ARTICLE

Journal of Circulating Biomarkers

Characterization of a Cell-culturing System
for the Study of Contact-independent
Extracellular Vesicle Communication
Original Research Article

Anne Louise Schacht Revenfeld1*, Evo Kristina Lindersson Søndergaard1, Allan Stensballe2,
Rikke Bæk1, Malene Møller Jørgensen1 and Kim Varming1

1 Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark
2 Department of Health Science and Technology, Laboratory for Medical Mass Spectrometry Fredrik Bajersvej, Aalborg University ,Aalborg,

Denmark
*Corresponding author(s) E-mail: anlor@rn.dk

Received 27 November 2015; Accepted 18 February 2016

DOI: 10.5772/62580

© 2016 Author(s). Licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.

Abstract

Appropriate and well-documented in vitro cell-culturing
systems are necessary to study the activity and biological
function of extracellular vesicles (EVs). The aim of this
study was to describe an experimental system, in which
dynamic, vesicle-based cell communication can be investi‐
gated. A commercially available cell-culturing system was
applied to study contact-independent cell communication,
which separated two cell populations using a membrane
with a pore size of 0.4 μm. The EV exchange characteristics
between the two compartments in the culture set-up was
preliminarily investigated in a cell-free set-up, and ana‐
lysed using the Extracellular Vesicle (EV) Array and
Nanoparticle Tracking Analysis. The application of the cell-
culturing set-up was demonstrated using co-cultures of
human primary cells. The effects of the relative placement
of the two cell populations on the phenotype of EVs found
in the cell supernatant were investigated. The results
indicate that this placement can be important for the
biological hypothesis that is being investigated. These
observations are relevant for short (<24h) as well as long
(several days) studies of vesicle-based cell communication.
Moreover, the introduced cell-culturing set-up and analyt‐

ical strategy can be used to study contact-independent
vesicle communication in a reproducible manner.

Keywords Extracellular Vesicles, Cell Communication,
Contact-independent, EV Array, Phenotype, Transwell

1. Introduction

It is currently well accepted that extracellular vesicles (EVs)
are released from a plethora of cell types in many biological
systems [1]. Furthermore, these vesicular entities can be
used as mediators of intercellular communication by a
cargo of proteins and RNAs [2]. In line with this, EVs play
an important role in many cellular processes in humans,
both in physiological and pathophysiological scenarios
[1-3]. However, uncovering the specific biological func‐
tions of EVs necessitates well-documented approaches
before the carrying out of valid functional studies. Many in
vitro experiments carried out to answer these biological
questions use two different approaches: i) EVs from one cell
population/condition are isolated and added to another cell
population [4-7]. Subsequently, the effect of the EVs on the

1J Circ Biomark, 2016, 5:3 | doi: 10.5772/62580



second cell population is investigated. ii) The two cell
populations are co-cultured, but are separated by a
membrane with a pore size that allows for the transport of
vesicles of a defined size range, as well as soluble factors [4,
8, 9]. The effects of the EV- and signal molecule-based
communication can thereafter be determined for either one
or two cell populations. The latter set-up represents the
most dynamic of the two approaches for studying vesicle-
based communication, since it is based on co-cultures.
Consequently, it incorporates the continuous communica‐
tion between the studied cells, with a consequent greater
resemblance to the in vivo conditions.

The cell-culturing set-up described in the current study is
based on the co-culture approach introduced above. The
principle of this set-up is depicted in Figure 1. Several
factors need to be taken into account when designing such
a study. A key factor, which this study investigates, is the
importance of which cell populations are placed in the
upper compartment (UC) and the lower compartment
(LC), relative to the biological hypothesis being tested. This
is relevant, since we were able to demonstrate that this
placement can affect the experimental outcomes, both for
short (<24h) and long studies (several days) of vesicle-
based cell communication. Additionally, the subsequent
analysis of the EVs must be reliable, reproducible and
provide as much information as possible. The aim of this
report is to present a combined cell culturing and analytical
set-up, which allows for an easy and reproducible detection
of differences in EV phenotypes caused by contact-
independent cellular communication.

