![Two simultaneous "skin-on-a chip" experimental setups. The system consists of a programmable syringe pump and a flow-through dynamic microfluidic device [30]. The samples are collected below the diffusion system in the collection bench.](https://www.researchgate.net/publication/355909760/figure/fig3/AS:1086425898450944@1636035527034/Two-simultaneous-skin-on-a-chip-experimental-setups-The-system-consists-of-a_Q320.jpg)
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pharmaceutics
Review
Skin-on-a-Chip Technology for Testing Transdermal Drug
Delivery—Starting Points and Recent Developments
Zsófia Varga-Medveczky, Dorottya Kocsis , Márton Bese Naszlady, Katalin Fónagy and Franciska Erd ˝o *
Citation: Varga-Medveczky, Z.;
Kocsis, D.; Naszlady, M.B.; Fónagy, K.;
Erd˝o, F. Skin-on-a-Chip Technology
for Testing Transdermal Drug
Delivery—Starting Points and Recent
Developments. Pharmaceutics 2021,13,
1852. https://doi.org/10.3390/
pharmaceutics13111852
Academic Editors: Romána Zelkóand
Istvan Antal
Received: 7 September 2021
Accepted: 27 October 2021
Published: 3 November 2021
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Copyright: © 2021 by the authors.
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Práter u. 50a,
H-1083 Budapest, Hungary; varga-medveczky.zsofia@itk.ppke.hu (Z.V.-M.); kocsis.dorottya@itk.ppke.hu (D.K.);
naszlady.marton.bese@itk.ppke.hu (M.B.N.); fonagy.katalin@ppke.hu (K.F.)
*Correspondence: erdo.franciska@itk.ppke.hu
Abstract:
During the last decades, several technologies were developed for testing drug delivery
through the dermal barrier. Investigation of drug penetration across the skin can be important in
topical pharmaceutical formulations and also in cosmeto-science. The state-of- the-art in the field
of skin diffusion measurements, different devices, and diffusion platforms used, are summarized
in the introductory part of this review. Then the methodologies applied at Pázmány Péter Catholic
University are shown in detail. The main testing platforms (Franz diffusion cells, skin-on-a-chip
devices) and the major scientific projects (P-glycoprotein interaction in the skin; new skin equivalents
for diffusion purposes) are also presented in one section. The main achievements of our research are
briefly summarized: (1) new skin-on-a-chip microfluidic devices were validated as tools for drug
penetration studies for the skin; (2) P-glycoprotein transport has an absorptive orientation in the
skin; (3) skin samples cannot be used for transporter interaction studies after freezing and thawing;
(4) penetration of hydrophilic model drugs is lower in aged than in young skin; (5) mechanical
sensitization is needed for excised rodent and pig skins for drug absorption measurements. Our
validated skin-on-a-chip platform is available for other research groups to use for testing and for
utilizing it for different purposes.
Keywords:
topical drug diffusion; skin-on-a-chip; microfluidics; Franz diffusion cells; skin equiva-
lents; drug delivery
1. State of-the-Art in Testing Topical Drug Absorption and Delivery
Transdermal drug delivery has a high importance as an alternative to traditional routes
of drug administration (e.g., per os and intravenous). It means that noninvasive drug
administration and transdermal drugs are able to bypass the liver first pass metabolism
and reduce the likelihood of side effects due to lower systemic exposure.
Topical drug administration and drug formulation has had a renaissance during the
last decades. The target organ of the topically applied drugs can be the skin itself, but
several other therapeutic indications are also possible (pain, inflammation, central nervous
system effects, humoral effects, etc.), which can be treated by transdermal drug delivery.
Testing the drug penetration through the dermal barrier is an important task in the develop-
ment of new drug formulations in pharmacokinetic/pharmacodynamic (PK/PD) profiling
studies and in dermatology. In addition to cosmeto-scientific research, there is a need to
test the transcutaneous absorption of active and cosmetic ingredients. To fully utilize the
potential of the topical administration route, it is important to optimize the delivery of
active ingredients/drugs into or through the skin. The optimization of carrier/vehicle
composition is very important at the early phases of product development. A rational
approach in designing and optimizing skin formulations requires well-defined skin models,
which are able to identify and evaluate the intrinsic properties of the formulation. Most
of the current optimization methods rely on the use of suitable ex vivo animal/human
Pharmaceutics 2021,13, 1852. https://doi.org/10.3390/pharmaceutics13111852 https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2021,13, 1852 2 of 17
models. However, increasing restrictions in the use and handling of animals and human
skin stimulated the research for suitable artificial skin models as well.
Currently, as a first approach, in silico studies have been performed as a substitute
of
in vivo
testing to simulate the drug absorption through the skin in the early phase. In
silico physiological modeling is critical for predicting dermal exposure to therapeutics and
assessing the impact of different formulations on transdermal distribution [
1
]. VeriSIM
Life (VSL) has developed BIOiSIM, a dynamic, biology-driven model which provides
a scalable computational solution through the use of machine learning (ML) integrated
physiological modeling to make fast predictions that can be applied to larger compound
datasets. Integration of ML with mechanistic modeling allows BIOiSIM to complete
biological datasets [1].
