Multi-chamber microfluidic platform for high-precision skin permeation testing

Abstract

The established in vitro tool used for testing the absorption and penetration of chemicals through skin in
pharmacology, toxicology and cosmetic science is the static Franz diffusion cell. While widespread, Franz
cells are relatively costly, low-throughput and results may suffer from poor reproducibility. Microfluidics
has the potential to overcome these drawbacks. In this paper, we present a novel microfluidic skin permeation
platform and validate it rigorously against the Franz cell by comparing the transport of 3 model
chemicals of varying lipophilicity: caffeine, salicylic acid and testosterone. Permeation experiments through
silicone membranes show that the chip yields higher sensitivity in permeant cumulative amounts and comparable or lower coefficients of variation. Using a skin organotypic culture, we show that the chip decreases the effect of unstirred water layers that can occur in static Franz cells. The validation reported
herein sets the stage for efficient skin permeation and toxicity screening and further development of
microfluidic skin-on-chip devices.

Full text

Lab on a Chip
PAPER
Cite this: Lab Chip,2017,17,1625
Received 23rd December 2016,
Accepted 3rd April 2017
DOI: 10.1039/c6lc01574c
rsc.li/loc
Multi-chamber microfluidic platform for high-
precision skin permeation testing
M. Alberti, †
a
Y. Dancik, †*
bc
G. Sriram,
b
B. Wu,
a
Y. L. Teo,
c
Z. Feng,
a
M. Bigliardi-Qi,
bc
R. G. Wu,
a
Z. P. Wang‡*
a
and P. L. Bigliardi‡
bc
The established in vitro tool used for testing the absorption and penetration of chemicals through skin in
pharmacology, toxicology and cosmetic science is the static Franz diffusion cell. While widespread, Franz
cells are relatively costly, low-throughput and results may suffer from poor reproducibility. Microfluidics
has the potential to overcome these drawbacks. In this paper, we present a novel microfluidic skin perme-
ation platform and validate it rigorously against the Franz cell by comparing the transport of 3 model
chemicals of varying lipophilicity: caffeine, salicylic acid and testosterone. Permeation experiments through
silicone membranes show that the chip yields higher sensitivity in permeant cumulative amounts and com-
parable or lower coefficients of variation. Using a skin organotypic culture, we show that the chip de-
creases the effect of unstirred water layers that can occur in static Franz cells. The validation reported
herein sets the stage for efficient skin permeation and toxicity screening and further development of
microfluidic skin-on-chip devices.
Introduction
High-throughput, reliable and cost-effective in vitro skin per-
meation assays are in high demand. In pharmaceutical sci-
ence, transdermal drug delivery (TDD) is a major area of re-
search and development due to the advantages of cutaneous
over oral drug administration for certain classes of com-
pounds. This includes drugs requiring a prolonged period of
delivery, high potency drugs with a short biological half-life
and those subject to a significant hepatic first-pass effect.
1
The sheer number of possible drug/vehicle interactions neces-
sitates fast and effective screening methods for drug formula-
tion design.
2
In toxicology and risk assessment, estimating
toxicity following skin exposure of consumer products, pesti-
cides or lipophilic industrial solvents is of major concern.
3–5
With the European Union's 2007 Registration, Evaluation, Au-
thorization and Restriction of Chemicals (REACH) regulation
having drastically increased the number of chemicals that
need to be evaluated for toxicity
6
and the European Union's
2013 ban on the use of animals in cosmetic product testing,
Lab Chip,2017,17, 1625–1634 | 1625This journal is © The Royal Society of Chemistry 2017
a
Singapore Institute of Manufacturing Technology, A*STAR, 2 Fusionopolis Way,
Level 10, Innovis, 138634 Singapore. E-mail: yuri.dancik@imb.a-star.edu.sg,
zpwang@simtech.a-star.edu.sg
b
Experimental Dermatology Laboratory, Institute of Medical Biology, A*STAR, 8a
Biomedical Grove, #06-06, 138648 Singapore
c
Clinical Research Unit for Skin, Allergy and Regeneration, Institute of Medical
Biology, A*STAR, 8a Biomedical Grove, #06-06, 138648 Singapore
†These authors contributed equally to this work.
‡Equal contribution as supervising authors.
Fig. 1 Schematic illustrations of (a) the static Franz diffusion cell and
(b) a permeation unit of the microfluidic permeation array (μFPA). In
the μFPA, skin or a synthetic membrane is placed in the permeation
chamber, and its edges sealed by an inset defining the diffusion area.
The receptor chamber is provided with inlet and outlet channels, and it
is perfused with a receiver solution into which the compound of
interest permeates.
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toxicology and cosmetic science have a similar need for the
development and validation of high-throughput, alternative
skin permeation testing methods.
The traditional systems for in vitro skin permeation test-
ing are the static Franz and flow-through diffusion cells, in
which excised skin or a skin substitute is sandwiched be-
tween a donor compartment and a receptor compartment
(Fig. 1a). Sampling of the receptor solution occurs at pre-
determined times following application of a donor solution.
