The following article originally appeared on March 1, 2021 in Biocompare here.
Adherent cells are routinely cultured on a planar surface, such as that provided by a cell culture dish or flask. Gas and nutrient exchange occur through the culture media, making it essential that conditions are thoroughly optimized to support cell growth. Problems inherent to adherent cell culture include poor attachment, compromised yield or viability, and the production of uneven cell monolayers, the effects of which can become more pronounced during cell expansion. This article highlights some common causes of these issues and provides helpful suggestions for preventing them, both during routine cell culture and scale up.
Challenges of Adherent Cell Culture
A major challenge of adherent cell culture lies in getting the cells to attach to the culture vessel. This can be especially problematic when growing cells in reduced-serum or serum-free medium because the attachment proteins in serum (vitronectin and fibronectin) are often required for cells to adhere and spread. Another problem is ensuring the cells are provided with sufficient oxygen and nutrients to avoid compromising viability or triggering phenotypic changes that could create issues further downstream. It is also critical that the cell monolayer be evenly distributed. Not only does even distribution promote consistent cell growth, but it also maximizes the number of cells that can be harvested from the culture vessel.
Preventing Common Growth Problems
The challenges just described are easily overcome by adopting best practices for adherent cell culture. Attachment can be encouraged by allowing cells several passages to adapt to reduced-serum or serum-free conditions or by using a culture vessel with a specially treated surface. Effective coatings, including poly-L-lysine, gelatin, collagen, and various proprietary formulations, should be carefully selected to suit the cell type. Bringing medium to the correct pH by pre-warming and pre-gassing also helps cells to adhere more quickly and is particularly beneficial when using larger culture vessels.
Because vessel size and media volume can have a significant impact on gas exchange and oxygen delivery to adherent cells, it is essential the media height is carefully optimized. Media height should be low enough to allow oxygen to travel freely from the headspace to the cells, yet high enough that sufficient nutrients are available to support healthy cell growth. It is generally recommended that adherent cell culture be performed using 0.2–0.4 mL growth media per cm2 of growth area.
To avoid generating an uneven monolayer, the cell inoculum should undergo adequate yet gentle mixing with the medium upon addition to the culture vessel. This can be achieved by carefully tilting culture dishes from side to side or by performing several slow aspiration and dispense steps with a wide-bore serological pipette. It is also vital to avoid generating shear forces when performing medium changes or moving cultures between the biological safety cabinet and the incubator. Shearing can occur as medium sweeps across the cells, causing parts of the monolayer to become detached. Gentle handling techniques eliminate these issues and can also help prevent the formation of bubbles that can stop freshly inoculated cells from adhering or lead to necrosis of cells that are already attached.
Applying Best Practices to Scale Up
There are numerous reasons for researchers to scale up adherent cell cultures. These include the generation of cell lysates for immunoassay applications such as western blot or ELISA, the expansion of antibody-producing clones during monoclonal antibody development, and the production of various cellular therapies. Irrespective of the end goal, scale up requires that growth remains consistent as the size of the vessel increases; this is key to ensuring cellular characteristics are not changed.
A typical scale-up operation might take cells from a multi-well plate, to a cell culture dish, to a flask, culminating in their growth in a multi-layer flask. The latter are designed to maximize yield without taking up excessive space in the CO2 incubator and models are now available that allow as many as ten layers of adherent cells to be grown in a vessel sharing the same footprint as a conventional 175 cm² flask. These include Falcon® Cell Culture Multi-Flasks (available in 525 cm2 and 875 cm2) and Corning® HYPERFlask® Cell Culture Vessels (1720 cm²). For even larger scale-up capacity, other options include roller bottles (up to 1,750 cm²), Corning CellSTACK® Culture Chambers (up to 25,440 cm²), and highly sophisticated closed perfusion systems that can be connected to a bioreactor (up to 85,000 cm²).
It is important to keep in mind that smaller vessels enable culture conditions to be thoroughly optimized prior to scale up. For example, multi-well plates or dishes can be used to compare different media formulations or to identify the best seeding concentration before cells are grown in larger vessels such as flasks. The culture media volume should be kept constant throughout the expansion process (mL medium per cm²) to avoid introducing variability that could have a more pronounced effect at larger scale.
Corning offers an extensive range of vessels for adherent cell culture, including multi-well plates and culture dishes, in addition to single layer and multi-layer flasks. To learn more, visit our cell culture solutions.