2. Materials and Methods

2.1 Transmembrane exchange of EVs

Cell culture conditions and EV collection: Cell-free and EV-
enriched conditioned cell media were obtained from the
human colon-cancer cell line LS180 (ATCC® CL-187™;
ATCC, Manassas, VA, USA) and cultured in a growth
medium containing RPMI 1640 (Gibco, Life Technologies,
Carlsbad, CA, USA), 10 % ultracentrifuged (100.000 xg, 24
h, 4 °C; Ti45 rotor, Beckman Coulter, Brea, USA) heat-

inactivated foetal bovine serum (FBS) (Gibco), 100 U/mL
penicillin and 10 μg/mL streptomycin (Ampliqon, Odense,
DK)). To remove the cells, the conditioned media were
centrifuged at 500 xg, 5 min, RT, after collection. To the cell-
free supernatant, a protease inhibitor cocktail was added
(EDTA-free, Roche, Basel, Switzerland, diluted 1:50 in
PBS). Subsequently, the EV-rich supernatant was reduced
in volume by using a 15 mL Amicon® Ultra filter unit with
a 100k MWCO (Merck Millipore, Darmstadt, Germany).
The volume-reduced, EV-rich supernatant was washed
twice with PBS prior to use. The final volume of the cell
supernatant was approximately 1/20 of the original
volume.

Dilution series to investigate transmembrane exchange of EVs:
The following dilutions of the EV-rich supernatant from
LS180 were included: undiluted, 1:10, 1:50, 1:100, 1:500 and
1:1000. All dilutions were made with the growth medium.
Initially, 800 μL of the supernatant was placed in a well in
a 24-well plate (Nunc, Thermo Scientific, Waltham, MA,
USA). This compartment was designated as the LC (Figure
1). Subsequently, 400 μL of growth medium was put in the
UC, constituted by a Millicell® Hanging Cell Culture Insert
(#PIHT 12R 48, Merck Millipore). With this set-up, the
exchange of EVs from the LC to the UC was investigated.
To describe the transmembrane EV exchange from the UC
to the LC, the reverse set-up was made (i.e., growth
medium in the LC; EV-rich supernatant in the UC). The
culture plate holding the inserts was placed in a CO2-
incubator (temperature: 37 °C; CO2-concentration: 5 %;
relative humidity: 90 %) for 24 hours with no agitation.
After this, the contents of each compartment was harvested
into separate tubes and stored at -40 °C until semi-quanti‐
fication of the vesicles by the EV Array. No further isolation
of the EVs was performed.

2.2 Contact-independent cell communication

Isolation of cells: Buffy coats were obtained from healthy
blood donors at the Aalborg University Hospital blood
bank. Each blood donor had signed a written consent form,
allowing the use of his or her blood for research purposes.
The procedure was approved by the local ethics legislation.

Figure 1. Principle of the cell-culturing system for contact-independent cellular communication. The use of cell culture inserts was applied in order to create
a two-compartment cell-culturing system. The insert was placed within a well of a standard 24-well culture plate, creating an upper compartment (UC) and
a lower compartment (LC). Multiple flanges at the top of the insert suspended it onto the edge of the culture plate well, ensuring the lack of direct contact
between the insert and the well. An incorporated membrane in the bottom of the insert facilitated the separation of the compartments. The membrane pore
size of the applied inserts was 0.4 μm in diameter, thus allowing for the passage of smaller vesicle subsets, as well as soluble factors (indicated by the arrows).
The pore density was 1 x 108 pores/cm2, while the effective membrane area was 33 mm2.

2 J Circ Biomark, 2016, 5:3 | doi: 10.5772/62580



Isolation of peripheral blood mononuclear cells (PBMCs)
was accomplished by using gradient centrifugation with a
Lymphoprep™ (Axis-Shield, Oslo, NO). A final washing
step (350 xg, 7 min, RT) was performed to reduce the
number of platelets in the final cell suspension. The PBMCs
were either used directly after the isolation or stored at -140
°C in a storage medium (RPMI 1640, 40 % heat-inactivated
FBS, 10 % dimethyl sulphoxide (Merck Millipore), 100
U/mL penicillin and 10 μg/mL streptomycin).