This yields profiles of the concentration or cumulative
amount over time of the compound of interest, from which
transport parameters can be derived. Though widely used,
Franz cells' typical diffusion areas of 1 to 3 cm
2
, receptor vol-
umes on the order of a few mL and time-consuming proce-
dures render them relatively costly and low-throughput.
7
In
addition, an unstirred water layer (UWL) may form in the
static Franz cell when highly permeable membranes and lipo-
philic chemicals are used. Supplementary experiments and
calculation are necessary to assess the effect of the UWL on
the chemical's permeability through the membrane.
8,9
Microfluidics has the potential to increase throughput
and improve reproducibility of experimental skin simulations
by providing cost-effective platforms that mimic the cutane-
ous physiological environment.
10
Microfluidic systems have
been designed to grow a keratinocyte and dendritic cells co-
culture,
11
integrate in vitro skin models, cultures or biopsies
and hair follicle units
12
or other tissues,
13,14
and to simulate
and test perspiration.
15–17
Systems focusing on skin perme-
ation include Mah et al.’s flow-through cell with rat or pig
skin,
18
Abaci et al.’s pumpless microfluidic platform housing
a human skin equivalent
19
and Provin et al.’s microfluidic
diffusion cell with a lipid-coated polycarbonate membrane as
a skin substitute.
20
Each of these permeability devices, how-
ever, was made of polydimethylsiloxane (PDMS), a material
unsuitable for industrial mass production, incompatible with
organic solvents and known to exhibit significant adsorption
of small hydrophobic compounds.
21–24
Differences in design
parameters between static and flow-through cell designs can
contribute to variability in the final results.
8,25–28
Hence,
there is a clear need for rigorous validation of novel micro-
fluidic devices for skin permeation against the conventional
systems. Given the growing number of organ-on-a-chip sys-
tems, such validation is also an important, often
underestimated step towards the development of reliable
skin-on-chips for in vitro permeation studies.
We present a novel microfluidic platform enabling high-
throughput skin permeation testing in a flow-through design
at a fraction of the material cost of Franz diffusion cells
(Fig. 1b). This microfluidic permeation array (μFPA) is made
of thermoplastic material, suitable for mass production. Syn-
thetic membranes, excised animal or human skin, and skin
organotypic cultures (skin-OTCs) can be easily integrated.
The device's current design allows for six permeation experi-
ments to be conducted simultaneously, important for repro-
ducibility. The flow rate can be tuned to guarantee minimal
permeant concentration in the receptor compartment in or-
der to mimic the in vivo sink condition due to cutaneous
blood flow.
29
We validated the μFPA against static Franz cells
by comparing the steady-state permeation of caffeine,
salicylic acid and testosterone. These chemicals cover a range
of lipophilicities as recommended by the OECD guideline
428.
30,31
In a first instance, silicone membranes were used to
avoid variability associated with skin. These experiments
show that the μFPA yields lower limits of detection as well as
lower coefficients of variation than the Franz cells. The μFPA
was further tested for permeation through full-thickness
skin-OTCs developed in-house. This latter experiment shows
that the μFPA lessens the effect of unstirred water layers, a
potentially significant pitfall of static Franz cells.
8
Materials and methods
μFPA design and fabrication
The μFPA is composed of a multi-chamber microfluidic chip
and a set of self-locking hollow cylindrical insets that define
six independent permeation units symmetrically arranged in
a50mm×75 mm format (Fig. 2a). Six synthetic membrane
disks or circular skin punches (7 mm in diameter) can be ac-
commodated in the open chambers of the microfluidic chip.
The edges of the membranes or skin pieces are sealed by the
self-locking insets, if necessary equipped with elastomeric an-
nuli. Each inset defines the donor chamber and the diffusion
area of the permeation unit. The assembled device is a
6-chamber μFPA with diffusion areas of 0.2 cm
2
, donor ca-
pacities of up to 300 μL and receptor volumes of 28 μL.
As shown in Fig. 2b, each microfluidic permeation unit is
composed of a cylindrical permeation chamber; a locking sta-
tion for the inset; a set of inlet and outlet channels; a de-
bubbling unit integrated along the inlet channel; and embed-
ded connectors. The receptor's inlet and outlet channels are
0.5 mm and 1 mm wide, respectively; all channels are 0.3
mm high. The inlet port is located in the upper surface of
the chip in order to connect the inlet channel to the inlet
tubing. The outlet port is located in the lower surface of the
chip so that the rim of the embedded connector protrude out
from the chip surface: this feature allows the outflow to drip
directly into the collecting well without touching the chip
surface, thus avoiding the spreading of the drops on the chip
lower surface and compromising the collection volumes. The
six outlet ports are regularly spaced as the wells of a 96-well
plate.
The microfluidic chips were fabricated by thermally bond-
ing together three microstructured thermoplastics layers, six
circular polytetrafluoroethylene (PTFE) filter membranes
(Fluoropore™, Merck KGaA, Germany), and twelve silicone
rubber connectors (Fig. 2c).