The next step in the complexity order of testing platforms is the use of
in vitro
skin
models. There exist different model membranes for testing drug permeability: (1) non-
lipid based membranes such as silicone membranes and the Strat-M
™
membrane, which
is composed of multiple layers of polyether sulfone, creating a morphology similar to
human skin, including a very tight surface layer (stratum corneum) and (2) lipid-based
model membranes, e.g., parallel artificial membrane permeability assay/PAMPA/and
phospholipid vesicle-based permeation assay/PVPA/etc.) [
2
]. The structure of the PAMPA
and PVPA systems are shown in Figures 1and 2.
Figure 1.
The structure of one-well of a 96-well plate of a parallel artificial membrane permeability
assay (PAMPA) system.
Figure 2.
The phospholipid vesicle-based permeation assay (PVPA), mimicking the stratum corneum.
It consists of a tight barrier of liposomes deposited on a cellular ester filter support.
Pharmaceutics 2021,13, 1852 3 of 17
In vitro
skin cell culturing has greatly developed over the last decades and it is
now a well-established technology in drug testing. Although many models have been
designed to assess tissue responses to the application of irritants or upon wound healing,
the absence of immune cells limits their physiological relevance, underlining the need
for more advanced models that better mimic human physiological responses that would
lead to the replacement of animal models.
In vivo
, the skin response to inflammation not
only involves tissue-resident cells, but also a range of immune cells that are recruited after
pro-inflammatory signals, which are released at the site of injury in the skin [
3
]. Depending
on the application, different cell types have been included in skin cultures (keratinocytes,
fibroblasts, melanocytes, macrophages, etc.), however, the inclusion strategies, scaffolds,
cell sources, culture media, and culture times are highly heterogeneous [
4
–
6
]. Keratinocyte
cultures serve as a skin model cultivated with collagen in different types of scaffolds.
HaCaT cells are immortalized human keratinocytes and have been extensively used to
study the epidermal homeostasis and pathophysiology [7].
To make more reproducible models, reconstructed human epidermis or full thickness
skin models have been developed. As cell-based techniques, these skin models are useful
tools for testing the phototoxicity, corrosivity, and irritancy, as well as drug permeability.
Recently, the models were also utilized in the optimization of vehicle composition of
different topical formulations.
2. Diffusion Studies
For the assessment of topical drug absorption through the skin, different technical so-
lutions (devices and equipment) are available. Additionally, for drug penetration testing,
various transport surface materials and biological systems have been developed. The different
methodological arrangements have advantages and weaknesses. The right selection of the
sufficient model is determined by the question to be answered. This can be related to the
number of compounds/formulations to be investigated, the amount of the test substances and
vehicle available, the dimensions of the diffusion surface materials (membrane, cell culture
monolayer, excised skin) and the time factor. In addition, the costs can be considered as a
determining factor in the decision. In the following Sections 2.1 and 2.2, the most widely used
technologies, diffusion platforms, and objects are discussed.
2.1. The Testing Platforms—Technologies
2.1.1. Diffusion Cells
One of the most common methodologies for studying transdermal drug delivery
in vitro
or ex vivo are the diffusion cell systems. The first equipment was developed
and described by Thomas J. Franz [
8
]—however, nowadays several subgroups can be
distinguished based on the different parameters such as the orientation (horizontal vs.
vertical), geometry, and volume of the compartments, or dynamics of the fluid flow in the
receptor chamber (static vs. flow-through).
In general, the diffusion cells consist of a donor and a receptor compartment, separated
by a barrier, i.e., an artificial membrane, cell culture, or skin sample. In the static diffusion
cells, the receptor chamber solution is continuously stirred with a magnetic bar to provide
the uniform distribution of the penetrated substance. The solution is sampled and replaced
with a new receptor fluid at each time point, which can be performed either manually or
automatically. The
µ
FLUX
™
diffusion cell (Pion Inc., Billerica, MA, USA) is a horizontal
diffusion cell system where the compartments are divided by a synthetic membrane. It is
mostly applied for studying the permeation and dissolution of poorly water soluble drug
candidates [
9
]. A further horizontal set-up is the Side-Bi-Side
™
(PermeGear, Hellertown,
PA, USA) diffusion cell system, which is suitable for studying the blood-brain barrier or
the nasal pathway [
10
]. A special chamber has been developed for imitating the surface of
the cornea, as well. Moreover, the real-time impedance measurement is also possible in
both chambers. The Navicyte Horizontal Diffusion Chamber System (Warner Instruments,
Holliston, MA, USA) is usually used for studying toxicology or transport mechanisms
Pharmaceutics 2021,13, 1852 4 of 17
in interfaces which are exposed to air (for instance dermal, pulmonary, corneal, or nasal
cells) [
11
]. The vertical arrangement of the Navicyte system has also been developed,
which is primarily designed for work with excised tissue segments and mostly used for
the characterization of the intestinal, corneal, or nasal permeability [
12
–
14
]. The vertically
oriented Franz diffusion cell system (Teledyne Hanson Research, Chatsworth, CA, USA) is
one of the “gold-standard” methods for the investigation of the transdermal drug delivery,
which is suitable for studying artificial membranes and ex vivo skin samples [
15
,
16
]. Beside
the transdermal route, several examples are known for Franz cell experiments, studying
the nasal, corneal as well as the transbuccal administration [10,17–19].