Cell culture set-up and EV collection: A co-culture was created
with PBMCs from two donors with known HLA serotypes.
The PBMCs from one donor were designated the stimulator
cells, while the cells from the second donor were entitled
the responder cells. Prior to the co-culture, the stimulator
cells were irradiated (1700 rad) to inhibit their proliferation.
A Millicell® Hanging Cell Culture Insert separated the
stimulator cells and responder cells (Figure 1). The UC of
the cell-culturing system contained 2.5x105 cells in a total
volume of 400 μL, while the lower chamber contained
5x105 cells in 800 μL. Control samples with monocultures
of both stimulator and responder cells were also included.
Here, 5x104 of either responder or stimulator cells were
seeded in a 96-well plate in a total of 150 μL culture
medium. The cells were placed in a CO2-incubator (tem‐
perature: 37 °C; CO2-concentration: 5 %; relative humidity:
90 %) for six days. On Day 6, the conditioned cell media
were harvested from each compartment separately and
centrifuged once at 500 xg, 10 min, RT to pellet cells. A
protease inhibitor cocktail (EDTA-free, diluted 1:50 in PBS)
was added to the cell-free supernatants prior to storage at
-40 °C until vesicle phenotyping by the EV Array or size
determination with Nanoparticle Tracking Analysis (NTA)
was carried out. No further isolation of the EVs was
performed.

2.3 EV Array analysis

Production of microarrays: Microarray printing was per‐
formed on a SpotBot® Extreme Protein Edition Microarray
Printer (Sunnyvale, ArrayIt, CA, US), as previously
described [10].

Antibodies/proteins for the phenotyping of vesicles: For the
phenotyping, a total of 10 anti-human antibodies and one
protein were used. They are listed in the following with the
corresponding product number (#) or clone. From R&D
Systems (Minneapolis, MN, USA): CD82 (#423524) and
TNFRI (#DY225). From Biolegend: CD63 (MEM-259) and
HLA-DR (L243). From LifeSpan BioSciences, Inc. (Seattle,
WA, USA): CD9 (#LS-C35418) and CD81 (#LS-B7347). From
Abcam (Cambridge, MA, USA): Flotilin-1 (#Ab41927).
From Haematologic Technologies, Inc. (Essex Juncton, VT,
USA): Lactadherin (#BLAC-1200) (protein). From BD
Biosciences: CD3 (Hit3a). From Abbiotec (San Diego, CA,
USA): CD11a (HI111). From eBioscience (San Diego, CA,
USA): ICAM-1 (R6.5). All antibodies/proteins for the
phenotyping were printed in triplicate at 200 μg/mL
diluted in PBS containing 5 % glycerol.

Antibodies for the semi-quantification of vesicles: For the semi-
quantification of vesicles, anti-CD9, anti-CD63 and anti-
CD81 were printed on the microarray slides, as previously
described [11]. In short, 18 repeated spots were printed
using a cocktail of the three antibodies, each in a concen‐
tration of 100 μg/mL.

Catching and visualization: The entire procedure was
performed as described previously [11]. In brief, the
printed slides were blocked, incubated with the EV-
containing sample, followed by the detection of bound EVs
with biotinylated anti-CD9, -CD63 and -CD81 and, subse‐
quently, Cy5-labelled streptavidin.

Data analysis: Data analysis and the creation of graphs were
carried out using SigmaPlot (version 11, Systat Software
Inc, San Jose, CA, USA) and Excel (version 2013, Microsoft,
Redmond, WA, USA). For a given antibody spot, the signal
intensity was calculated as the mean signal of the triplicate
spots (18 spots for semi-quantification) in relation to the
sample signal of the negative spot (PBS) in triplicate. For
each spot, the signal intensity was calculated by subtracting
the mean of the background (no sample/blank, washing
buffer) from the mean of the foreground (spot signal).
Before visualization and the calculation of linearity, the
antibody signal intensities were converted to log space by
log2 transformation. The transmembrane EV exchange,
evaluated from the semi-quantitative data (Figure 2), was
calculated as: EV Array signal in compartment with growth
medium/ EV Array signal in compartment with added EV-
rich cell supernatant.

