Method of culturing cell, method of manufacturing cell support complex, cultured cell, and cell support complex
Inventors
Assignees
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Publication Number
US-12221597-B2
Publication Date
2025-02-11
Expiration Date
Abstract
A method of culturing cells includes culturing the cell in a cell suspension under repetitive cycles of stirring and rest while keeping the cell non-adherent to a culture vessel, so as to form at least one aggregate of the cells.
Core Innovation
The invention provides a method of culturing cells by culturing the cells in a cell suspension under repetitive cycles of stirring and rest while keeping the cells non-adherent to a culture vessel. The culture vessel is treated by cell-repellent treatment or composed of a cell-repellent material so that aggregates form without applying a support in the cell suspension. After forming at least one aggregate, the method cultures the cells in a state of aggregate while keeping the cell suspension stirred.
The method enables restoration of physiological function of renal/proximal tubule epithelial cells and maintains pluripotency in iPS/ES-derived cells during aggregated cultivation. The culture includes intermittent stirring/rest that tunes uniform aggregate geometry and gene expression, followed by continuous stirring to culture cells in an aggregated, stirred state. Gene-expression comparisons indicate that aggregates show upregulation of renal-associated markers, including AQP1 and OAT1, while continuous stirring alone prevents aggregates.
The document further describes quantitative culture conditions and aggregate characteristics for the repetitive stirring/rest cycling, including a rest time greater than 5 minutes and less than 480 minutes, a culture period of 5–14 days, and aggregate size and cellularity ranges. Stirring parameters are constrained by a stirring blade rotating speed and a shear stress absolute value of 0.3 N/m2 or less. It also includes a cell support complex manufacture involving deaggregating cultured cells into discrete cells, coating a base with an adhesion-molecule layer, seeding the cells, and culturing to form a stable confluent monolayer.
Claims Coverage
The document identifies one independent claim directed to a culturing method based on non-adherent suspension culture with repetitive stirring/rest to form aggregates without a support, followed by continuous stirred aggregate culture. The claim set includes dependent refinements that add inventive features such as cell-type limitation and quantitative constraints for rest time, stirring speed, shear stress, aggregate cellularity, and sequencing related to adhesion-based monolayer formation.
Non-adherent repetitive stirring/rest aggregate culture
Culturing the cells in a cell suspension under repetitive cycles of stirring and rest while keeping the cells non-adherent to a culture vessel treated by cell-repellent treatment or composed of a cell-repellent material, and forming at least one aggregate of the cells without applying a support in the cell suspension.
Stirred aggregate state after aggregate formation
Subsequent to the steps of forming at least one aggregate and culturing the cells under repetitive cycles of stirring and rest, culturing the cells in a state of aggregate with the cell suspension kept stirred.
Rest time range for stirring/rest cycling
The repetitive cycles of stirring and rest are performed with a rest time longer than 5 minutes and shorter than 480 minutes.
Stirring blade rotating speed constraint
Stirring a cell suspension with a stirring blade rotating at a speed greater than 20 rpm.
Shear stress absolute value limit
Stirring applies a shear stress to the cells with an absolute value of 0.3 N/m2 or less.
Aggregate cellularity range
The cell aggregate contains between 500 and 5000 cells.
Renal cell limitation
The method is limited such that the cells are renal cells.
Overall, the claim coverage centers on keeping cells non-adherent in a cell-repellent culture vessel, using repetitive stirring and rest to form aggregates without a support, and then culturing those aggregates in a stirred state. Dependent claims further specify a rest-time range, stirring blade speed, shear stress limit, aggregate cellularity range, and restriction to renal cells.
Stated Advantages
Restoration of physiological function of renal/proximal tubule epithelial cells during aggregated cultivation.
Maintenance of pluripotency in iPS/ES-derived cells during aggregated cultivation.
Enabling aggregate formation without applying a support in the cell suspension.
Tuning uniform aggregate geometry and gene expression using intermittent stirring/rest, while continuous stirring alone prevents aggregates.
Documented Applications
Bioartificial kidney use, including drug evaluation using modules such as Transwell, hollow fibers, microchannels, microcarriers, and well plates.
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