The Pros and Cons of Adherent Versus Suspension Cell ...

When considering scaling adherent cultures versus suspension, there are some additional limitations to factor in: media usage and open versus closed systems. Some adherent culture vessels may have fixed or more restrictive media volume requirements. Nevertheless, incorporating circulation with in-process monitoring can help to get media usage per growth area to a more favorable level for specific dynamic adherent platforms. Finally, many traditional adherent vessels are handled as open systems. While open system handling of dishes and flasks on a small scale might not be problematic, there is risk of contamination associated with manual open handling of roller bottles and larger stacked vessels (2). Closed system configurations and accessories are becoming increasingly available for some adherent vessels to mitigate contamination risk for large scaled-out processes.

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Labor also becomes a factor in the scalability of adherent platforms. At some point with some vessel types (e.g., multilayer stacked vessels), greater surface area can only be achieved by scaling out the process by adding multiple units of the same vessel versus scaling up through progressively larger vessels. Scaled-out processes generally require more manual labor and have a much larger footprint per growth area compared to suspension platforms (2).

However, adherent platforms are limited in scalability. Because adherent cells are attached to the growth substrate, culture expansion is limited by surface area. When cells have covered the growth surface, the cells must be subcultured by either chemical (e.g., ethylenediaminetetraacetic acid), enzymatic (e.g., trypsin), or mechanical dissociation (e.g., scraping) to disrupt the cell&#;cell and cell&#;substrate bridges (15,16). Many cell types tolerate dissociation well. However, care must be taken to minimize degradation to surface proteins, which could disrupt adherence to subsequent culture vessels and/or negatively impact downstream applications, such as surface marker identification (17). There are also added process steps and labor involved in subculturing adherent cells&#;wash steps, incubation, quenching, and/or centrifugation to remove the dissociation reagent (15,16).

Adherent platforms offer versatility not only in simply surface modifications, but also in size range, making them adaptable to diverse experimental needs. Traditional dishes, flasks, and microplates are widely used for a broad spectrum of research applications and drug screening. For larger-scale production, roller bottles continue to be one of the major platforms for vaccine production. Stacked platforms (e.g., CellSTACK Culture Chambers, Corning) and more advanced technologies amplify conventional planar adherent culture by packing multiple layers into a standard footprint. Fixed-bed bioreactors combine an adherent substrate in a bioreactor vessel with media conditioning and process automation to deliver high yield and larger scale. In short, there are a breadth of offerings at different scales, from R&D through to clinical production, for a range of budgets and accounting for different spatial footprints.

Adherent culture is essential in applications requiring closely controlled cell physiology and function (1,2). For regenerative medicine and tissue engineering applications, structural scaffolds are employed to support cell growth and differentiation. Examples include MSCs expanded on ECM hydrogels for bone and cartilage repair (10&#;12) and hiPSCs cultured on adherent substrates to produce cardiomyocytes for heart tissue repair (13). Additionally, skin grafts for wounds are created by culturing fibroblasts and keratinocytes on collagen matrices (14), highlighting the necessity of adherent culture for tissue architecture fidelity.

The intracellular signaling cascades that regulate many cellular functions, including cell cycle progression, cell differentiation, and responses to growth factors are governed by cell&#;cell and cell&#;matrix interactions (3). Adherent cell culture provides a more natural environment because it can recapitulate these interactions. Vessel surfaces can be modified with various treatments and biologically relevant coatings to optimize cell adhesion for specific cell types and experimental conditions. In this way, adherent cell culture vessels can be tailored to specific applications to achieve or maintain a particular cellular phenotype. In particular, iPSCs often exhibit changes in morphology depending upon the surface they are cultured upon. Gelatin-coated surfaces promote endothelial differentiation (8), whereas natural ECM-based hydrogels provide the signaling cues to maintain pluripotency (9).