2.4 Nanoparticle tracking analysis (NTA)

Instrument details: For EV-size determination, NTA was
performed with a NanoSight LM10-HS system equipped
with a finely tuned 405 nm laser (NanoSight Ltd., Ames‐
bury, UK), supplied with the NTA 3.0 0060 analytical
software, which was used for capturing and analysing the
data. The camera type was EMCCD and the camera level
was set to 11.

Size determination: The NanoSight was calibrated with 100
nm polystyrene latex microbeads (Thermo Scientific,
Fremont, USA) prior to analysis. Dulbecco's phosphate
buffered saline (DPBS) without Ca2 + and Mg+, and filtered
using a 0.22 μm filter prior to use (Lonza, Verviers,
Belgium), was used to dilute the microbeads (1:1000
dilution) and the EV-rich supernatants (1:40 dilution). The
samples were manually injected into the sample chamber
and a temperature-measuring device inserted directly into
the sample chamber was applied to record the temperature
of the sample for each run. Samples were measured with a
slide shutter of 600 and with a slider gain of 300 for 60 s.
The applied dilutions yielded between 20-100 particles/
frame and each sample was measured in triplicate. Subse‐
quently, the integrated software automatically processed
the data, yielding values such as the mean, the median, the

3Anne Louise Schacht Revenfeld, Evo Kristina Lindersson Søndergaard, Allan Stensballe, Rikke Bæk, Malene Møller Jørgensen and Kim Varming:
Characterization of a Cell-culturing System for the Study of Contact-independent Extracellular Vesicle Communication



mode particle size, the value of the highest point of the peak
and the corresponding standard deviations. The detection
threshold was set to three and the blur setting was 9 x 9.

3. Results

3.1 Transmembrane EV exchange between the two compartments
of the cell-culturing set-up

Initially, the exchange characteristics of EVs between the
two compartments of the cell-culturing system (Figure 1)
were investigated using a cell-free approach. This was done
to evaluate whether the experimental outcome was affected
by the relative position of the cells in the two compart‐
ments. For this purpose, a volume-reduced, EV-rich
supernatant from the human colon-cancer cell line LS180
was placed in one of the compartments and a growth
medium was placed in the opposite compartment. Conse‐
quently, the transport of EVs from the UC to the LC, and in

the reciprocal direction, was studied. After 24h, the extent
of transmembrane EV exchange was evaluated in each
compartment by using the EV Array to semi-quantify the
contents of CD9-, CD63- and/or CD81-containing EVs. The
results from this analysis can be seen in Figure 2. Here, the
transmembrane EV exchange illustrated in Figure 2A
designates the ratio between the EV Array signal from the
compartment, which initially only contained growth
medium, and the signal from the EV starting compartment.
From Figure 2A, it can be deduced that the transmembrane
EV exchange was greater from the UC to the LC in the
investigated time frame, as compared to the reciprocal
direction. This difference was already noticeable with the
first dilution. Here, the signal from the UC of the lower to
upper set-up (orange) was reduced to roughly 50 % of that
from the LC, while the signals from both UC and LC of the
reversed set-up were almost similar (blue). Furthermore,
with the 1:50 dilution, practically no signal could be
detected in the UC for the lower to the upper combination.

Figure 2. Transmembrane exchange of EVs between the two compartments of the cell-culturing set-up. The exchange of EVs from the UC to LC of the cell-
culturing set-up, and vice versa, was evaluated. A) An EV-rich, cell-free supernatant from the human cell line LS180 was placed in one of the two compartments,
while growth medium was placed in the opposite compartment. This was done with six dilutions of the supernatant, as indicated on the x-axis. After 24h, the
contents of each compartment were harvested, and the amount of CD9, CD63 and/or CD81 was semi-quantified using the EV Array for the EVs that had
bound to the array. The transmembrane EV exchange shows the relationship between the EV Array signals from the compartment initially containing growth
medium and the compartment, to which the EV-rich supernatant was originally added. B) The size distribution of the EVs in the UC and LC of the 1:50 dilution
was determined by NTA. Here, the upper to lower samples are shown, while a summary of the remaining samples can be found in Table 1. In addition, the
applied growth medium was also analysed separately. For each sample, the histogram indicates the mean of the triplicates. The numbers in blue indicate the
particle size at the peak measurements in the histogram, while the red bars indicate +/- 1 standard error of the mean. The x-axis has been truncated from 1000
nm to 700 nm, since no particles were detected in the largest size range.