All the chips used for permeation tests on silicone mem-
branes were made of polycarbonate (PC). One chip made of
PC and one made of polyIJmethyl methacrylate) (PMMA) were
used for the permeation tests on skin-OTCs to check for vari-
ability due to the material. The microfluidic features were
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microstructured in the polymer layers by micromilling. The
PTFE filter membranes (8 mm in diameter) were placed be-
tween the second and the third layers, aligned with the
microstructured features of the de-bubbling unit. The de-
bubblers prevent bubbles from reaching the receptor cham-
bers and affecting permeation. The tubing connectors were
cast in silicone rubber (XIAMETER®RTV-4130-J, Dow
Corning, USA) from a micromilled PMMA mould. The de-
bubblers and connectors were fabricated according to de-
signs and processes developed by SIMTech Microfluidic
Foundry.
32
The self-locking insets were fabricated in PC by micro-
milling. The inset has a hollow cylindrical body and four thin
teeth (0.6 mm thick, 1.6 mm wide) protruding 1.8 mm from
its lateral surface, perpendicularly to the cylinder axis
(Fig. 2a). The locking station on the chip is composed of four
docking slots arranged in a geometry corresponding to the
inset's teeth. Each slot is composed of a vertical entrance for
the tooth and a lateral (1 mm high) locking chamber.
The base of the inset is meant to clamp and seal the edges
of the membrane or the skin tissue to avoid undesired flow
of the donor solution through eventual gaps between the
membrane and the walls of the diffusion chamber. When
using skin-OTCs, a gasket annulus made of silicone (HT-6240
BISCO®transparent, Rogers Corporation, USA) is placed be-
tween the inset and the skin tissue in order to guarantee an
adequate sealing. The inset may also allow placing a protec-
tive material on the membrane or skin and studying its influ-
ence on the permeation results.
Working principle
Once the membrane (or the skin tissue and a gasket annulus)
is placed in the permeation chamber, the inset is mounted in
the locking structure of the chip by aligning its teeth with the
corresponding vertical entrances in the locking station, press-
ing it down and turning it counterclockwise to position and
maintain the teeth inside the lateral slots (Fig. 2b). Once in
the locking position, the inset compresses the edges of the
membrane. This compression seals the system and at the
same time generates a counterforce from the inset's teeth
against the upper surface of the locking chambers that keeps
the inset in place.
The inlets are connected to six 2-stop SC0002 Tygon®ST
R-3607 tubings (Cole-Parmer GmbH, Germany) of an
Ismatec®IPCN-12 peristaltic pump (Cole-Parmer GmbH, Ger-
many) to supply the receptor buffer to each diffusion unit.
The μFPA is positioned above a UV transparent 96-well plate
so that each outlet port aligns with one row of microplate
wells. The outflow is collected directly in the wells and, at the
end of each time interval, the μFPA is slid above the plate un-
til the outlet ports align to the wells of the next row (Fig. 3).
The pump flow rate and collection time intervals are
tuned so that, unlike conventional flow-through systems, the
Fig. 2 (a) Top view image of the 6-chamber μFPA. (b) 3D illustration of a single permeation unit composing the μFPA and of the self-locking inset
mechanism. The inset enables the sealing of the skin tissue or membrane into the permeation chamber. (c) Exploded view of the permeation
microfluidic unit composing the chip.
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entire perfusate is fractioned and collected directly in 96-well
plates and easily assayed for concentrations.
Skin organotypic cultures
Immortalized human N/TERT-1 keratinocytes
33
were
maintained in keratinocyte serum free medium (K-SFM,
Gibco, ThermoFisher Scientific) supplemented with 0.09 mM
calcium, 0.2 ng mL
−1
epidermal growth factor (EGF, Gibco,
ThermoFisher Scientific), 25 μgmL
−1
bovine pituitary extract
(Gibco, ThermoFisher Scientific) and 1% penicillin–strepto-
mycin (PAN Biotech GmbH). Human primary foreskin-
derived dermal fibroblasts were maintained in Dulbecco's
modified Eagle medium (DMEM) supplemented with 10% fe-
tal bovine serum and 1% penicillin–streptomycin.
Full-thickness skin-OTCs were fabricated using a fibrin-
based dermal matrix as previously described by Toh et al.