Adherent cell culture describes the method for propagation of anchorage-dependent cell types on a growth substrate. Anchorage-dependent cell types require physical attachment to a substrate or extracellular matrix (ECM) for proliferation and survival (1&#;3). Many common primary and continuous cell lines are adherent, including mesenchymal stem cells (MSC), induced pluripotent stem cells (iPSC), human embryonic kidney 293 (HEK293), Vero, and Chinese hamster ovary (CHO). Adherent cell types fall into several groups based on their origin and morphological features such as nuclei size, cytoplasm ratio, and shape ( Figure 1 ). For example, fibroblast-like cells are elongated with a spindle-shaped morphology (4), epithelial cells exhibit a polygonal shape (5), and endothelial cells elongate and assemble in a ductal fashion (6), whereas neuronal cells spread with projecting processes (7). Many adherent platforms enable visualization of cells to monitor cultures, which is valuable because cell morphology is an important phenotypic indicator of cellular biology and function.

Cell culture is fundamental to biomedical research and can be categorized into two major platforms: adherent and suspension. Both approaches are suitable for a range of diverse research applications, including basic cellular biology and disease modeling as well as for biopharmaceutical manufacturing, including biologics production, cell and gene therapies, and vaccine production. Each approach has advantages and limitations to be considered along with other factors, such as scalability, resource requirements, facility space, and process monitoring. This article provides an overview of these considerations.

Suspension is a method for cultivation of cells suspended in growth medium (Figure 1). Suspension cells do not require attachment to a substrate; they grow free-floating as single cells or as multicell clumps or clusters (16). This growth paradigm is the native state for hematopoietic stem cells (18) and immune cells (e.g., Jurkat [19]). Various insect cell lines, such as Sf9, also grow in suspension. CHO and similar production lines have been adapted for growth in suspension (20). However, not all adherent cell lines are amenable to the process of suspension culture adaptation. Consider epithelial cells which lose polarity and the ability to form junctions in suspension culture, affecting their functionality (21).

Generally, suspension cells require some form of agitation during culture to keep them from settling to the bottom of the vessel and to facilitate gas and nutrient exchange. They can be cultured in a few different dynamic culture platforms. Shaker flasks, which resemble Erlenmeyer flasks fitted with a gas-permeable cap, swirl media as flasks are rotated on an orbital shaker. Spinner flasks and bioreactors use impellers to stir cultures to keep them aerated and in suspension. Suspension cultures can also be grown in culture bags on a rocking-motion bioreactor platform. Depending on scale and vessel type, gas exchange can occur passively by diffusion of the culture incubator atmosphere&#;or actively by delivery of a controlled gas mixture.

Because suspension culture is dynamic, there is greater shear stress exerted on these cultures compared to static adherent cultures. Some cell types, in particular, primary cells and stem cells, are more sensitive than others (2). Frequently, suspension culture media is supplemented with pluronic or other surfactants to protect cells from shear stress (22,23). Likewise, different suspension platforms exert varying levels of shear. Design variations can lessen shear stress&#;for example, flasks with or without baffles (24), different impeller designs (25), and traditional stirred versus vertical wheel bioreactors (e.g., Vertical-Wheel, PBS Biotech [26]). Gas sparging method and rate can also affect shear stress for actively gassed cultures (23,27). Suspension culture requires a careful balance between sufficient agitation to provide media aeration and prevent culture settling and minimizing the shear stress on cells.

In addition to shear stress limitations on cell expansion, media volume and cell concentration can constrain suspension cultures (15). However, suspension cultures are simply diluted with fresh medium to subculture, maintaining a specific density of viable cells per unit volume to sustain growth and keep the cells in a logarithmic growth phase (15). Provided that culture vessel minimum and maximum working volumes are met, suspension cells can be grown in as little or as much medium as desired. With significantly high yield for the spatial footprint, especially in the production stirred-tank reactors, suspension cell culture is substantially more scalable than adherent cultures. These platforms operate as closed systems, with in-process monitoring and controls. With enough process development to characterize and understand how each suspension line grows, in-process monitoring can accurately track progress of cell expansion in the absence of direct cell visualization.