4 J Circ Biomark, 2016, 5:3 | doi: 10.5772/62580



For the reversed combination, this tendency was observed
later, at the 1:500 dilution.

As an additional investigation, the size distributions of the
EVs present in each compartment were determined by
Nanoparticle Tracking Analysis (NTA) after 24h of trans‐
membrane EV exchange. With this investigation, it was
possible to evaluate whether both small and large EVs were
present in the EV-rich supernatants. The analysed samples
included those from the 1:50 dilution (Figure 2). The
histograms of the EV size distribution for the upper to
lower combination can be seen in Figure 2B. The remaining
results from the NTA are summarized in Table 1, in which
they can be compared to the corresponding EV Array
signals. The samples presented in Figure 2B contained
smaller vesicles, with 90 % being smaller than approxi‐
mately 100 nm and 150 nm for the LC and UC, respectively.
Moreover, no larger vesicles/particles (>400 nm) were
present in any of the samples (Figure 2B), including the
growth medium analysed prior to the investigation of the
transmembrane EV exchange. From the mean and mode
values found in Table 1, it is not possible to accurately
distinguish the transferred EVs from those inherently
present in the growth medium. However, two points can
be deduced when comparing the NTA data with the
matching EV Array signals. First, it appears that even
though vesicles were present in the growth medium,
despite using UC FCS, these EVs do not elicit a specific and
interfering EV Array signal. This consequently leads to the
second point, indicating that the EV Array signals detected
in the compartments initially only holding growth medium
come from a transmembrane exchange of LS180 EVs. The
EV Array signal from that compartment was 8.6 fold higher
in the upper to lower scenario than in the opposite direction
(Table 1). This accordingly supports the tendency deduced
from Figure 2A, in which the transmembrane EV exchange
was seemingly greatest from the UC to the LC.

EV array
signal

Mean size (SEM)
[nm]

Mode (SEM) [nm]

EV transport: Upper to lower

UC 7.8 105.9 (5.5) 88.6 (2.6)

LC 4.3 79.3 (2.7) 87.3 (9.1)

EV transport: Lower to upper

LC 7.7 102.6 (0.6) 91.8 (5.8)

UC 0.5 97.1 (3.3) 85.3 (5.1)

Growth medium 0 103.9 (3.1) 102.1 (3.7)

Table 1. Size distribution of the vesicles after 24h of transmembrane EV
exchange. An EV-rich, cell-free supernatant was placed in either the UC or
LC of the cell-culturing set-up, while growth medium was placed in the
opposite compartment. After 24h, the size of the EVs present in each
compartment was determined by nanoparticle tracking analysis (NTA). The
data presented are from the 1:50 dilution shown in Figure 2. For each
compartment, the signal from the EV array is given along with the results
from the NTA. The mean and the mode, and the corresponding standard
error of the mean (SEM), are given for triplicate measurements of each
sample.