34
Briefly, dermal equivalents were generated by encapsulating
human dermal fibroblasts (2.5 ×10
5
cells per mL of matrix)
within a fibrin-based matrix in a 6-well Falcon®cell culture
insert (polyethylene terephthalate (PET) membrane, pore size
1μm, Corning Life Sciences, MA, USA). The dermal equiva-
lents were cultured in serum-free OTC medium-A comprising
of Opti-MEM (Gibco, ThermoFisher Scientific), supplemented
with 0.1% bovine serum albumin (BSA), hydrocortisone (50
μgmL
−1
), ascorbic acid (10 mg mL
−1
), 1% SITE supplement
containing selenium (5 ng mL
−1
), insulin (10 μgmL
−1
), trans-
ferrin (5.5 μgmL
−1
), and ethanolamine (2 μgmL
−1
), 200 KIU
mL
−1
aprotinin (MP Biomedicals, CA, USA) and 1% penicil-
lin/streptomycin. After 4 days, N/TERT-1 keratinocytes (4 ×
10
5
cm
−2
) were seeded on top of fibroblast-populated dermal
equivalents and cultured under submerged conditions using
serum-free OTC medium-B comprising of K-SFM
supplemented with 0.1% BSA, hydrocortisone (50 μgmL
−1
),
ascorbic acid (10 mg mL
−1
), 1% SITE supplement, 0.2 ng
mL
−1
EGF, 200 KIU mL
−1
aprotinin, 1.2 mM calcium chloride
and 1% penicillin/streptomycin. After 2 days, the organotypic
cultures were moved to deep-well plates (Corning Life Sci-
ences, MA, USA) and cultured at air–liquid interface for 2
weeks to allow differentiation, stratification, and cornifica-
tion. During the air–liquid interface culture, serum-free OTC
media-C comprised of K-SFM supplemented with 0.1% BSA,
hydrocortisone (50 μgmL
−1
), ascorbic acid (10 mg mL
−1
), 1%
SITE+3 supplement containing selenium (5 ng mL
−1
), insulin
(10 μgmL
−1
), transferrin (5.5 μgmL
−1
), ethanolamine (2 μg
mL
−1
), linoleic acid (4.7 μgmL
−1
) and oleic acid (4.7 μg
mL
−1
), 200 KIU mL
−1
aprotinin, 1.2 mM calcium chloride
(MP Biomedicals, CA, USA) and 1% penicillin/streptomycin.
All the media supplements were obtained from Sigma-Al-
drich, unless otherwise specified.
Chemicals
Caffeine, salicylic acid, testosterone, isopropyl myristate
(IPM), propylene glycol (PG) were obtained from Sigma-Al-
drich, Singapore; ethanol (EtOH) from Merck KGaA (Ger-
many), Dulbecco's phosphate buffered saline (PBS) from GE
healthcare Life Sciences (USA). Solvents and concentrations
(Table 1) were selected to obtain solutions corresponding to
∼90% saturation, ensuring infinite applied doses and the on-
set of steady-state transport while allowing for mass balances
to be measured.
35
Silicone membrane and organotypic skin preparation
Prior to the permeation experiments, 2 ×2cm
2
pieces of 0.05
cm-thick silicone membrane (HT-6240 BISCO®transparent,
Rogers Corporation, USA) were equilibrated overnight in IPM
for the caffeine and testosterone experiments, and in PBS for
the salicylic acid experiments. The skin-OTCs attached to
their support membranes were carefully cut out from the cell
culture inserts using a scalpel on the day of the permeation
experiment. Thicknesses of the silicone membrane and skin-
OTCs pieces were measured using a digital caliper. Penetra-
tion experiments were also run on the PET membranes of the
cell culture inserts to account for any diffusive resistance of
the support membrane. For each compound, permeation ex-
periments in the μFPA and in the Franz cells were conducted
on the same day under the same environmental conditions.
Permeation experiments
μFPA. The permeation experiments with silicone mem-
branes were conducted using PC chips. Disks of 7 mm in di-
ameter were punched out of the pre-equilibrated silicone
membrane pieces and clamped into the μFPA's permeation
chambers.
The skin-OTCs experiment was performed with two differ-
ent chips, one made of PC, the other of PMMA. In this way,
the effect of material on inter-chip variability was assessed.
Six circular pieces of skin-OTCs (7 mm in diameter) attached
to support membranes were punched out of two cell culture
inserts and integrated into the μFPAs such that each chip
contained skin-OTCs from each insert. Six PC support mem-
brane disks alone were inserted into the remaining
Fig. 3 Schematic illustration of the μFPA's working principle. The μFPA
is placed on a 96-well plate such that the 6 outlet connectors are
aligned with 6 wells of a given row. At the end of each time interval,
the plate is moved until the outlet ports align with the next row of
wells. The receptor solution is collected in each well from time t(i−1)
to tIJi). As the outlet connector protrudes slightly from the chip's bot-
tom surface, contact between the connector and the edge of the well
at time tIJi), when the plates is moved, prevents undesired spillage of
the perfusate outside of the well.
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permeation chambers. In each experiment, 275 μL of donor
solution was applied to the silicone membrane or skin. To
minimize evaporation during the experiments, the top open-
ings of the self-locking insets (Fig. 3) were occluded with
sealing tape (Petri-Seal™, Diversified Biotech, USA). Immedi-
ately after each experiment, the solutions remaining in the
donor and in the receptor were collected for the mass bal-
ance. The receptor compartment was emptied by occluding
the de-bubbler with sealing tape and infusing air into it. The
sealing tape, the silicone membranes or skin pieces, the sili-
cone annulus used in the skin experiments as well as the PBS
used to wash the μFPA components were assayed.
Franz cells. Static Franz cells (PermeGear Inc., USA) with
0.64 cm
2
diffusion areas and 5 mL receptor volumes were
used. In each experiment, 900 μL of donor solution was ap-
plied to the silicone membrane or skin, thus ensuring the
same dose per cm
2
as in the μFPA. The receptor solution was
continuously stirred with magnetic stirrers. To minimize
evaporation, the donor compartment was occluded with para-
film. At each time point, a 200 μL sample was collected from
the receptor compartment, which was then topped up with
200 μL of fresh receptor solution. Following each experiment,
the donor solution and silicone membranes or skin pieces
were collected, and the parafilm and all Franz cell compo-
nents were washed with PBS for a mass balance.
