density measurements from optical sensors are used to follow cellular growth on-

line. Although seemingly simple, these measurements have many limitations. First,

they are extremely sensitive to the presence of bubbles, cellular debris, and other

particles in the medium. Thus, their performance decreases over the process time.

Another disadvantage is that changes in cellular size and morphology can strongly

influence cell counts, limiting the validity of the results. Additionally, both methods

do not allow to discriminate viable cells. In contrast, dielectric spectroscopy takes

advantage of the unique electrical properties cells exhibit upon exposure to an

electrical field. Cells behave as microscopic charge containers or capacitors. The

changes in cell permittivity at different frequencies describe a sigmoidal curve

designated as β-dispersion. At the inflection point of the curve, the permittivity

measurement correlates with the viable cell concentration. This method is practi-

cally insensitive to the presence of non-cellular material or non-viable cells [13].

Capacitance probes consist of platinum electrodes embedded in a resin and housed

in a stainless steel body connected to a pre-amplifier. The additional option to

perform conductivity measurements is another useful feature that could be exploited

to track changes in the culture medium that arise from by-product excretion (lactate,

ammonium) or pH control such as addition of NaHCO3 or NaOH. For their routine

verification, signal simulators and electrolyte conductivity standard solutions are

used, but calibration requires specialized service. Another option for cell growth

monitoring is the use of online imaging systems that allow to carry out cell counts

(total and viable cell concentration, cell diameter and viability) through differential

digital holographic microscopy. For example, the iLineF systems consist of a

single-use closed loop tube mounted in the bioreactor, where an integrated pump

recirculates continuously the cell culture through the measurement chamber for

image acquisition [14]. Finally, flow cytometry is increasingly used to obtain a

deeper insight into the composition of cells populations. For instance, the use of this

system allows online monitoring of cell size, apoptosis, and cell cycle [15].

Precise determination of the cell concentration and cell viability are key for

process monitoring, optimization and control. It directly influences medium feeding

to provide a sufficient supply of nutrients, and allows to determine the optimum

time of infection (TOI) and the required ratio of the number of infectious viruses to

the number of viable cells (MOI). Furthermore, many derived parameters calculated

to evaluate process performance directly relate to cell concentration measurements.

This concerns, in particular, the cell-specific virus yield (CSVY) or the cell-specific

substrate feeding rate in perfusion cultures.

5.4.4

ADHERENT VERSUS SUSPENSION CELLS

As most animal cells used in research laboratories and for biologics manufacturing

are tissue-derived, they will primarily grow as adherent cells and, thus, require a

growth surface for proliferation. This surface can be plastic materials as for T-flasks

(coated or uncoated, charged or uncharged) or carriers (porous or non-porous).

Many viral vaccine production processes still rely on adherent cells due to ease of

handling, high CSVY, well-established production facilities, and years of experi-

ence concerning regulatory approval. Moreover, recent improvements regarding

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Bioprocessing of Viral Vaccines