3.2 Demonstration of the applicability of the cell-culturing set-
up and analysis platform

In addition to the cell-free set-up, the presented cell-
culturing system was further investigated using a co-
culture of human peripheral blood mononuclear cells
(PBMCs). In this co-culture, one cell population was
designated as the stimulator cells, since these cells could
induce an immunological response in the responder cells,
constituted by the second cell population. The stimulator
cells were not subject to a reciprocal activation, since they
had been irradiated, making the cell communication one-
way. Both combinations of the stimulator and the respond‐
er cells in the UC and LC were made and, after six days of
co-culturing, the phenotype of the cell-derived EVs was
determined, as shown in Figure 3. It can be seen for both
combinations of the co-culture that all the reported protein
markers were present on EVs above control levels (grey
bars). Moreover, the presence of the more EV-specific
markers, including CD9, CD81 and CD82, were the most
abundant, when compared to the remaining markers
included in the EV phenotyping. One exception to this
related to CD63, which was only marginally detected in
comparison to CD9, CD81 and CD82. The EV Array signals
for the majority of the more cell-specific markers were at
the lower level of detection for the applied array, with the
exception of CD11a and TNFRI. Nonetheless, the relative
placement of the two cell populations in the co-culturing
set-up affected the obtained EV Array signals for both the
vesicle- and cell-specific markers, although to a varying
degree. From the responder cell compartments (Figure 3,
top panel), some of the most pronounced differences were
observed for ICAM-1, with an almost 3.5 fold higher signal
for this marker from the EVs harvested from the UC
(orange) than for the LC (blue) of the responder cells.
Moreover, CD3 could only be detected from EVs in the
responder cell compartment in one of the settings. Finally,
the detection of CD81 was almost 1.5 times higher in the
UC sample, when compared to the LC sample. In contrast,
comparable signals for CD81 were observed in both
stimulator cell compartments (Figure 3, bottom panel). For
these stimulator cells, a differential EV Array signal was
particularly observed for HLA-DR. This marker was
preferentially present, when analysing the EVs in the UC
sample (blue), with an approximate 2.5 fold enrichment. A
similar observation was demonstrated for CD63.

As a final notion, the reproducibility of the presented cell-
culturing set-up and analysis platform was evaluated.
Accordingly, the contact-independent co-culture was
repeated several weeks apart, using cells from the same
individuals. In Figure 4, the results of three selected
markers are presented for these two technical replicates. It
can be seen that, for CD9 and CD81, the detected EV signals
predominantly correlated from replicate to replicate, with
10 of the 12 obtained %CV values ranging from 1.7%-15.6%
(Figure 4). The last two %CV values for CD9 and CD81 were
30% and 36.9%, and they were both calculated from the

5Anne Louise Schacht Revenfeld, Evo Kristina Lindersson Søndergaard, Allan Stensballe, Rikke Bæk, Malene Møller Jørgensen and Kim Varming:
Characterization of a Cell-culturing System for the Study of Contact-independent Extracellular Vesicle Communication



stimulator cells in the lower compartment. For CD63, four
of the six %CV values could not be determined, as either
one or both EV Array signals from the two technical
replicates were below the lower limit of detection (LOD).
The last two %CV values for CD63 were 6.9 and 100.7. For
the latter sample, the detected log2 signals were very close
to the lower LOD (log2 values: 0.13 and 0.75).

4. Discussion and Conclusion

In the research field of EVs, much effort is put into deci‐
phering the biological functions of these vesicular entities.
Consequently, useful experimental and analytical plat‐
forms are of great interest. In this technical report, we have
investigated a commonly used cell-culturing system for the
study of dynamic, contact-independent cell communica‐
tion, focusing on the ability of the EVs involved to enter
each compartment (depicted in Figure 1). In the applied set-
up, a membrane with 0.4 μm pores separated the cells, but
other pore sizes between 1-8 μm are also available. Initially,
we wanted to investigate whether the transmembrane
exchange of EVs were similar for both compartments. With
the results presented in Figure 2, it is apparent that for this
short study (<24h), the exchange of EVs was greater from
the UC to the LC than in the opposite direction. The
observed differential EV exchange is relevant because there

are many studies investigating selected features of contact-
independent cellular communication that have applied
short incubation times [4, 8, 9, 12, 13]. Our study also
suggests that this is important, since for short studies it may
be advantageous to place the primary EV donor cells in the
UC, while the primary recipient cells should be placed in
the LC. These suggestions are based exclusively on the
observations derived from Figure 2 and Table 1, since it was
not determined which contributing factors could have
affected this differential transmembrane EV exchange,
such as diffusion, sedimentation or hydrostatic pressure.
Moreover, the transmembrane EV exchange could have
been investigated using a cell-containing set-up. Neverthe‐
less, a cell-free approach was chosen since it incorporated
the possibility of demonstrating any concentration depend‐
ency of the transmembrane EV exchange by making precise
dilutions of the EV-rich supernatant. This option was not
given when using cells, as it has not been confirmed that
there is a linear correlation between the number of cells and
the amount of EVs produced. One element that may
alleviate any possible effects of the observed differential EV
transport is agitation of the culturing set-up. However, this
was not investigated in the current study, as agitation has
not been employed in several other studies investigating
contact-independent cell communication [4, 8, 9, 12, 13].
Consequently, the presented data relate to the practice of

