Modern therapeutic biotechnology is characterized by blockbuster products, mostly monoclonal antibodies, produced in mega-cultures of mammalian cells or microorganisms, of which CHO lines and E. coli are just two examples. Mammalian cell cultures of 1,000 liters and higher volumes would be impractical were CHO cells not adapted specifically for suspension culture.

Nevertheless most biological research involves adherent cells, as do some niche manufacturing-scale processes. Research scientists have exploited cells’ innate desire to attach to surfaces and other cells by developing three-dimensional cell cultures, some of which consist of two or more cell types. Early biopharmaceutical development may include plating cells into culture dishes to test them for viability or productivity, followed by systematic adaption of these cells to more convenient suspension culture.

On the manufacturing side, vaccines are an ideal application where the use of adherent cells is likely to persist. Compared with therapeutic proteins, vaccines are dosed in minute quantities, so manufacturing focuses more on producing an effective, viable product than in generating tons of material.

It’s all in the surface

Nevertheless, vaccine makers who rely on adherent cells are interested in consistency, productivity, and process reliability, which to a great degree depends on culture conditions, particularly the cell growth surface.

To Cindy Neeley, senior global applications scientist for cell culture at Thermo Fisher Scientific, selecting the right growth surface depends on the end goals of the culture.

“From the perspective of cell biologists, cell culture is a gateway to life science research and discovery. We run cell cultures as models that recreate a natural event.” For the model to succeed two things are necessary: physiologic relevance and systematic consistency. For example a cell-based breast cancer model is useless if it does not mimic, to some degree, what breast cancer cells naturally do. Cell-line authentication, it turns out, is a big thing these days given the irreproducibility of so much biomedical research.

Experimental consistency is in large part a function of cell culture systems ...

Experimental consistency is in large part a function of cell culture systems, which for adherent cultures is a reflection of the culture surface, whether that happens to be a microcarrier or the inside surfaces of a flask or roller bottle.

To paraphrase the real estate mantra of location, location, location, Neeley notes the significance of consistency, consistency, consistency. “That means consistency within and between wells, dishes, or flasks, and consistency among formats. If you begin with a small-format culture and scale up to a larger culture device or a different format, the surface and coating must remain consistent to eliminate that variable.”

Adherent 2D cultures are not ideal, even though most animal cells are inherently attachment-dependent. “These cultures don’t mimic nature because tissues within our bodies do not exist in two dimensions,” Neeley says. “Adherent cell culture is a shortcut, and not always the best way to obtain physiological data. 3D cell culture is more physiologically relevant but it’s not easy and has its own shortcomings. But the strength of 2D cultures is consistency because it is more controllable.”

Biocompare’s Cell Culture Equipment Search Tool
Find, compare and review cell culture
equipment from different suppliers Search

Numerous options exist for adherent culture scale-up, for example the Thermo Scientific™ Nunc™ TripleFlask™ and the Nunc Cell Factory™ which holds up to 53 layers of cell-adherent surfaces. Microcarrier cultures, another scalable format, are often run in single-use bioreactors. In theory, microcarriers can be made with the same material and modified with the same surface treatment as those of the regular culture formats for adherent cell cultures. But because the shape and dimensions of microcarriers are so different from flasks or Cell Factories, reproducing the exact conditions—consistency—is difficult.

Suspension cultures can achieve much higher cell densities than adherent cultures where cell expansion is limited by the available surface. But high cell numbers aren’t everything, Neeley says. “For vaccine manufacturing, for example, the yields of cellular byproducts in the supernatants as well as the downstream purification processes are more important and should afford careful considerations.” For cell therapy, where one batch equals one dose, cell numbers and the functionality of the cells are both critical to the efficacy and success of the treatment.

Counting adherent cells

Whether the goal is research or manufacturing, organizations involved in cell culture need to keep track of their cells’ health and productivity. Monitoring cells is more involved for adherent compared with suspension cultures, and even more difficult for microcarrier-based cultures. The NucleoCounter® instrument and method, developed by ChemoMetec, simplifies the workflow and reduces errors from pipetting and other hands-on operations.

The NucleoCounter quantifies total cells and viable cells through fluorescent staining of cell nuclei. The technique uses a non-enzymatic lysis and DAPI, a dye highly specific to DNA, providing accurate detection even in the presence of cellular debris. Cell sampling, fluorescent staining, and counting chamber loading are combined into a single workflow by ChemoMetec’s Via1-Cassette™.

Compared with more traditional methods employing trypsin digestion, NucleoCounter eliminates several centrifugation, pipetting, and incubation steps. Cell counting involving trypsin workflows take about 40 minutes, which NucleoCounter cuts to less than 5 minutes.

Adherent culture is normally associated with research applications, a market ChemoMetec had in mind when it designed its instrument. However, the company has been working with bioprocess companies on applications for vaccine manufacturing and cell therapy.

“Vaccine makers, who primarily rely on adherent cells, are looking for ways to scale out vs. scale up,” says Christian Berg, product manager.

He mentions Pall’s iCELLis bioreactor as an example. iCELLis is a single-use, microcarrier-based, fixed-bed system that allows scaling a process within a relatively small volume. The bed matrix, consisting of medical grade polyester microfibers, provides a cell growth area of 500 m2, equivalent to 3,000 roller bottles—all within a 25-liter volume. iCELLis is also available in bench-scale volume.

“When you grow cells in flasks it’s common to release them with trypsin,” Berg says, “but with microcarriers trypsin-based methods do not give reproducible results.”

By using DNA and not nuclei (or cells themselves) NucleoCounter eliminates many artifacts of conventional cell counting. “”We count cells using DNA we get more specific signals,” Berg explains. Because it does not remove dead cells and cell debris, microscopic cell counting generates false positives. “When we get a signal we know it’s definitely a cell.”

Also eliminated are signals from cell clumps or aggregates, which are difficult to distinguish from individual cells under a microscope. By staining only nuclei the image analysis algorithm in the NucleoCounter counts every cell, even if it exists within an aggregate.

Image courtesy of Dreamstime.