Unstirred water layers. To interpret the difference in the
skin-OTC permeation experiment with caffeine, additional ex-
periments were conducted on both the μFPA and on Franz
cells to check for the effect of an unstirred water layer (UWL).
Based on Miller and Kasting's protocol,
8
the permeation of
the same caffeine solution through 1, 2, and 3 dialysis mem-
branes (Bel-Art Products, USA) mounted in series on Franz
cells was assessed. Because it was not possible to accommo-
date and properly clamp 3 dialysis membranes in the μFPA
diffusion chamber, the caffeine permeation through only 1
and 2 dialysis membranes was assessed on the microfluidic
chip. The dialysis membranes have a molecular weight cut-
off of 6000 Da and a thickness of 0.073 mm.
Analytical method
Concentrations were determined by UV spectroscopy using a
microplate reader (Synergy™H1, BioTek Instruments Inc., VT,
USA). Caffeine, salicylic acid and testosterone peak absorbance
readings were at 272 nm, 296 nm and 245 nm, respectively.
Data analysis
Transport model. Fick's second law of diffusion relates
the permeant concentration in the membrane (silicone mem-
brane or OTC skin), c
m
, to depth xin the membrane, over
time,
(1)
where D
m
is the permeant diffusion coefficient in the mem-
brane. The initial condition expressing a lack of permeant in
the membrane and boundary conditions expressing a con-
stant donor concentration in equilibrium with the membrane
concentration and receptor sink conditions are:
(2)
The solution to eqn (1) and (2) is often expressed as the
cumulative amount of permeant in the receptor, QIJt):
(3)
In eqn (3), h
m
designates the membrane thickness, K
m/d
the membrane/donor equilibrium partition coefficient and A
the diffusion area. At steady state, the cumulative amount is
given by
(4)
or, in terms of the steady state permeability coefficient (k
p
),
flux (J
ss
=k
p
c
d
) and the lag time to steady-state (t
lag
),
Q
ss
(t)=k
p
c
d
A(t−t
lag
)=J
ss
A(t−t
lag
) (5)
The receptor solution concentrations obtained from the
Franz cell and μFPA permeation experiments were converted
to cumulative amounts. In the case of the μFPA results, this
conversion accounts for the receptor flow rate. Regression of
eqn (5) against the linear part of the experimental Q
ss
IJt) pro-
files yields k
p
,t
lag
and J
ss
.
The steady state 1D model can be extended to incorporate
unstirred water layers (UWLs) above and below the mem-
brane by considering the membrane and UWL steady state
Table 1 Experimental conditions for the permeation tests on silicone membranes
Permeant Donor concentration 10
3
[μgmL
−1
] Vehicle Membrane treatment Receptor solution
Caffeine 13.5 PBS IPM PBS
Salicylic acid 2.92 PG : PBS (20 : 80) PBS PBS
Testosterone 7.07 EtOH : PBS (50 : 50) IPM EtOH : PBS (50 : 50)
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permeability coefficients as inverses of serial diffusive resis-
tances. The total diffusive resistance is thus the sum of the
resistances due to the membrane and the UWLs:
R
total
=R
m
+R
uwl
(6)
In the UWL experiments, linear regression of R
total
against
resistances from idialysis membranes is conducted to obtain
R
uwl
according to eqn (7):
8
R
total
=iR
dialysis m
+R
uwl
(7)
The total UWL thickness h
uwl
is estimated from R
uwl
and
the diffusivity of the permeant of interest in dilute aqueous
solution, D
∞
w
:
h
uwl
=D
∞
w
R
uwl
(8)
To estimate D
∞
w
, the Wilke–Chang correlation
36
is used,
(9)
where Tis the temperature in Kelvin, ϕ
w
= 2.6 is the water as-
sociation factor, MW
w
is the solvent molecular weight, η
w
=
0.89 cP is water viscosity at 25 °C and V
p
is the permeant's
molecular volume estimated from Schroeder's group contri-
bution method.
37
Numerical simulation
In the μFPA, permeant accumulation in the receptor chamber
may be expected. It is described by the left-hand term of the
general mass balance equation for flow-through diffusion
cells:
26,28,38
(10)
where V
rec
is the receptor chamber volume, c
rec
the permeant
concentration in the receptor chamber, Jthe permeant flux
from the membrane into the receptor chamber, Athe diffu-
sion area and F
rec
the receptor solution flow rate.
To evaluate the relevance of the accumulation for the
three compounds, a finite element analysis of the 3D trans-
port in the μFPA was implemented in COMSOL
Multiphysics®(COMSOL Inc., USA). The membrane diffusion
and partition coefficients (D
m
,K
m/d
) were estimated from the
Franz cells experiments.
