evaluated a novel seed train strategy termed “cooled bead-to-bead,” designed to enable uniform cell transfer without enzymatic treatment while reducing unit operations.
[Method]
A thermo-responsive block copolymer was synthesized and coated onto MC surfaces to fabricate trMCs. Several adherent cell lines were cultured on trMCs to evaluate attachment, proliferation, and viability. For thermo-triggered detachment, cell-attached trMCs were subjected to low-temperature treatment under agitation, and recovery efficiency and viability were assessed.Serial passaging experiments were performed using the “cooled bead-to-bead” method, which involves adding fresh trMCs and pre-cooled medium to cultures containing cell-attached trMCs, followed by agitation at ambient temperature to promote cell detachment as a single cell. Proliferation kinetics and cumulative cell yields were compared with those obtained using a conventional bead-to-bead strategy.
[Results]
Cells cultured on trMCs exhibited proliferation comparable to conventional MCs. Low-temperature treatment enabled efficient detachment from trMCs with high viability, whereas minimal detachment occurred on conventional MCs. Recovered cells appeared as single cells or small aggregates depending on intrinsic adhesion properties.In expansion studies, the “cooled bead-to-bead” strategy demonstrated higher proliferation rates and cumulative cell yields than the traditional bead-to-bead method. The process eliminated enzymatic treatment and washing steps, resulting in a simplified workflow.
[Consideration]
Temperature modulation reversibly alters the surface properties of trMCs, enabling non-enzymatic control of cell attachment and detachment. Controlled detachment prior to reseeding facilitates improved cell redistribution onto fresh MCs. Compared with enzymatic approaches, this strategy reduces or avoids contamination risk and removes enzyme washout steps. In contrast to conventional bead-to-bead transfer, prior detachment promotes more uniform reattachment. These features may contribute to improved process robustness, shortened process time, and reduced CoGs.
[Conclusion]
This study presents trMCs platform combined with a “cooled bead-to-bead” seed train strategy for expansion culture. The approach enables non-enzymatic cell transfer, supports efficient proliferation, and simplifies operational procedures. The proposed strategy may provide a scalable and robust framework for MC-based upstream bioprocessing and contribute to improved manufacturing efficiency in cell culture-based production systems.
In upstream bioprocessing, microcarrier (MC)-based seed train strategies typically involve bead-to-bead transfer or enzymatic subculture. These approaches can lead to non-uniform cell distribution, increased operational complexity, process variability, and elevated cost-of-goods (CoGs). To overcome these limitations, we developed thermo-responsive microcarriers (trMCs) and evaluated a novel seed train strategy termed “cooled bead-to-bead,” designed to enable uniform cell transfer without enzymatic treatment while reducing unit operations.
[Method]
A thermo-responsive block copolymer was synthesized and coated onto MC surfaces to fabricate trMCs. Several adherent cell lines were cultured on trMCs to evaluate attachment, proliferation, and viability. For thermo-triggered detachment, cell-attached trMCs were subjected to low-temperature treatment under agitation, and recovery efficiency and viability were assessed.Serial passaging experiments were performed using the “cooled bead-to-bead” method, which involves adding fresh trMCs and pre-cooled medium to cultures containing cell-attached trMCs, followed by agitation at ambient temperature to promote cell detachment as a single cell. Proliferation kinetics and cumulative cell yields were compared with those obtained using a conventional bead-to-bead strategy.
[Results]
Cells cultured on trMCs exhibited proliferation comparable to conventional MCs. Low-temperature treatment enabled efficient detachment from trMCs with high viability, whereas minimal detachment occurred on conventional MCs. Recovered cells appeared as single cells or small aggregates depending on intrinsic adhesion properties.In expansion studies, the “cooled bead-to-bead” strategy demonstrated higher proliferation rates and cumulative cell yields than the traditional bead-to-bead method. The process eliminated enzymatic treatment and washing steps, resulting in a simplified workflow.
[Consideration]
Temperature modulation reversibly alters the surface properties of trMCs, enabling non-enzymatic control of cell attachment and detachment. Controlled detachment prior to reseeding facilitates improved cell redistribution onto fresh MCs. Compared with enzymatic approaches, this strategy reduces or avoids contamination risk and removes enzyme washout steps. In contrast to conventional bead-to-bead transfer, prior detachment promotes more uniform reattachment. These features may contribute to improved process robustness, shortened process time, and reduced CoGs.
[Conclusion]
This study presents trMCs platform combined with a “cooled bead-to-bead” seed train strategy for expansion culture. The approach enables non-enzymatic cell transfer, supports efficient proliferation, and simplifies operational procedures. The proposed strategy may provide a scalable and robust framework for MC-based upstream bioprocessing and contribute to improved manufacturing efficiency in cell culture-based production systems.
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