Suspension platform advantages of scalability and operational efficiency may come at the cost of time and work upfront. On the one hand, suspension cell cultures are integral to biopharmaceutical production due to their scalability. CHO cells grown in suspension are widely used for production of monoclonal antibodies (mAbs), treating conditions from cancer to autoimmune diseases (20,28,29). Furthermore, suspension cultures of insect cells facilitate rapid vaccine production, as seen with recombinant protein vaccines against viruses such as human papillomavirus (30). However, other cell types require adaptation to suspension culture. Time and resources are necessary for cell line and application development, though the boundaries between adherent and suspension cell cultures are becoming blurred because of new technological advances. There has been a shift toward more scalable suspension culture for some traditionally adherent applications. For instance, adeno-associated viral vectors for cell and gene therapy applications, once reliant on adherent HEK293 cultures, are shifting toward suspension cultures for improved yields with suspension HEK293 lines and suspension-ready transfection reagents now commercially available (31,32).

Advanced platforms offer hybrid approach

Microcarrier technology is a hybrid approach that enables growth of anchorage-dependent cells in a scalable suspension environment. Microcarriers are small beads used to provide a growth substrate for adherent cells to grow in suspension (33). Microcarriers are manufactured from a variety of materials including polystyrene and dextran-based microcarriers (34&#;36). Other microcarriers are made from biodegradable polymers that simplify downstream processing by eliminating the need for separation steps (35). Surface modifications on microcarriers can range from simple protein coatings, such as collagen or fibronectin, to more complex synthetic peptides that mimic extracellular matrix components (37), each designed to optimize the growth and functionality of specific cell types. Importantly, microcarriers are enabling traditionally adherent-dependent cells to be cultivated in suspension for mAb production (38).

Another hybrid approach that lies between adherent and suspension culture is the cultivation of spheroids and organoids, otherwise known as 3D cell culture. There are methods that utilize extracellular matrix scaffolding to support 3D growth and scaffold-free methods that encourage cells to self-organize into structures that mimic the form and function of tissues in vivo (39). For example, human iPSCs (hiPSCs) cultured in suspension form spheroids that can be differentiated into hiPSC-derived cardiomyocytes (40). Regardless of method, cell&#;cell connections are formed in three dimensions that provide the contextual clues for more natural cell behavior, with improved gradients of oxygen, nutrients, and signaling molecules that are critical for cellular differentiation and function (39). The resulting tissue-like structures present in 3D cultures offer advanced models for studying disease mechanisms, tissue regeneration, and drug responses with higher physiological relevance compared to 2D systems.

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Conclusion

The evolution of cell culture technology continues to revolutionize biological research and industrial applications. While adherent platforms that facilitate natural cell&#;cell and cell&#;matrix interactions characteristic of many cell types exemplify this endeavor, this advantage is tempered by limited scalability and higher labor requirements. In contrast, suspension cultures excel in scalability, particularly for high-yield production, provided that the cell type thrives in suspension. Advanced platforms that merge the advantages of both, such as microcarrier technology and 3D cultures, are leading the charge towards more advanced and efficient culture systems. Choosing between these platforms requires a deep understanding of their respective advantages and limitations, ensuring a strategic fit for the intended application and research objectives. Looking forward, continued innovation is expected to further enhance these cell culture technologies, broadening their application in the fields of general bioproduction, regenerative medicine, and cell and gene therapy. This progress emphasizes the critical role of strategic platform choice to leverage the full potential of cell culture advancements.

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About the author

Ann Rossi Bilodeau, PhD, is senior bioprocess applications scientist at Corning Life Sciences.

Article Details

BioPharm International
Volume 37, No. 1
January
Pages: 18&#;22

Citation

When referring to this article, please cite it as Bilodeau, A. R. The Pros and Cons of Adherent Versus Suspension Cell Culture. BioPharm International , 37 (1), 18&#;22.