Caffeine and salicylic acid diffusivities in the receptor so-
lution (PBS) were estimated using the Wilke–Chang correla-
tion
36
(eqn (9)). Testosterone diffusivity in the 1 : 1 v/v etha-
nol : PBS (water) mixture (D
∞
m
) was estimated from the
Leffler–Cullinan correlation:
39
(11)
with x
w
,x
e
designating the mole fractions of water and etha-
nol in the mixture, D
∞
w
and D
∞
e
the diffusion coefficients of di-
lute testosterone in water and ethanol (calculated from the
Wilke–Chang correlation) and η
e
= 1.1 cP and η
m
= 2.4 cP are
the viscosities of ethanol and the mixtures, respectively, at
25 °C.
40
Results and discussion
Flow rate
Flow-through system parameters such as receptor cell vol-
ume, flow rate, sampling frequency and collector tube vol-
ume have been reported to modify the apparent flux
41
and
the onset of steady state.
26
The flow rates in the μFPA were
set at 4 μL min
−1
in the silicone membrane experiments and
8μL min
−1
in those with skin-OTCs, where higher fluxes were
expected. These flow rates maintained receptor sink condi-
tions over the course of the experiment, avoided excessive
permeant dilution in the outflow volumes, and allowed for
collection volumes smaller than the maximum 96-well plate
capacity (<300 μL) and minimal lower limits of detection
(LOD). In order to collect the outflow during each time inter-
val in a single well, maximum time intervals of 1 h and 30
min were allowed with receptor flow rates of 4 and 8 μL
min
−1
, respectively. These flow rates determine an outflow
volume of 240 μL at these maximum time intervals, which
enables LODs of 0.1 μgmL
−1
for caffeine and salicylic acid,
and 0.4 μgmL
−1
for testosterone. The limits of quantitation
(LOQ) are 0.4 μgmL
−1
and 1.2 μgmL
−1
, respectively.
Permeation in μFPA vs. Franz cell
Permeation through silicone membranes. Fig. 4 shows the
receptor cumulative amount profiles of caffeine, salicylic acid
and testosterone in the μFPA and in the Franz cells. Mass
balance calculations show that recovery of the applied drug
in the μFPA is as good as in the Franz cells and within the
recommended guidelines of (100 ± 10) % (Table 2). The
steady state fluxes (J
ss
), permeability coefficients (k
p
) and lag
times (t
lag
) obtained from the cumulative amount profiles are
reported in Table 2. Differences in the mean lag times be-
tween the two systems are consistently less than 10 min. The
differences in the estimated cumulative amounts and fluxes
can be due to variations in the nominal diffusion areas of the
two systems; these can be related to manufacturing toler-
ances for the Franz cell orifice diameter (±0.3 mm) as well as
to small deformations of the membranes caused by the
clamping mechanisms.
28
The transport of caffeine and
salicylic acid in the μFPA and in the Franz cells are essen-
tially identical. Steady state flux and permeability coefficients
agree within 10% of their mean values. For testosterone, the
μFPA flux and permeability coefficient are 11% lower com-
pared to the Franz cells. This difference is due to testosterone
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accumulation in the μFPA receptor (Fig. 5a). As verified by nu-
merical simulations, accumulation was significant only in the
testosterone experiment. Fig. 5b compares the simulated
mass flowing out of the receptor (M
out,sim
) with the mass pen-
etrating into it from the membrane (M
m/r,sim
). At steady state,
the ratio between these two mass flows (M
out,sim
/M
m/r,sim
) sta-
bilizes to a value of 1.09. Multiplication of this accumulation
factor to the experimental mean testosterone flux and perme-
ability coefficient yields corrected values of 62 μg (cm
−2
h
−1
)
and 0.0089 cm h
−1
for the μFPA, in agreement with the Franz
cell results (Table 2). This accumulation factor is confirmed
by the testosterone mass recovered from the μFPA receptors at
the end of the experiment (∼9μg). This adds ∼46 μgcm
−2
to
the total penetrated amount at time 8 h, yielding a final μFPA
cumulative amount that more accurately reflects the Franz cell
values. The relevant accumulation of testosterone in the μFPA
is due to the lower diffusivity of testosterone in the receptor
solution. Small volume flow-through cells have been reported
to decrease accumulation
28
compared to high volume flow-
through cells. In a small volume system such as the μFPA, ac-
cumulation can be minimized by increasing the flow rate and
the outlet channel size. To demonstrate the possibility to limit
accumulation, we increased the μFPA's outlet channel size
from 0.3 to 0.75 mm and scaled the receptor chamber height
accordingly. Fig. 5c shows good agreement between the testos-
terone cumulative amounts obtained from the modified μFPA
and Franz cells. Differences in the steady state fluxes and per-
meability coefficients are about 4% (Table 3).
Fig. 4 Cumulative amount profiles of (a) caffeine, (b) salicylic acid and (c) testosterone through silicone membranes in the μFPA and the Franz cells.
