Adherent Cell Culture Research: From Start-Up to Scale-Up | Corning

Adherent cell culture research has contributed to significant advances in modern medicine, improving understanding of disease states and supporting the development of safe and effective interventions. In 2022, over 2,000 clinical trials in regenerative medicine used cell therapy, cell-based immuno-oncology, gene therapy, or tissue engineering.

For an adult human, a therapeutic dose might require hundreds or millions of cells. Multiply that by numerous doses and hundreds of patients per clinical trial, and billions of cells can quickly become necessary. To complicate matters, the vast diversity of cell types and their many possible uses means there's no one-size-fits-all approach for work with adherent cells. Nonetheless, a growing list of established protocols in published literature can serve as a good starting point for projects in the research and development phase. In addition, recent advances in cell culture platforms have simplified the process of scaling up to millions or billions of adherent cells.

Mapping Out the Seed Train

For any research project, it makes sense to consider the end goals early in the process, including the estimated number of cells needed for a certain number of patients. From there, work backward to create a plan for efficiently getting from the research stage to the pilot stage to the commercial production stage. The path to get from a frozen cryovial to billions of cells for patient treatment is called the seed train. It should evolve as you learn how to effectively seed, expand, and harvest your cells without compromising the quality of the cells or the cellular products you need.

What you can realistically achieve will also be determined by the time available for process development, as well as the budget, space, and number of personnel you can devote to production. In general, faster solutions will require more space, a larger workforce, and more consumables (including media and flasks) per number of cells. However, technological innovations have led to out-of-the-box solutions that are attractive for many applications over a range of scales. These solutions use multilayer and stacked vessels that pack more surface area into smaller footprints. For research groups that can devote more time to process development, fixed bed reactors (FBRs) offer additional control, and dissolvable microcarriers can be used with disposable spinner flasks or traditional bioreactors for adherent cell culture in a suspension environment.

Adherent Cell Culture Scale-up: Transitioning to Larger Vessels

For many research groups, the first step in scaling up will involve transitioning from a typical T-150 flask to a larger vessel such as the Corning® CellSTACK®HYPERFlask®, or HYPERStack® vessels. CellSTACK culture chambers are available in 1-, 2-, 5-, 10-, and 40-layer sizes with up to 25,440 cm² of growth area. All CellSTACKs can be converted to closed systems using a variety of closed system caps and tubing offered through Corning. Transitioning to a closed system will facilitate the transition to GMP (good manufacturing practice), which is essential for the production phase.

Corning HYPERFlask and HYPERStack use an innovative gas-permeable film to offer more compact solutions. These vessels have an outer layer of rigid polystyrene, but the growth surface is ultra-thin polystyrene that allows for gas exchange. The HYPERFlask is similar to a traditional T-175 flask in overall size and shape but has 10 layers of ultra-thin polystyrene for 1,720 cm² of growth area. HYPERFlask is available for manual or automated handling.

HYPERStack vessels come out of the box as a closed system and are available with 12 or 36 layers offering 6,000 or 18,000 cm² of growth surface respectively. The footprint of a HYPERStack vessel is similar to that of a CellSTACK chamber, but the HYPERStack-36 is just 28 cm tall compared to CellSTACK-40 at 72 cm tall. To reduce handling time, HYPERStack vessels can be joined together with single-use manifolds. A five-arm manifold can be used to join stacks of four HYPERStack-36 vessels.

To facilitate liquid handling and improve consistency, the Corning Automated Manipulator Platform can support six HYPERStack-36 vessels for a total of 108,000 cm² (or 10.8 m²) of growth area. Alternatively, it can support three CellSTACK-40 vessels for a total of 76,320 cm² (or 7.632 m²) of growth area.

Ensuring Compatibility in Early Adherent Cell Culture Scale-up

Using CellSTACK, HYPERFlask, and HYPERStack vessels requires relatively modest changes to traditional 2D cell culture techniques. While some verification and modification are necessary as the scale of production increases, the process tends to be fairly straightforward and thus a good choice when process development time is limited. In the research and development phase, cells are typically grown on a 2D polystyrene surface that has been treated for tissue culture and perhaps given an additional coating to promote cellular attachment. Early in the research process, check whether the surface coatings, cells, medium, and additional components (including medium, serum and supplements) being used are compatible with later steps in the process, including clinical trials. Also, consider how cells or cellular products are being harvested. If any changes need to be made, consider making them sooner rather than later.

Gaining Additional Control

For CellSTACK, HYPERFlask, and HYPERStack vessels, the cell culture medium and other components are added and removed intermittently. For continuous control, a bioreactor and appropriate gas lines are needed. If you want to retain the convenience of 2D cell culture, Corning CellCube® chambers are single-use modules of different sizes that provide up to 85,000 cm² (or 8.5 m²) of treated polystyrene growth surface. CellCube modules can be paired with a bioreactor for perfusion-based culture of adherent cells. The constant flow of fluid mimics in vitro conditions and allows for continuous monitoring and control of temperature, pH, dissolved oxygen, nutrients, and metabolites.

Another option is the Corning Ascent® FBR System, with a dedicated controller and single-use bioreactors that are currently available with 1m², 2.5m², or 5m² of growth surface. Larger "Pilot" and "Production" systems are in development to support bioreactors with up to 100m² and 1,000m² of growth surface respectively. The growth surface consists of layers of specially treated polyethylene terephthalate (PET) polymer mesh that enables uniform, low-shear fluid flow through the bioreactor bed for high-yield cell growth, transfection efficiency and viable cell harvest.

Adherent Cell Culture Scale-up: Maximizing Production

Finally, microcarriers allow adherent cells to grow in traditional bioreactors. The microcarriers are beads that remain suspended in media due to constant agitation and thus provide a growth surface for adherent cells in a 3D environment. Standard microcarriers are available in polystyrene with different surface treatments. Dissolvable microcarriers are a recent innovation, made of polyglycolic acid (PGA) polymer chains cross-linked with calcium ions. When cells are ready for harvest, the calcium can be chelated with EDTA, and the PGA degraded with pectinate. This gentle harvest procedure protects cell health and opens up endless possibilities.

Using microcarriers and bioreactors requires substantial process development time, which can be rewarded with space savings and improved efficiency and production. Preliminary experiments might start in a disposable 125 mL spinner flask and scale up from there.

Plan Your Path

For adherent cell culture scale-up, many good options are available. Mapping out your seed train early will make the most efficient use of time and resources, and set your project on the path to success.