2

Materials

2.1

Equipment

Laminar flow hood.

Shaking CO2 incubator.

Autoclave.

Cell counting system (either automatized or microscope).

Single- or multi-use bioreactor (see below).

Bioreactor control unit (see below).

Metabolic analyzer (see Note 2).

Dissolved oxygen and pH probes (see below).

Weldable and silicone/bioprene tubing, clamps, connectors (see

Note 3).

Tubing welder (see Note 4).

A source of pressurized air, oxygen and CO2.

pH meter.

Gas analyzer (pCO2 measurement).

2.2

Bioreactor

Glass, stainless steel as well as single-use (plastic) bioreactors are

available in a large range of sizes and designs from various manu-

facturers, which has to be considered when many runs are planned

in a short time frame [10]. The exact design characteristics of a

bioreactor combined with the particular setpoints (rpm of the

impeller, gassing strategy) have a significant impact on various

parameters influencing the behavior of cells from shear stress to

mass transfer coefficients and potential dead zones [11, 12]. Litera-

ture on the subject is extensive with the described findings needing

to be verified for the particular setup, application, and/or clone.

Bioreactors using pitched blade impellers and macrosparger are

often successfully used in bench top (i.e., less than ~10 L working

volume) setups used for mammalian cell cultivation. See Fig. 1 for a

representation of a benchtop bioreactor.

2.3

Cell Culture

Media, Feeds, and

Supplements

Basal media formulations allow the expansion of the cells from the

cryovial to inoculation of the bioreactor. They also allow a short-

term cultivation of cells in the bioreactor. Feeds are concentrated

nutrient solutions added during the process to replenish consumed

media components. They allow to cultivate cells to high cell den-

sities of tens of millions of cells per mL but also aim to drive cells

towards a metabolism favorable for production and secretion of the

protein of interest [13].

Highly potent cultivation media and feeds are nowadays avail-

able from a number of different suppliers. Small quantities are

usually ordered in form of chemically defined liquid formulations.

Benchtop Bioreactors in Mammalian Cell Culture: Overview and Guidelines

3