The other group of diffusion cells are the “flow-through” cells, which require a pump
to ensure the continuous fluid flow through the receptor chamber. It maintains the sink
conditions during the experiment, which is beneficial in the case of drugs having large
permeability coefficients [
20
], moreover this feature simulates the
in vivo
conditions better
than the static mode. The In-Line Cells from PermeGear (Hellertown, PA, USA) are
flow-through vertical diffusion cell systems available on the market. Various types of
experiments using in-line cells can be found in the literature, such as examination of
synthetic membranes mimicking human skin, ex vivo animal, and human skin or even nail
samples or buccal membranes [
21
–
23
]. A vertical, flow-through diffusion cell system has
also been developed by our research group, which is described in details in Section 4. The
different diffusion cell types are summarized in Table 1.
Table 1. Comparison of different diffusion cell types.
Trade Name of
the Device Manufacturer Structure Applications References
µFLUX™
Pion Inc.,
Billerica, MA,
USA
Static,
horizontal
Determination of the
intrinsic permeability
coefficients,
dissolution-permeation
study of poorly
water-soluble candidates
through artificial
membranes
[9,24]
Side-Bi-Side™
PermeGear,
Hellertown,
PA, USA
Static,
horizontal
Studying the transport
mechanism in the
blood-brain-barrier and in
the cornea, investigation of
the nasal dosage forms
[10,25]
Navicyte
Horizontal
Diffusion
Chamber System
Warner
Instruments,
Holliston, MA,
USA
Static,
horizontal
Investigation of the
toxicology or transport
mechanisms in interfaces
exposed to air (dermal,
pulmonary, corneal, nasal
cells)
[10,26]
Navicyte
Vertical
Diffusion
Chamber System
Warner
Instruments,
Holliston, MA,
USA
Static,
vertical
Characterization of the
intestinal, corneal, or nasal
permeability
[12–14]
Franz-Diffusion
Cell
Teledyne
Hanson
Research,
Chatsworth,
CA, USA
Static,
vertical
Investigation of the
transdermal, nasal, corneal,
or transbuccal
administration
[10,17–19]
In-Line Cells
PermeGear,
Hellertown,
PA, USA
Flow-
through,
vertical
Studies for artificial or ex
vivo skin or nail samples, or
buccal membranes
[21–23]
Pharmaceutics 2021,13, 1852 5 of 17
2.1.2. Organ-on-a-Chip Systems
Predicting the pharmacokinetic and dynamic properties of the candidate drugs is
resource- and time-consuming, moreover the translational possibilities of the 2D cell culture
and animal models are limited.
A novel, alternative approach is provided by the so-called organ-on-a-chip systems. It
utilizes the advanced techniques of microfabrication and tissue engineering, which also
enables to eliminate the discrepancy between the human and animal organs, as well as the
problems of the low availability of human samples. These systems overcome the problem
of the homogenous cell cultures, since the complex interactions of cell and tissue types can
be reproduced.
Moreover, tissue barriers, parenchymal tissues, and interorgan interactions can be
mimicked as well [
27
]. There are organ-on-a-chip methods for imitating the lung, heart,
liver, kidney, intestine, muscle and placenta [
27
,
28
]. Models showing both physiological
and non-physiological conditions can be designed, which are proper disease models, such
as various tumors [27] or skin diseases described in Section 5as well.
The ultimate goal is to fabricate a “body-on-a-chip” device, which includes multiple
microscale cellular environments simulating the systematic function of a human organism.
Such a kind of chip could be used to study a complex pharmacokinetic profile of drugs,
covering the absorption, distribution, metabolism and excretion. However, the proper,
physiologically relevant scaling of the different organs remain a challenge, moreover the
cost of manufacturing is relatively high [27,28].
Currently there is only a few examples for the application of different drug formula-
tions in skin-on-a-chip systems [
29
,
30
]. The most used forms are creams and gels, but other
solid, semisolid or liquid formulations can also be used in such devices. For a summary
of conventional and novel topical drug dosage forms that can be tested in skin-on-a-chip
device, see Tables 2and 3. [31].
Table 2. Different topical dosage forms that can be used in miniaturized diffusion cell device.
Conventional Topical Dosage Forms
Solids Powders, plasters
Liquids Lotions, solutions, suspensions, collodions, liniments, emulsions
Semi-solids Ointments, creams, pastes, gels, suppositories
Miscellaneous Transdermal delivery systems, alcohols, medical tapes
Table 3. Novel topical dosage forms that can be used in miniaturized diffusion cell device.
Novel Topical Dosage Forms
Novel gels
Aerosol foams
Microsponges
Muco- and bio-adhesives
Novel vesicular carriers
Nano-emulsions and nano-emulgels
Proteins and peptides
Polymers
Emulsifier-free formulations
Fullerenes
2.2. The Diffusion Objects (Diffusion Surfaces)
2.2.1. In Vitro Cell Culture Models
A huge milestone was achieved in the field of skin models by Rheinwald and
Green in 1975. They used lethally irradiated 3T3 fibroblasts as feeder layers to create
cultures of human keratinocyte colonies from keratinocyte from human skin biopsy.
