What is the right cell cultivation system for emerging cell and gene therapies? Exploring the roles, advantages, and limitations of cell culture flasks, multilayer flasks, and roller bottle cultures in bioprocessing
The main types of adherent cell cultivation systems – culture flasks, roller bottles, and multilayer flasks support diverse bioprocessing needs but do not fully address the challenges of scalability and footprint. © Green Elephant Biotech
As an alternative to large-scale bioreactors, compact cell cultivation systems such as flasks, roller bottles, and multilayer stacks remain essential for growing 2D adherent cell cultures and Cell and Gene Therapy (CGT). From the proof-of-concept studies to the seed train expansion or the stepwise amplification of cells, cultivation systems form a backbone of manufacturing workflows.
With researchers and manufacturers carefully orchestrating the cultivation processes based on the desired scale and efficiency, certain criteria are required for the systems to perform effectively. For instance, stable access to the key elements – namely, oxygen, carbon dioxide, and a pH of 7.2-7.4. Shear forces, influential for cell morphology, differentiation, and viability, are also regulated to ensure the quality of adherent cells. Lastly, adherent cell expansion is associated with a substantial footprint, making its reduction a key objective in modern bioprocessing (1).
A growing interest in the approved CGTs, such as Zolgensma, Luxturna, and Kymriah, reinforces the challenges present in research and development. With the successful commercialization of high-quality CGTs depending heavily on adherent culture expansion platforms, this article highlights the role, advantages, and limitations of each cultivation system.
Researchers see the T-flasks as unsophisticated but irreplaceable
Even though viewed as simplistic, the role of T-flasks or cell culture flasks remains foundational in cell culture workflows and extends beyond small batch maintenance. In large-scale biomanufacturing and bioprocessing, T-flasks are commonly used in the early stages of seed train expansion for adherent cells. For over 100 years, researchers have favored these disposable cell culture vessels because they are easy to handle, inexpensive, and efficient in localized experiments (2).
However, with the maximum number of adherent cells per flask reported as 1 x 10⁷ and 1.5 x 10⁸ cells in suspension, the practicality of T-flasks decreases with the growing batch size (3). To achieve larger volumes, facilities must expand the number of flasks in use, demanding additional incubator space and labor, which represents some of the most expensive factors in adherent cell expansion and modern biomanufacturing.
Automation of conventional cultivation systems and roller bottles is costly
Roller bottle culture was originally developed to support the growth of larger quantities of anchorage-dependent cells, providing a dynamic environment that improves nutrient distribution and gas exchange. Since their first successful use in viral vaccine production, roller bottles have been widely implemented for cultivating adherent cell lines requiring mechanical agitation (2, 4). Each roller bottle can yield 1 x 10⁹ adherent cells and 1 x 10⁸ cells in suspension (3), making them a valuable tool for bioprocessing and offering a potential for partial automation.
Yet, the ability of this cell cultivation system to address the manufacturing capacity crunch in CGT production is its main limitation. Automation of roller bottle cultures is known to be costly, reducing their appeal for large-scale commercial applications (3). Similarly, improved nutrient and oxygen availability supplied by roller bottles come at the cost of incubator space and labor, encouraging more flexible solutions.
Multilayers flasks demonstrate that larger is not better
A multilayer flask (also known as CellSTACK or cell factory system) provide a labor-saving solution for the large-scale cultivation of adherent cells under conditions like standard T-flasks. Widely used in biopharmaceutical production, these vessels enable researchers to expand cell cultures in a more space-efficient manner. Accommodating 2 x 10⁸ of attached cells per stack, multilayer flasks offer a more economical use of incubator space while supporting greater process control (3,5).
Larger vessels, however, carry a higher risk of leakage, contamination, and establishment of a heterogeneous environment throughout the stacks due to the design (6). Along with these characteristics, multilayer flasks present handling challenges at larger scales. For example, 10-stack systems require around 1,900 mL of media, while 40-stack systems demand as much as 7,200 mL (2). Although these volumes increase the available growth surface, they also result in extensive manual handling and mechanization, limiting the facilities to accommodate the multilayer flasks.
The limitations of others are the advantages of CellScrew®
In comparison, CellScrew® drastically improves the growth of adherent cell cultures by addressing the limitations of traditional systems such as T-flasks, roller bottles, and multilayer stacks. Available in multiple sizes, including mini (850 cm²), 6K (6,000 cm²), and 10K (10,000 cm²), CellScrew® enables scalable adherent cell expansion and provides the right size for every scale. Unlike a T75 cell culture flask, CellScrew® offers an 80-times larger growth surface within its 6K cultivation vessel, optimizing the spatial footprint required for expansion. For instance, a single CellScrew® 10K unit provides a growth area of 10,000 cm² and the volume comparable with approximately 12 roller bottles, while maintaining compatibility with roller devices for a more streamlined and cost-effective workflow.
Designed to enhance nutrient and gas exchange, CellScrew® combines a maximized growth surface area with 3-times lighter weight in g/cm² and easier handling compared to multilayer flasks. These advantages make CellScrew® an attractive platform for starter cultures, CGT manufacturing, and batch bioprocessing, supporting a more scalable and environmental solution for scaling up adherent cell expansion.
The spectrum of cell culture systems provides solutions for diverse needs, ranging from small-scale research with T-flasks, to mid-scale expansion in roller bottles, and high-density production using multilayer flasks. Emerging platforms such as CellScrew® represent the next step, offering a more efficient and sustainable approach to scale up adherent cell production, and helping to address the manufacturing bottlenecks faced by CGTs. At Green Elephant, our mission is to enable broader patient access to CGTs while reducing the environmental footprint of bioprocessing. Through innovative platforms like CellScrew® mini, 6K, and 10K, we aim to deliver scalable, cost-effective, and environmentally conscious solutions for the future of life-saving therapies and biomanufacturing.
Facts before promises
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References:
(1) Bellani, CF, Ajeian, J, Duffy, L, Miotto, M, Groenewegen, L, Connon, CJ, Scale-Up Technologies for the Manufacture of Adherent Cells (2020), Front Nutr, 7; 575146: 1-14. doi: 10.3389/fnut.2020.575146.
(2) Growing More Cells: A Simple Guide to Small Volume Cell Culture Scale-Up (2008). Ryan, J. A. Available at https://novabio.ee/uploads/Corning-scale-up-brochure.pdf (Accessed 16 July 2025).
(3) Alternative High-Yield Cell Culture Systems (2016). ECACC Laboratory Handbook 4th Edition. Available at https://www.sigmaaldrich.com/IE/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/alternative-culture (Accessed 16 July 2025).
(4) Mansbridge, JN. (2013) Biomaterials Science: An Introduction to Materials in Medicine. Chapter II.6.12. San Diego, CA: Academic Press. doi: 10.1016/B978-0-08-087780-8.00119-4
(5) Advantages of the Multilayer Structure Design of Cell Factories (2024). Luoyang Fudau Biotech Co., Ltd. Available at https://www.fdcell.com/news/advantages-of-the-multilayer-structure-design-of-cell-factories.html#:~:text=Compared to expanding the area,Culture Flask in Vaccine Development (Accessed 16 July 2025).
(6) Strecanska, M, Sekelova, T, Smolinska, V, Kuniakova, M, Nicodemou, A, Automated Manufacturing Processes and Platforms for Large-scale Production of Clinical-grade Mesenchymal Stem/Stromal Cells (2024), Stem Cell Rev Rep, 21; 2: 372-289. doi: 10.1007/s12015-024-10812-5.
