Supplementary Notes:
The In vitro Sedimentation, Diffusion and Dosimetry model (ISDD) has been a subject of much debate
recently as toxicologists argue the relevance of effective dosage rather than absolute exposure as the
parameter to compare toxic potentials. (Hinderliter et al., 2010) In usual in vitro models, adherent cells
exposed to a suspension of nanoparticles. While concentration is an appropriate parameter to measure
dosage for soluble toxins (or drugs), it is not an accurate measure of the true exposure for insoluble
particles, especially those prone to sedimentation. Since adherent cells are at the bottom of cell culture
wells, they do not immediately gain exposure to the totality of nanoparticles suspended in the medium.
Instead, the amount of nanoparticles exposed to the cells increase over time as the particulates deposit to
the bottom of the well. ISDD simulates this sedimentation profile to reduce the inaccuracy in dosimetry
when absolute exposure concentrations are used.
The ISDD considerations are relevant for TiO2 nanoparticles (TNP) which are insoluble. Figure S1A
shows the deposition profile of TNP over time in 6-well and 96-well plates. Since the both well plates
have different base growth area and media volumes, the media height which effectively is the deposition
path length is different (96-well plate > 6-well plate). This causes a difference in the deposition profiles.
The volumes used for the assessment are in agreement with standard recommended volumes as well as
other published studies (1-2mL for 6-well plates; 0.1-0.2mL for 96-well plates). It is interesting to note
that due the two well plate formats incur a different TNP deposition profile. It takes roughly 4 hours for
all TNP to be deposited in 6-well plates while the same is achieved after roughly 10 hours in 96-well
plates. This is in agreement with the deposition path length.
The profile mapped in Figure S1A can be integrated to calculate the area under the curve. This number is
a factor which is dependent on the deposition profile as well as the incubation time. This factor is called
the Time-Weighed Average Factor (TWA Factor) and figure S1B shows how it increases as the
incubation times are increased. Table S1A shows one set of data which was generated for the TWA factor
and corresponding TWAD values. Interestingly, the TWA Factor is higher in 96-well plates at all times,
which is another indication of quicker deposition in this well plate format.
The utility of the TWA Factor lies in its ability to be transformed into a time-weighted average dosage.
Multiplying this factor with the starting dosage (referred to as absolute exposure) will yield a timeweighted average dosage. Since the deposition is over a limited surface area in well plates, it makes sense
to use the mass per surface area as the starting dosage where surface area is the well format dependent
growth area. Figure S1C and S1D show the time-weighted average dosage in terms of mass per surface
area. Unsurprisingly, the TWAD increases linearly with absolute exposure. In 96-well plates as exposure
time is increased from 2 to 24 hours, the TWAD is increased by 2 orders of magnitude (pay attention to
units in figures). The same is true for 6-well plates. However, when the two plate formats are compared to
each other, 96-well plates demonstrate TWAD higher than 6-well plates by one order of magnitude.
While this is still a repercussion of different media heights, TWAD considerations provide a measure of
how stark the difference may be across various well plate formats.
In the case of TNP, this difference may not create a huge discrepancy across well plates because of the
inertness of TNP even at higher concentrations. However, in the case of moderately toxic nanoparticles
(or any other particulate toxin) this phenomenon may cause a sharp difference in toxicity rendered to cells
in different well formats. Most studies perform cytotoxicity analysis in 96-well plates due to its ease of
culture and high throughput nature. However, for more in-depth analysis e.g. protein measurements,
microscopy, flow cytometry, etc., 6-well formats are more commonly used because more sample is
needed. If absolute exposure values, e.g. concentration, are not translatable across well-plates, then the
results obtained would also not be comparable. A serious consideration using appropriate nanoparticles is
needed to validate this possibility. If true, researchers would need to tune their experimental designs
accordingly in order to keep the TWAD constant across the well formats.
References
Hinderliter, P., Minard, K., Orr, G., Chrisler, W., Thrall, B., Pounds, J., and Teeguarden, J. (2010). ISDD:
A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity
studies. Particle and fibre toxicology 7(1), 36, 10.1186/1743-8977-7-36.