Pharmaceutics 2021,13, 1852 6 of 17
This discovery made it possible for the scientists to generate large quantities of ker-
atinocytes for
in vitro
cell culture studies and gave a perspective for the treatment of
burning injury patients [
32
–
35
]. Rheinwald and Green were also the first to describe
that such monolayer cultures can differentiate and form multilayered structures [
36
].
Their method was further developed by many research groups, for a recent application
see Wufuer et al., 2016 [
37
]. Simplicity and reproducibility are major advantages of
monolayer keratinocyte cultures. However, many features of epidermis are lacking in
this model and keratinocytes are forced to adapt to artificial circumstances [38].
2.2.2. Human Reconstructed Tissues Models
In the past 10 years, several tissue culture-based 3D human skin models have been
developed and become commercially available [
39
]. They are usually classified as hu-
man reconstructed epidermis models (e.g., EpiSkin, SkinEthic, EpiDerm) and living full
thickness skin equivalent models (GraftSkin, EpiDermFT, Pheninon). The models are
composed of the human cells grown as the tissue culture and matrix equivalents normally
present in the skin [
40
,
41
]. These models have numerous application possibilities: skin
irritation, corrosion, hydration, drug delivery, anti-aging, UV protection, anti-psoriatic,
and anti-melanoma drug diffusion testing, etc. For a summary of the applications of the
marketed products, see Table 4[42–48].
2.2.3. Ex Vivo Excised Skin Models
Numerous articles reported on the evaluation of skin formulations based on the use
of ex vivo models of either human or animal origin. The choice of an appropriate ex vivo
model can be influenced by the storage, sample handling, preparation technique, and
accurate experimental setup for the evaluation of the drug permeability. The selection of
species is also an important factor to be considered. The comparison of some features of
rat, mouse, pig, and human skins is summarized is Table 5. The strength and weaknesses
of different skin model diffusion objects are summarized in Table 6.
Table 4. Application possibilities of marketed RHE and LSE products.
Reconstructed Human Epidermis Models (RHE)
Product Manufacturer Main Application Areas
EpiDerm MatTek Corporation, Ashland, MA, USA
Skin irritation, skin corrosion, skin
hydration, dermal drug delivery,
phototoxicity, dermal genotoxicity,
epidermal differentiation
EpiSkin L’Oréal, Lyon, France
Skin irritation, skin corrosion, UV
protection, bacterial adhesion, DNA
damage, dermal drug delivery
SkinEthic SkinEthic, Lyon, France
Skin irritation, skin corrosion, UV
protection, bacterial adhesion, DNA
damage, dermal drug delivery, medical
devices
SkinEthic RHPE SkinEthic, Lyon, France UV exposure, OMICS, depigmentation
EpiCs CellSystems, Troisdorf, Germany
(HENKEL), Phenion Skin irritation, skin corrosion
EpiCs-M CellSystems, Troisdorf, Germany
(HENKEL), Phenion
Skin irritation, skin corrosion,
pigmentation, environmental effects
open source reconstructed epidermis
model Phenion, Düsseldorf, Germany Skin irritation, skin corrosion
Straticell Straticell, Les Isnes, Belgium
Skin aging, barrier function, damage
related to light, acute inflammation,
pigmentation, pollution,
Pharmaceutics 2021,13, 1852 7 of 17
Table 4. Cont.
Reconstructed Human Epidermis Models (RHE)
Product Manufacturer Main Application Areas
EPI MODEL Labcyte, Gamagori, Japan Skin irritation, skin corrosion
Full-Thickness (living) Human Skin Models (LSE)
EpiDermFT MatTek Corporation, Ashland, MA, USA Anti-aging, wound healing, skin
hydration, UV protection
Phenion Full-Thickness Skin Phenion, Düsseldorf, Germany
Skin physiology and biochemistry,
clinical dermatology, transdermal drug
delivery studies, skin penetration studies,
wound healing, toxicological assessment
of chemicals, analysis of environmental
effects on skin physiology, e.g., UV and
IR irradiation
Phenion FT-AGED Phenion, Düsseldorf, Germany For testing skin aging
GraftSkin AApligraf; Organogenesis, La Jolla, CA,
USA
Wound and injuries, pressure ulcers,
varicose ulcers, etc.
Vitrolife-Skin Kyoto, Japan Skin irritation, skin corrosion
Table 5.
Comparison of the dermal barrier properties of four different species (modified from
Liu et al., 2009 [49]).
Mouse Rat Porcine Human
Full skin thickness
(average) 0.4–1 mm 1–2 mm 1.5–2 mm 2–3 mm
Epidermal thickness 9.4–13.3 µm 21.7 µm 52–100 µm 50–100 µm
Stratum corneum 2.9 µm 5 µm 12.28 µm 10–12.5 µm
Fixed skin no no yes yes
Average hair density 658 hairs/cm2289 hairs/cm211 hairs/cm211 hairs/cm2
Table 6.
Comparison of strengths and weaknesses of different
in vitro
and ex vivo skin models used in permeability studies
(modified from Flaten et al., 2015 [2]).
