Measurement of Nanoparticle Uptake by Alveolar Macrophages - Safety of Nanomaterials along Their Lifecycle - page 137

analyzing the supernatant for remaining nanoparticles, for example, by tracking
analysis. The combination of both methods delivers a fairly complete picture of par-
ticokinetics in cell culture experiments and allows us to calculate the mass of settled
or even ingested particles in ideal cases, that is, if the end point of sedimentation is
known. Then the measure “particle mass per mL” can be converted into “particles
per area.” The development of the computer program was based not only on particle
and macrophage experiments but also on numerous simulations to define a sedi-
mentation model to correct for particle overlap (Schippritt et al. 2010). The major
features of the software, its development as well as some results will be described
in this chapter.

7.2 MATERIALS AND METHODS

7.2.1 C ells
Experiments were carried out with two macrophage cell lines namely the murine cell
line RAW 264.7 and the rat AM-derived cell line NR8383. RAW 264.7 macrophages
are fast growing cells, which are strongly dependent on serum and virtually show
no locomotion. They were cultivated in DMEM supplemented with 10% fetal calf
serum (FCS), 2 mM glutamine, penicillin (100 U/mL), streptomycin (100 µg/mL),
and passaged twice a week. In contrast, NR8383 cells are highly motile cells, which
actively collect particles while moving around. They were cultured in F12-K supple-
mented with 15% FCS, 2 mM glutamine, penicillin (100 U/mL), and streptomycin
(100 µg/mL). Cultures were passaged once a week.

7.2.2 s etting uP o Bservation e xPeriments
Observation experiments were carried out with a fully automated, inverted micro-
scope (Nikon, BioStation IM) operated under cell culture conditions (37°C, 5%
CO 2 , 100% humidity). To achieve highest optical resolution, glass bottom dishes
(Ibidi, Munich, Germany) were used. The filling height of the Petri dish was a criti-
cal parameter and was set to 6 mm (3.88 mL total volume). This equaled the filling
height obtained with 200 µL pipetted into a well of 96-well plate, which is routinely
used for cell culture experiments. Since inhaled particles inside alveoli are not pri-
marily covered with proteins, experiments with macrophages were conducted in the
absence of serum. Particle concentrations were 11.25-180 µg/mL as indicated in the
following and reflected the conditions hitherto used for biological testing. For a typi-
cal experiment, cells had been cultivated in a Petri dish at low density. The medium
was then replaced by serum-free F12-K and the dish was transferred into the obser-
vation chamber. The filling height was adapted to in vitro testing conditions (6 mm).
All media and suspensions were used at 37°C, which was found indispensable to
keep the focal plane stable. Time-lapse imaging was started using a z-stack option
to take micrographs at 1 µm intervals. Images were captured at—three to four sites
every 30 s with a 20-fold objective. This allowed observation of—three to six cells
per image and optical recording of settling particles in cell-free regions. Particles
were added by replacing half of the fluid with the prewarmed, double concentrated