Table 2 Steady state flux (J
ss
), lag time (t
lag
) and permeability coefficient
(k
p
) obtained from the cumulative amount profiles in the silicone mem-
brane permeation experiments (data shown as mean ±standard devia-
tion). The mean testosterone J
ss
and k
p
adjusted for accumulation in the
μFPA are reported in parentheses
μFPA Franz cells
Caffeine n=6 n=6
J
ss
[μgcm
−2
] 13 ± 0.13 13 ± 0.36
t
lag
[h] 0.43 ± 0.022 0.45 ± 0.072
k
p
[10
−3
cm h
−1
] 0.93 ± 0.03 0.94 ± 0.01
Permeant recovered [%] 97.1 ± 1.1 100.8 ± 1.3
Salicylic acid n=4 n=5
J
ss
[μgcm
−2
] 32 ± 0.60 30 ± 0.34
t
lag
[h] 0.43 ± 0.03 0.32 ± 0.11
k
p
[10
−3
cm h
−1
] 11 ± 0.21 10 ± 0.12
Permeant recovered [%] 99.8 ± 0.8 103.2 ± 2.2
Testosterone n=6 n=6
J
ss
[μgcm
−2
] 57 (62) ± 4.9 64 ± 2.0
t
lag
[h] 0.36 ± 0.11 0.22 ± 0.16
k
p
[10
−3
cm h
−1
] 8.2 (8.9) ± 0.70 9.2 ± 0.29
Permeant recovered [%] 98.5 ± 4.5 103.4 ± 1.7
Fig. 5 Simulation results for the determination of testosterone accumulation in the μFPA's receptor chamber. (a) Simulated testosterone
concentration in the μFPA: the mass accumulates in the receptor towards the outlet channel. (b) Comparison of the simulated testosterone mass
flowing from the membrane into the receptor (dashed line) with the mass flowing out of the outlet channel (continuous line). (c) Cumulative
amount profiles of testosterone through silicone membranes in the μFPA modified to eliminate permeant accumulation in the receptor
compartment and Franz cells.
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The precision of the two systems was assessed from the
coefficients of variation (CV) of the steady state cumulative
amount values. Sources of variations may be related to the
manufacturing tolerances for the Franz cell orifice diameter
and to small deformations of the clamped membranes. For
caffeine and salicylic acid, the steady state cumulative
amount CVs range from 1 to 2% in the μFPA, and 3 to 8% in
the Franz cells, indicating higher measurement precision in
the former (Fig. 4a and b). For testosterone, CVs are 6 to 8%
in the unmodified μFPA vs. 3 to 6% in the Franz cells
(Fig. 4c). The somewhat lower precision in the μFPA's testos-
terone test is due to cumulative amounts obtained from one
of the six permeation chambers. In the comparison of the
modified μFPA to Franz cells (Fig. 5c), CVs are 5 to 8% in the
μFPA, comparable to the 5 to 10% obtained in the Franz
cells.
Khan et al. reported high CVs for caffeine (64.9%) and
for testosterone (32.3%) permeation through PDMS mem-
branes in Franz cells.
42
Provin et al. tested their microfluidic
diffusion cell with caffeine through a lipid coated mem-
brane, reporting an average CV of 52.3%.
20
Ng et al.
achieved a CV of 6% for ibuprofen permeation through
PDMS membranes on Franz cells.
43
Bosman et al. obtained
CVs around 10% in a flow-through cell for transport of [
3
H]
dexetimide through Silastic®membranes.
44
None of these
studies reached CVs as low as the ones achieved by our
μFPA. Overall, the μFPA enables higher precision when com-
pared directly to the Franz cell or to other systems for per-
meation testing.
A possible explanation for the lower variability observed in
the μFPA is the fact that the whole perfusate is assayed, while
in the static Franz cell only samples of the receptor solution
are used for quantification of the permeate. Moreover, the
higher reproducibility of the geometrical features in the μFPA
due to the computer numerical control machining aids mini-
mizing the variability. It is expected that fabrication by injec-
tion molding may enhance this effect.
The cumulative amount profiles in Fig. 4 also show that,
with the analytical method used in this study, concentrations
of the initial permeation time points can be measured only
in the μFPA experiments. This higher sensitivity is due to the
smaller volumes in which the permeated compounds are di-
luted. Since the initial permeation typically follows an expo-
nential profile, the μFPA may allow a more accurate estimate
of the lag time if the entire experimental cumulative amount
curve is fit directly to eqn (3). Consequently the use of the
μFPA can also be advantageous for finite dose experiments,
for which sharper changes in the flux need to be detected
and smaller permeation rates need to be quantified.
Permeation through skin-OTCs. A histological section of
the skin-OTC in shown in Fig. 6a. Fig. 6b shows the receptor
cumulative amounts of caffeine following permeation
through the skin-OTCs. The steady state cumulative amounts
in the μFPA are about 20% greater than in the Franz cells.
The resulting mean permeability coefficients are 0.051 cm
h
−1
and 0.045 cm h
−1
, a 13% difference (Table 4). The perme-
ability values suggested an UWL might be affecting transport
into the receptor solutions.