In Vitro/Ex Vivo Skin Models Strengths Weaknesses
Silicone membranes
Low cost
No storage problems
Reproducible
Non-lipid based
Not good model of stratum corneum
Non-biological origin
PAMPA
Low cost
Storage for longer time
Reproducible
High throughput
Synthetic lipids or non-lipid based
Not good model of stratum corneum
Non-biological origin
PVPA
Relatively low cost
Storage for longer time
Reproducible
Lipid composition can be modified
Parallelization
Lipid structure is not characterized
Non-biological origin
Skin cell cultures
Cell lines or primary cultures are
available,
Monolayers and multiple layers can be
developed
High permeability
Not sufficient barrier function
High costs, different pH
Pharmaceutics 2021,13, 1852 8 of 17
Table 6. Cont.
In Vitro/Ex Vivo Skin Models Strengths Weaknesses
Reconstructed human skin equivalents Good reproducibility
Wide spectrum of applications
High permeability
Not sufficient barrier function
High costs, different pH
Ex vivo skins
Snake
Single animal provides repeated sheds
Multiple samples from one shed
Storage at room temperature
No hair follicles
Not relevant skin metabolism
No living epidermis and dermis
Mouse
Easy handling
Convenient size
Hairless species also available
Very thin skin,
High permeability
High density of hair follicles
Hair removal results in damage
Ethical issues, storage issues
Storage issues
Rat
Easy handling
Convenient size
Hairless species also available
Thin skin, high permeability
High density of hair follicles
Hair removal results in damage
Ethical issues
Storage issues
Pig Ears are easily obtained, similarity with
human skin
Age and the anatomical region of the
animal influence the skin thickness, Hair
removal results in skin damage
Storage issues
Rabbit Ears are easily obtained, similarity with
human skin
High density of hair follicles
Hair removal results in damage
Ethical issues
Storage issues
Guinea pig Similarity with human skin
Hairless species also available
High density of hair follicles
Hair removal results in damage
Ethical issues
Storage issues
Human The most relevant model
Inter- and intra-individual variability,
differences with age, sex, race, origin,
anatomical region
3. Franz Diffusion Cell Studies at PPCU
3.1. Filter Paper and Artificial Membranes
In our initial pilot experiments, filter paper (Whatman 50) and cellulose-acetate mem-
brane (Sartorius) permeability was tested and compared to rat skin in Franz diffusion cells
using caffeine cream as a hydrophilic model drug [
50
]. The degree of diffusion of the active
molecule was proportional with the pore size of the diffusion object (filter paper: 2.7
µ
m,
cellulose acetate membrane 0.45 and 0.8
µ
m) and the excised ex vivo rat skin permeability
was comparable to 0.45 µm membrane.
3.2. Excised Skins of Different Species
The next question to be discussed was the optimization of the degree of mechanical
sensitization of the excised skin surface before the permeability experiments. This inter-
vention helps to get higher drug concentrations in the receiver chamber of the diffusion
cells to make the detection possible by different analytical techniques. For this purpose,
different species (mouse, rat, pig, and human) were investigated and various numbers
of tape stripping processes were applied (0, 5, 10, 20, 30). The thinner skins (mouse, rat)
needed less sensitization (5, 10) while the thicker skins expected more tape stripping
Pharmaceutics 2021,13, 1852 9 of 17
(20, 30) for the proper permeability. Based on the results for the routine topical transport
and absorption experiments, 10-fold sensitization was utilized in excised epilated rat skin.
Different topical drug formulations (e.g., erthromycine and Aknemycine creams or
quinidine cream and gel) were also compared in Franz diffusion cells. The effect of skin
sample freezing and thawing and the role of the age of the excised skins were also tested in
some experiments. Both the freezing and the aging (2–3 months young and 16–22 months
old rats were compared) influenced the drug absorption in a statistically significant manner.
However, the lipophilic character of the test compounds was also a determining factor in
this respect in transdermal delivery.
4. Skin-on-a-Chip Technology at PPCU
Skin-on-a-chip devices offer innovative and state-of-the-art platforms essential to
overcome the limitations of other diffusion cell techniques [51]. In our laboratory, a novel
microfluidic device concept has been designed for testing drug penetration through the skin
(Figure 3). The generalized design is optimized for manufacturing with rapid prototyping
techniques; CNC milling, polymer casting, laser cutting and 3D printing. The developed
Microfluidic Diffusion Chamber (MDC) can be used for
in vitro
/ex vivo monitoring of the
transdermal delivery of topical drugs.
Figure 3.
Two simultaneous “skin-on-a chip” experimental setups. The system consists of a programmable syringe
pump and a flow-through dynamic microfluidic device [
30
]. The samples are collected below the diffusion system in
the collection bench.
The generalized device design consists of three main parts: a polymer-based microflu-
idic channel assembly, a frame that surrounds the microfluidic channel system and a sample
holder that holds and inserts the membrane (diffusion well or integrated skin sample) into
the MDC. The device is fabricated using rapid prototyping technologies, which allow us to
create multiple MDCs that have slight variations in their design according to the needs of
the experiments. Such variations can be the presence or absence of a temperature control
unit, the geometry of the microfluidic channel system, the construction of the membrane
receiving area, or the configuration of the fluid inlet and outlet ports.