Figure 3. EV phenotype after the co-culture of human peripheral blood mononuclear cells. A six-day, contact-independent co-culture between peripheral
blood mononuclear cells (PBMCs) from two different individuals was created. The phenotypes of the cell-derived EVs were evaluated using the EV Array to
detect differences caused by the relative placement of the two cell populations. Consequently, both combinations of responder (resp) and stimulator (stim)
cells in the lower compartment (LC) and upper compartment (UC) were included. For non-specific reactions, monocultures of both stimulator (stim ctrl) and
responder cells (resp ctrl) were included. Antibodies targeting the listed markers on the x-axis were used for the capturing of the EVs. The panel of antibodies
targeted both immunological markers, which were related to the different leukocyte subsets within the PBMCs, and more general vesicle-related markers.
The observed signal for each of the markers implies the simultaneous presence of CD9, CD63 and/or CD81, since a cocktail of antibodies against these three
EV markers was used for detection. For each combination of responder and stimulator cells in the UC and the LC, the bars show a mean value ± SEM from
two independent experiments using cells from the same two donors. EV Array measurements were performed in triplicate.

6 J Circ Biomark, 2016, 5:3 | doi: 10.5772/62580



many relevant studies. As a technical note, it can be seen in
Table 1 that the mean size of vesicles detected in the
compartment, which initially only contained growth
medium, was variable (79.3 nm versus 97.1 nm, respective‐
ly). When addressing the corresponding EV Array signals
(4.3 versus 0.5, respectively), it has previously been
demonstrated that vesicles that bind to the array are
primarily <100 nm [11]. However, despite this reported size
maximum, the mean size of the eluted EVs from the array
in reality had a mean size <85 nm. Hence, for the present
study, this correlates with the observation that the smaller
sized vesicles yielded a higher amount of detected CD9,
CD63 and CD81.

The differential transport of EVs across the insert mem‐
brane was observed in a simplified version of the cell-
culturing system, which does not incorporate the dynamics
of a cell-based set-up, where EVs are continuously pro‐
duced and taken up by cells. However, the observed
difference may be a contributing factor in the cell-based
system as well. This might possibly be connected to the
differences detected for the phenotype of the cell-derived
EVs, which was affected by the relative placement of the
two cell populations (Figure 3). Biological variations alone
could, most likely, not account for this phenotypic differ‐
ence, since the set-up was reproducible (Figure 4). Hence,
this points to other determining factors, such as a differen‐
tial transmembrane EV exchange. Moreover, the induced
EV phenotypes in the co-cultures were not artefactual, as
all investigated markers were detected above control
levels. The cellular phenotype, which was investigated for
a subset of the responder cells, was not affected to the same
degree as the vesicular phenotype by the relative placement
of the cell populations (data not shown). Any biological
relevance of these observations has not yet been investi‐
gated in more detail. However, the EV phenotype may be
more sensitive to the experimental design, with a possible
importance of the functional consequences of the EV-based
communication between the involved cell populations.
Consequently, this stresses the point that it may not be
trivial how the cell populations are placed relative to each
other, both for short and long studies of contact-independ‐
ent cell communication. Several studies do not account for

their choice of the relative placement of the cells in the two
compartments [4, 8, 9, 12, 13], which our study indicates as
being relevant. As a final note, the recommendations,
which are made using the presented data, are based on a
co-culture of PBMCs. This cellular population consists
predominantly of suspension cells with a smaller fraction
of adherent cells. Therefore, applying other cell types with
different characteristics could entail other affecting factors,
such as an increased blocking of the membrane pores.
Nevertheless, this yet again underlines the significance for
investigating any influence that the relative placement of
the cells has on the experimental outcome, regardless of the
cell types used.

