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Fabrication of biomimetic placental barrier structures within a microfluidic device utilizing two-photon polymerization

1. introduction used. This technique takes advantage of two-photon
absorption, which results in true 3D structuring and
Research, in the field of placental biology, represents a spatial resolution below 100 nm [8,11,12] . Due to this very
challenging topic. Although a variety of in vivo animal high resolution, structural parameters such as external
models, ex vivo placental perfusion models and in vitro shape, pore size and internal porosity of fabricated
models have been described, current approaches are structures can be manufactured in a precise manner [8,13] .
difficult to perform, time-consuming and often carry the In this study, GelMOD-AEMA was combined with 2PP
[1]
risk of harming the unborn fetus . However, progressive to produce high-resolution structures with micrometer-
research and the increasing provision of human tissue precision, to closely mimic the native microenvironment
samples for study purpose enable the advancement of placental tissue, as there are currently no publications
of current methods and the establishment of new about utilizing 2PP techniques for fabrication of
approaches. In recent years, microfluidic methods have placental barrier models. Firstly, cellular response of
[2]
gained increasing attention in this respect . This novel the GelMOD-AEMA biomaterial as well as the applied
and highly interdisciplinary research field combines photoinitiators was tested in a two-dimensional approach
microfabrication with bioengineering and material to ensure maximal biocompatibility. Secondly, a study
sciences. Microfluidic barrier models are of particular on biomaterial composition was performed to find the
interest, as the culture conditions resemble the dynamics best-suited, photosensitive material compatible with
similar to native human tissue. The complexity of highly placental trophoblast cells. Because the establishment
specialized organs, such as, for example the placenta, of a placenta-on-a-chip model to recreate an in vivo-like
can be recapitulated using these micro-engineered cell villous membrane structure was envisaged, resolution
[3]
culture systems thereby allowing human cells to grow and stability of the GelMOD-AEMA 2PP processing
[4]
under physiologically relevant conditions . With respect were further evaluated. Finally, as proof-of-principle the
to placental biology, this controllable microenvironment presented villous placental membrane model was used to
can be engineered to reflect the multi-layered mem- study the transport of glucose-sized molecules.
branous structure of the placenta in combination with
native conditions, regarding media flow and media 2. Experimental
[3]
composition . With a high-resolution 3D-printing
technique, the membranous structure of the placental 2.1 Cell Culture
membrane can be mimicked precisely. To simulate the
barrier function of the placenta, aside from selection of For this study, human umbilical-vein endothelial cells
cell model also the influence of the membrane material (HUVEC) and human choriocarcinoma cells (BeWo
2
has to be considered. A promising material in this context B30) were grown in 75 cm cell culture flasks (Greiner)
is for instance gelatin, as it is derived from collagen, as monolayer cultures at 5% CO and 37 °C. Cells were
2
which is the main component of placental connective sub-cultivated before reaching confluence, using 1-fold
tissue, extensively used in tissue engineering [5,6] . The trypsin-EDTA (Sigma) solution. BeWo B30 cells were
main benefit of gelatin is that it can be modified to grown in DMEM Ham nutrient composition F12 media
enhance functionality and versatility of the biomaterial. (Sigma) supplemented with 10% fetal bovine serum (FBS
For instance, the incorporation of methacrylamide - Lonza) and antibiotics (10,000 units/mL penicillin and
groups onto the amine-containing side groups results in 10 mg/mL streptomycin in 0.9% sodium chloride, Sigma
a biopolymer (GelMOD), which can be used for photo- Aldrich). HUVECs were maintained in supplemented
[7]
crosslinking processes at room temperature with high endothelial growth media (EGM2, Lonza) including 2%
stability at 37 °C after polymerization [7–9] . FBS, 0.04% hydrocortisone, 0.4% human fibroblastic
In addition, the mechanical properties of the material growth factor (hFGF), 0.1% vascular endothelial growth
can be improved further by modifying GelMOD with factor (VEGF), 0.1% long-insulin-like growth factor-1
additional methacrylates (GelMOD-AEMA), thereby (R3-IGF-1), 0.1% ascorbic acid, 0.1% human endothelial
creating more functional groups for the cross-linking growth factor (hEGF), 0.1% heparin and a mixture of
process, which outperforms the mechanical properties of 30 mg/mL gentamicin and 15 µg/mL amphotericin.
[10]
GelMOD and performs better in aqueous environment . 2.2 Evaluation of Membrane Material
Over the last decades, the ongoing trend of miniatur-
ization and multiplexing tissue engineering entails Biocompatibility
new demands on manufacturing techniques as well as To evaluate the biocompatibility of the structure material
biomaterial compositions and functionalities, especially cellular response of seeded HUVECs and BeWo B30
regarding micrometer-scale resolution. To achieve sub- cells was evaluated in a two-dimensional setting. The
micrometer spatial resolution a 3D printing technique material under investigation was a 15 wt% GelMOD-
called two-photon polymerization (2PP) can be AEMA solution containing 0.6 mM of UV sensitive

2 International Journal of Bioprinting (2018)–Volume 4, Issue 2