In all device generations, the polymer-based channel assembly is made from poly-
dimethylsiloxane (PDMS) (SYLGARD
™
184 Silicone Elastomer Kit, Dow Consumer Solu-
tions, Los Angeles, CA, USA). The microfluidic channel is created by mixing the PDMS
liquid monomer and its treating agent at a 10:1 ratio and incubating it at 70
◦
C for 2 h in
a CNC milled casting mold made from acrylic or aluminum. The polymerized PDMS is
cut to shape and punched through at the inlet and outlet ports. If the channel system is
Pharmaceutics 2021,13, 1852 10 of 17
created from multiple PDMS parts, then the layers are held together by sandwiching them
between laser-cut (Epilog Zing 16 Laser) poly(methyl methacrylate) (PMMA) plates. The
microfluidic channel assembly is then inserted into the frame which is held together with
bolts and nuts.
4.1. The Device (First- and Second-Generations)
The first-generation MDCs were designed and manufactured by Lukács et al. [
29
].
The chip was designed to be used with skin samples that are stretched at the bottom of
the membrane holder. In this design, the membrane holder is merged with the donor
compartment, creating a box-like shape that is conical in the inside. The examined
substance (e.g., a cream) can be loaded into the cone-shaped funnel where at the bottom
it is separated from the peripheral perfusion fluid (PPF) filled channels with the skin
sample acting as the membrane. This generation of the device used a holder and
frame made from polylactic acid (PLA) using 3D printing (CraftBot Plus 3D printer,
CraftUnique Ltd., Budapest, Hungary).
A variation of this device included a combined heater-thermistor unit that could be
used for the temperature control of the MDC. The heater unit was placed directly beneath
the PDMS layers to ensure good thermal conductivity.
In some experiments, the PLA-based clamping device turned out to not be durable
enough, the tensile strength and heat resistance of the PLA material was not sufficient;
and the frame cracked under the stress. In these cases, the PLA material was substituted
by polyethylene terephthalate glycol (PETG), which has better mechanical properties in
this application.
The second generation was developed to correct the design flaws of the first generation
as well as to implement a new sample insertion feature. The second-generation MDCs
support two types of clamping devices, the one seen in the first generation and a new one
that can be used for the insertion of transwell inserts into the chamber. The wells are placed
directly onto the PDMS gel through a window in the acrylic sheet and then the well is
pressed down using the well holder (Figure 4).
Figure 4.
Exploded schematic view of the Microfluidic Diffusion Chamber (MDC) generations.
Left
(blue): Cross-sectional
and layer-by-layer view of the first-generation temperature-controlled device with the skin sample holder.
Right
(red):
Layer-by-layer and cross-sectional view of the second-generation device with a diffusion well holder.
The frames of the second-generation MDCs were made from PETG or Onyx, the
latter is a micro carbon fiber filled nylon material with a very good tensile strength
and heat resistance, manufactured by Markforged, Inc., Watertown, MA, U.S.A. Parts
created from Onyx were printed on a Mark Two 3D printer (Markforged, Inc.). In some
applications the frame and holder were further reinforced with embedded fiberglass or
Kevlar (aramid) fibers.
Pharmaceutics 2021,13, 1852 11 of 17
5. Drug Penetration Studies
During drug development, in addition to the development of the appropriate drug
formulation, the penetration of a given drug is studied according to strict regulations (e.g.,
OECD, Guidance Notes on Dermal Absorption (No. 156) etc. [
52
–
54
]). Diffusion cells are
most commonly used to study the penetration of drugs through the skin, many variants
of which are known and available on the market (e.g., open, closed, vertical, horizontal
cell, etc.) [
55
] (see also Table 1), but during the last years, skin-on-a-chip devices are also
increasingly used for drug penetration studies [
56
]. The great advantages of these devices
is that they mimic the skin microcircular perfusion by dynamic arrangement, they are
simple to use and small in size (less tissue, cell, test substance, and vehicle demand).
At the laboratory of PPCU, drug penetration studies are performed with microfluidic
chips, the structure of which is presented in details in Section 4.1. The topical drug
absorption studies were preceded by device validation. In our experiments, caffeine was
used as a hydrophilic model drug. Caffeine is widely used in topical formulations and
is popular in the cosmetic industry for its beneficial effects on skin microcirculation and
hair growth [
50
]. Moreover, caffeine is easily available, inexpensive, and measurable with
a UV-VIS spectrophotometer at 273 nm. During validation assays, cut-to-size, properly
prepared filter paper, or cellulose acetate membranes of various pore size were placed in
the middle compartment of the device [
50
,
57
], and then caffeine cream (cream composition
described in Lukács, 2019 [
29
]) was placed in the microchip donor compartment with a
Micromen device (positive replacement pipette). Peripheral perfusion fluid (PPF) was
passed through the system at a rate of 4
µ
L/min. Samples were collected and immediately
placed onto dry ice and stored at
−
75
◦
C until the analytical investigation. The caffeine
concentration of the samples was examined with a NanoDrop spectrophotometer. The
intra- and inter-device differences were evaluated and compared. A good reproducibility
was observed in the repeated experiments. The validation experiments were summarized
in the BSc thesis of Lilla Friedreich [57].