As mentioned previously, it is essential to have reliable
assays to delineate the biology and functionality of EVs.
Therefore, the reproducibility of the cell-based experiments
was evaluated by repeating the co-cultures and the
subsequent analysis after several weeks, using cells from
the same individuals. As presented in Figure 4, the ob‐
tained %CV values for CD9 and CD81 ranged from 1.7 to
36.9. However, 10 of these 12 samples had a %CV below 16,
whereas the two highest values were calculated for the
same sample, namely the stimulator cells in the LC,
pointing to an isolated trend for this sample. It has been
established that the EV Array yields %CV values below 10
when working with plasma samples [10], and %CV values
below 25 have been proposed as an acceptable limit for
immunoassays [14]. However, this limit can be expanded
if an experimental rationale is present [14], which may be
the case when both technical and biological variations exist,
as in this study. Nonetheless, one of the %CV values for
CD63 differed to a somewhat greater extent, amounting to
100.7 (Figure 4). The log2 intensities from the relevant
replicates, forming the basis of the %CV calculation, were
0.13 and 0.75. Hence, the standard deviation was very large
compared to the mean of the two intensity values, with a
consequent large impact on the calculated %CV. This is an
inherent issue for small intensities close to the lower LOD,
which is difficult to completely circumvent. A third
technical replicate may serve to improve the %CV. How‐
ever, several other results from our work point to the fact
that CD63 is a poor marker for EVs in general since it occurs

Figure 4. Reproducibility of the cell-culturing set-up and analytical platform. To demonstrate the reproducibility of the presented methodology, two
independent experiments of the contact-independent co-culture were performed (using one biological replicate). The plots display the results from three
selected EV markers; CD9, CD63 and CD81 (also shown in Figure 3] from the EV Array analysis of the resulting cell supernatants from the UC and the LC.
The corresponding %CV values are noted below each sample, and were calculated based on the two technical replicates. The %CV values were not calculated
for the samples with log2 signal intensities below the lower limit of detection (marked by a -).

7Anne Louise Schacht Revenfeld, Evo Kristina Lindersson Søndergaard, Allan Stensballe, Rikke Bæk, Malene Møller Jørgensen and Kim Varming:
Characterization of a Cell-culturing System for the Study of Contact-independent Extracellular Vesicle Communication



in relatively small amounts, as compared to CD9 and CD81
(unpublished data and [10, 11, 15-17]).

As part of the combined experimental set-up presented
here, the determination of the EV phenotype plays a major
role. Using the EV Array to phenotype the vesicles provides
the opportunity to gain much information about the system
that is being studied. In the context of vesicle-based cell
communication, the EV phenotype may be used for several
purposes. First, the use of an extensive EV phenotype may
aid in fine-tuning the biological hypothesis that is being
investigated. In this study, we targeted 11 protein markers.
However, 60 analytes (in this case, antibodies) can current‐
ly be used simultaneously for each sample when pheno‐
typing EVs with the EV Array [10]. Consequently, the EV
phenotype may not only provide a large amount of
information but it can also be used to optimize the experi‐
mental design in an iterative fashion. Furthermore, when
applying the EV Array, this can be achieved without having
to resort to extensive EV isolation procedures. Currently,
the designed EV Array does not provide direct information
about which cells produce the EVs and their absolute
quantities. However, by linking the extensive EV pheno‐
type to a number of additional experimental outcomes,
such as the cellular phenotype, or the EV RNA and
subproteome cargo, unravelling the biological functions of
the EV-based communication becomes substantial and
highly relevant.

5. Compliance with Ethical Research Standards

The authors declare no conflicts of interest. All research on
human subjects presented in this paper was conducted in
accordance with the local ethics legislation. Each human
subject signed a written consent form, allowing for the use
of his or her blood for research purposes.

6. Acknowledgements

The authors thankfully acknowledge Dr. Shona Pedersen
and Clinical Professor Søren Risom Kristensen (Depart‐
ment of Clinical Biochemistry, Aalborg University Hospi‐
tal, Aalborg, Denmark) for their support in the NTA
experiments. The authors also acknowledge technician
Anne Elbæk (Department of Clinical Immunology, Aal‐
borg University Hospital, Aalborg, Denmark) for excellent
technical assistance.

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9Anne Louise Schacht Revenfeld, Evo Kristina Lindersson Søndergaard, Allan Stensballe, Rikke Bæk, Malene Møller Jørgensen and Kim Varming:
Characterization of a Cell-culturing System for the Study of Contact-independent Extracellular Vesicle Communication