8
The UWL experiments confirm
this, with mean diffusive resistances of 2.54 and 6.13 h cm
−1
estimated in the μFPA and the Franz cells, respectively
(Fig. 6c). These are due to approximately 0.062 and 0.15 cm-
thick UWLs, likely forming mainly in the unstirred donor
compartments of the μFPA and Franz cells. Factoring in the
effect of the UWLs, the mean μFPA and Franz cell permeabil-
ity coefficients increase to 0.062 and 0.059 cm h
−1
, reducing
the difference to 6.1% (Table 4). The presence of a UWL and
its impact in an in vitro transport study depend on the diffu-
sion cell's design, the permeant, the membrane, and the sol-
vent (receptor solution) under consideration.
8,45,46
For a given
membrane in contact with an aqueous solution, a greater
Fig. 6 (a) Representative hematoxylin and eosin-stained section of the full-thickness skin organotypic culture (skin-OTC) used in the caffeine
permeation experiment. (b) Cumulative amount profiles of caffeine through the skin-OTC in the μFPA vs. Franz cells. (c) Total resistances to caf-
feine diffusion in the unstirred water layer (UWL) experiments.
Table 3 Testosterone steady state flux (J
ss
), lag time (t
lag
) and permeabil-
ity coefficient (k
p
) obtained from the cumulative amount profiles in the
silicone membrane permeation experiments conducted with the μFPA
modified to eliminate receptor chamber accumulation (data shown as
mean ±standard deviation)
Modified μFPA (n= 6) Franz cells (n=6)
J
ss
[μgcm
−2
] 70.4 ± 2.8 68 ± 7.2
t
lag
[h] 0.28 ± 0.19 0.33 ± 0.16
k
p
[10
−3
cm h
−1
] 10.2 ± 0.40 9.8 ± 0.10
Permeant recovered [%] 91.4 ± 5.2 101.7 ± 2.1
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fraction of the diffusional resistance can be attributed to a
UWL for more lipophilic permeants.
8
The CV of the cumulative amounts in the steady state re-
gime stabilizes around 3% in the μFPA experiments, and
around 5% in the Franz cells, indicating higher measurement
precision in the former. These CV values are calculated from
the combined results of both μFPAs and of both skin tissues
used in this test. The μFPA's inter-chip and inter-OTC vari-
ability for caffeine permeating through the skin-OTC is very
low. Similarly, the bulk materials of the chip (PC and PMMA)
did not affect the reproducibility or the precision, with steady
state cumulative amounts in the PC and PMMA chips differ-
ing by less than 4% and the derived flux and permeability co-
efficients by less than 2%.
Conclusions and outlook
In this study, we presented and validated a microfluidic plat-
form for improved, cost-effective in vitro skin permeation
testing. This μFPA yields comparable or lower variability in
the results, allows quantitation of lower permeation rates
and reduces the problem of UWLs. In contrast to other mini-
aturization attempts,
9–11
the μFPA was developed following
the principles of design for manufacturing, with a standard
format size and a body made of a thermoplastic material;
thus it is suitable for mass production techniques. The possi-
bility of using PC, PMMA or cyclic olefin copolymer for the
bulk of the μFPA overcomes issues related to adsorption of li-
pophilic compounds usually associated with PDMS. The six-
chamber version of the μFPA described here allows for an ad-
equate number of replicates as recommended by OECD
guidelines for skin absorption
30
and by the FDA's Guidance
for Industry for in vitro release testing of semisolid dosage
form.
47
The throughput can be easily increased by using
more μFPAs in parallel and is only limited by the number of
available perfusion channels in the pumps. The design of the
μFPA can also be modified to integrate more permeation
chambers, further reducing the cost of a replicate. In addi-
tion, maintenance of the skin surface temperature at 32 °C
can be achieved by either placing the μFPA on a hotplate or
pre-heating the receptor solution.
The cost of the experiment is significantly reduced as the
μFPA requires ten-fold smaller skin tissues and lower
amounts of tested drugs compared to Franz diffusion cells.
Six glass-made Franz diffusion cells with large fabrication tol-
erances are replaced by an equivalent multi-chamber dispos-
able plastic chip. As the proposed analytical method only re-
quires a microplate reader, the μFPA is a convenient option
for those laboratories where expensive and sophisticated in-
strumentation such as high-performance liquid chromatogra-
phy or mass spectroscopy is not available.
Because of the simplicity of the μFPA, the fraction collec-
tion process and the procedural steps required for a valid
mass balance can be automated by means of a downstream
collecting system and an upstream fluidic control system, re-
spectively. The downstream collecting system will automati-
cally slide the 96-well plates placed under the chip at
predetermined time intervals, enabling long-term experi-
ments with minimal manual operations. The upstream flu-
idic control system should be programmable and its opera-
tion coordinated with the downstream collecting system. For
finite dose protocols
48
involving liquid or gaseous sub-
stances, exposure to the skin could be automated in the μFPA
by integrating microfluidics on the donor side.
The validation of this μFPA is a critical step towards the
development of a skin-on-chip device for reliable, standard-
ized and high-throughput skin permeation and toxicity
assays.
Acknowledgements
This research was financially supported by Singapore
A*STAR's Joint Council Office, grant no. 1334 K00081. We
thank Dr. Bhimsen Rout (Institute of Medical Biology,
A*STAR) and Matthew A. Miller (James L. Winkle College of
Pharmacy, University of Cincinnati) for helpful discussions.
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