In the first-generation skin-on-a-chip devices, the penetration of various drugs (e.g.,
caffeine, erythromycin, quinidine) was examined in mouse, rat and human excised skin
samples. The differences between the species on freshly excised and frozen samples, as
well as on native and mechanically sensitized samples (various numbers of adhesive tape-
stripping) were investigated [
29
]. In addition, young and old rat excised skin samples were
also examined (both functionally and morphologically) in fresh and frozen samples, thus
studying the changes that take place during the process of aging [
30
]. These studies were
recently expanded to include the investigation of pathological skin samples in collaboration
with the research group of Prof. Rolland Gyulai at University of Pécs. In these experiments,
the drug permeability properties of imiquimod-induced psoriatic mouse skin samples
were examined relative to samples from healthy animals and vehicle-treated controls
(unpublished data). Our drug penetration studies with Franz diffusion cells as well as
the skin-on-a-chip experiments were complemented by various microscopic techniques
(scanning electronmicroscopy, two-photon microscopy) to analyze the morphology and
structure of the uppermost layer and cross-sectional view of stratum corneum.
6. Efflux Transporter Interaction Studies (Fresh and Frozen Tissue, Erythromycin, Quinidine)
The transdermal delivery of efflux transporter substrates has also been examined
using the Franz-diffusion cell and the MDC system, which are described in detail in our
previous paper [30].
P-glycoprotein (P-gp, MDR1, ABCB1) is an extremely widespread transporter pro-
tein, it is expressed in almost all eukaryotic cell (including the different cell types of the
human skin), and in parallel its substrate specificity is also exceedingly broad. We could
demonstrate that P-gp is functional in the skin
in vivo
and ex vivo, moreover its transport
mechanism contributes to the transdermal absorption.
In this project, the transdermal penetration profile of two P-glycoprotein substrates,
erythromycin and quinidine, was investigated in the presence and absence of a P-gp
Pharmaceutics 2021,13, 1852 12 of 17
inhibitor (PSC-833). The achievements showed the inhibitory effect of PSC-833 on the
absorption of both tested compounds in the Franz diffusion cells and the MDC system.
However, these effects were present only on freshly prepared skin samples, since the protein
was damaged during the freezing and thawing of the skin samples, which increased the
tissue permeability. Therefore, the use of frozen tissues is not recommended when studying
the dermal barrier.
A further examined aspect is the effect of aging on the transporter function. It is
known from the literature that the thickness and the water, collagen, and extracellular
matrix contents of the aged skin are reduced, which might be the reason of the measured
higher permeability rate of the aged skin for lipophilic drugs and lower permeability for
special hydrophilic components.
It was also demonstrated that the magnitudes of the penetrated substrate concentra-
tions are comparable in studies on the Franz-diffusion cells and on the MDCs, and only the
shapes of the curves were different. While they are continuously ascending in the case of
the Franz-diffusion cell system, the MDC results have an absorption-plateau-elimination
three-phase profile. It corresponds to the differences in the dynamics of the fluid flow
of the two systems. As mentioned before, the Franz cell is a static diffusion system, i.e.,
the penetrated compound is accumulated in the receptor fluid. Although in our 5 h long
experiments only the absorption phase could be reached in case of erythromycin cream,
when running the experiment for a longer time, after the absorptive period, a plateau phase
is outlined (Figure 5A). However, by investigating the quinidine transport from the gel
formulation, the three-phase concentration-time profiles have been achieved (Figure 5B).
In the MDC system, a third elimination phase is also present because of the flow-through
technique, where the drug might be washed out from the receptor compartment.
Figure 5.
Skin penetration of erythromycin cream (ERY) and quinidine gel (QND) (Panel (
A
,
B
), re-
spectively) across the freshly prepared excised young rat abdominal skin in the microfluidic chip.
Concentration data are shown as means +/
−
SE, n= 3–5 [
30
]. The P-gp inhibitor (PSC-833) significantly
reduced QND and ERY penetration through the skin.
Pharmaceutics 2021,13, 1852 13 of 17
In conclusion, it was demonstrated that the MDC system can be utilized for the
investigation of transporter interactions at the dermal barrier.
7. Skin-Equivalent on a Chip Studies
In recent years, there has been a gradual increase in social pressure to reduce the
number of animal experiments. In modern societies, many different stringent laws and
regulations have been introduced to meet this demand, including a ban on the use of
animals in cosmetic experiments in the European Union since 2013. As a result, there has
been a significant increase in the demand for alternatives that can fully elicit the role of
animals in pharmaceutical and cosmetic research [58].
Skin is a large, heterogeneous, multilayered organ [
59
]. Due to its complexity, it is
difficult to create a proper alternative that can model all the properties of the skin. In
collaboration with a research group of Dr. András Czirok at Eötvös Loránd University
(Budapest), a measurement system has been created in which the transport of caffeine
was successfully examined through a skin equivalent placed in a validated microfluidic
chamber. The skin substituent was based on a polycaprolactone (PCL) membrane made by
electrospinning [
60
,
61
]. Polycaprolactone was used as a scaffold for artificial skin due to its
biocompatibility and slow biodegradation [
60
]. Electrospinning is a versatile technique
with a wide range of applications, one of which is biomedical use [
61
]. The technique
makes it possible to produce fibers not only in the micrometer but also in the nanometer
range by adjusting the parameters, thereby creating structures that are able to mimic
the natural cellular matrix. Our PCL mesh was placed on a 3D printed sample holder
consisting of two elements and then the main cylindric element of the sample holder was
loaded with collagen-1 gel. HaCaT immortalized human keratinocytes were implanted
on the mesh and placed in the sample holder, which attached to the membrane to form
a confluent monolayer. During the measurements, the transport of caffeine across the
artificial skin equivalent was compared with the human excised skin samples, the results
of which showed similar transport kinetics [58] (Figure 6).
Figure 6.
Transdermal transport measurements using skin equivalent (SE) and human skin. Caffeine
concentration in the collected fractions was measured by spectrophotometry and is shown as a
function of time [
58
]. The difference between concentration readings from the marked mesh-only
and SE samples is significant (p= 1.2 ×10−7,n= 12 in each group).
8. Discussion
As it was demonstrated in the previous paragraphs, the physiological relevance
of dermal diffusion models has been improved by various technologies to obtain more
accurate and reproducible results in drug and cosmetic research. The current article
intended to show some recent advancements in the field of skin-on-a-chip technology
achieved at Pázmány Péter Catholic University, a new player of Hungarian medical-
Pharmaceutics 2021,13, 1852 14 of 17
biotechnology research. Some details of design, engineering, and manufacturing of the
new microfluidic platforms were described and also a few examples of the validation
experiments and the major running projects for the utilization of the microchip device were
presented. At this phase of the developments, mainly excised human and animal skins
and HaCaT cell-based human skin equivalents are used as diffusion platforms. For future
directions, more relevant biological platforms are planned to be created and mounted on
the chips (e.g., human reconstructed skin and organ substituents with parallelization) [
62
].
Additionally, organ–organ interactions should be considered at the design and fabrication
of novel miniaturized investigational platforms in the pharmaceutical and cosmetical
testing laboratories [63].
As the properties of
in vitro
skin diffusion testing platforms are improving, they have
many advantages contrary to the use of
in vivo
systems. However, they still have some
cons as well (Table 7). Therefore, at the selection of the proper permeability model, the
purpose of the study and all these aspects need to be considered.
Table 7.
Advantages and limitations of the use of
in vitro
skin assays contrary to the
in vivo
models.
In Vitro Skin Permeation Studies
Pros Cons
Reduce the number of experimental animals Physiologically limited relevance
No ethical issues Lower complexity of the test system
Good reproducibility Focuses only on one target
High throughput Not all dermal cell types are included
Relatively fast No proper circulation
Lower costs than the in vivo No immunological reactions
Lower standard errors
Can be more specific
Mechanistic approach
Human cells/tissues can be used, high relevance
To further generate more reliable
in vitro
skin-on-a-chip models, there is a growing
interest in integrating different additional skin components, such as microvasculature,
immune cells, and hair follicles into the complexity of cell cultures on the diffusion surface
of microfluidic devices. On the other hand, some additional factors such as the skin of
different populations (pediatric, adult, elderly, different ethnicity, diseased) or different
anatomical regions (face, neck, scalp, forehead, upper arm, lower leg, upper leg, inner
forearm, outer forearm, back, abdomen, etc.) [
64
,
65
] should also be considered when ex
vivo models are evaluated.
These days in silico models greatly help the planning of
in vitro
/ex vivo testing and
contribute to the reduction of
in vivo
models. On the other hand, the in silico methods
are often supported by strong
in vitro
data and can provide timely results, bringing down
costs and the need for extensive biological studies. The
in vivo
testing can be limited and a
large proportion of the required criteria can be planned and met in silico [66,67].
The microfluidic devices and the novel testing technology worked out at Pázmány
Péter Catholic University will be further developed, but they are available in the current
form for other laboratories and for interested research groups as well.
Author Contributions:
Conceptualization, writing—original draft: F.E., Z.V.-M., D.K., M.B.N.
and K.F.; funding acquisition F.E. All authors have read and agreed to the published version of
the manuscript.
Funding:
Project no. ED_17-1-2017-0009 has been implemented with the support provided by the
National Research, Development, and Innovation Fund of Hungary, financed under the National
Bionics Program funding scheme.
Pharmaceutics 2021,13, 1852 15 of 17
Institutional Review Board Statement:
The excised human tissue was purchased from the plastic
surgery clinics (Révész Plasztika, Budapest, Hungary) and used in the experiments based on the
ethical permission 6501-6/2019/EKU of TUKEB, Budapest, Hungary.
Data Availability Statement: The data of our experiments can be found in the laboratory archive.
Acknowledgments:
The authors are grateful to Kristóf Iván for the funding acquisition and to Rózsa
Molnár, Lilla Friedreich, and Orsolya Berezvai, who were involved in the validation studies of Franz
diffusion cells and skin-on-a-chip device. Many thanks to Bence Lukács for manufacturing the first
prototype of the microfluidic diffusion cell.
Conflicts of Interest: The authors declare no conflict of interest.
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