Inspired by the human placenta: a novel 3D bioprinted membrane system to create barrier models

Abstract

Barrier organ models need a scaffold structure to create a two compartment culture. Technical filter membranes used most often as scaffolds may impact cell behaviour and present a barrier themselves, ultimately limiting transferability of test results. In this work we present an alternative for technical filter membrane systems: a 3D bioprinted biological membrane in 24 well format. The biological membrane, based on extracellular matrix (ECM), is highly permeable and presents a natural 3D environment for cell culture. Inspired by the human placenta we established a coculture of a trophoblast-derived cell line (BeWo b30), together with primary placental fibroblasts within the biological membrane (simulating villous stroma) and primary human placental endothelial cells-representing three cellular components of the human placental villus. All cell types maintained their cell type specific marker expression after two weeks of coculture on the biological membrane. In permeability assays the trophoblast layer developed a barrier on the biological membrane, which was even more pronounced when cocultured with fibroblasts. In this work we present a filter membrane free scaffold, we characterize its properties and assess its suitability for cell culture and barrier models. Further we show a novel placenta inspired model in a complex bioprinted coculture. In the absence of an artificial filter membrane, we demonstrate barrier architecture and functionality.

Figure 1

Scheme of placental barrier (gestation week 5), and its model. A placental villus (a) contains fetal blood vessels (fb), which are lined with endothelial cells (HPVEC) and surrounded by a basal lamina (bl). Mesenchymal stroma (st) with fibroblasts (HVMF) surround the vessels. The outermost layer of the villus is built out of villous cytotrophoblast cells (vCTB), with a basal lamina (bl), which fuse into one syncytiotrophoblast (STB) that is in direct contact with maternal blood (mb) in the intervillous space. At this stage of development the placental barrier consists of endothelial cells (with basement membrane), stromal tissue, cytotrophoblast cells (with basement membrane) and syncytiotrophoblast. Analogous the scheme of the placental barrier model on the Membrick, (b) the biological membrane is based on methacrylated gelatine (GelMA), containing human villous mesenchymal fibroblasts (HVMF). Human placental vascular endothelial cells (HPVEC) are cultured on the basolateral side, while the trophoblast cell model BeWo is cultured on the apical side of the biological membrane.

Figure 2

Biological membrane system (Membrick) and its characteristics. The cell culture insert is designed for hanging cultivation in a 24 well plate (a). Depiction of cell culture insert scheme (b): biological membrane (1) separating two compartments (2, 3). The Membrick body has arms for a hanging configuration in 24 well; the bottom of the cylindrical body includes a pipette rest for facilitated media exchange (4). The biological membrane is transparent in bright-field microscopy (c, d). Cells seeded on top of the biological membrane can be observed continuously through the membrane (e). Scale bars represent $1000\mathrm{\mu }$m (c), and $100\mathrm{\mu }$m (d, e). Permeability of the biological membrane (f) compared to PET (g) with $1\mathrm{\mu }$m pore size for Dextran-FITC and -Texas Red (70 kDa and 3 kDa) and Lucifer Yellow (457 Da). Dextran permeability for biological membrane $\text{n}=6$, and LY $\text{n}=5$, permeability for PET $\text{n}=7$, and LY $\text{n}=5$. Median and error are displayed.

Figure 3

Monotypic culture of placental endothelial cells (HPVEC) and BeWo cells cultured on biological membrane. Cell morphology of HPVEC in bright-field microscopy at the beginning of experiment and after 14 days of culture on the Membrick (a), scale bar $100\mathrm{\mu }$m. Immunofluorescence staining of HPVEC culture on PET and biological membrane for endothelial specific marker CD 31, on day 12 (b). Cross sections (c) on day 14, stained for CD31 and von Willebrand factor (vWF), scale bar $50\mathrm{\mu }$m. BeWo b30 cells stained for beta-catenin ($\beta$-cat) at day 5 of culture on Membrick (d), scale bar $25\mathrm{\mu }$m. Comparison of vascular endothelial cadherin (VE-Cad) localization in HPVEC cultured on PET and Membrick (e), scale bar $50\mathrm{\mu }$m.

Figure 4

Bioprinted biological membrane with primary human fibroblasts HVMF. Light microscopic image of cells right after printing process and after 3 days in culture (a), scale bars $100\mathrm{\mu }$m. Cell viability of HVMF over four weeks of culture (b), for each time point individual experiments ($\text{n}=3$) with range are displayed.

Figure 5

Cell (co)culture on Membrick. Primary placental fibroblasts (HVMF) were included into the biological membrane and viability was investigated throughout four weeks ($\text{n}=3$ for each time point), a; average and range are displayed. Barrier formation of monotypic culture (BeWo on or HVMF in biological membrane) or coculture of both represented by TEER measurements (b, $\text{n}=3$) and permeability assay with Lucifer Yellow (c, $\text{n}=3$, empty control $\text{n}=2$). Median and individual samples are displayed.

Figure 6

Immunofluorescence staining of coculture on biological membrane at day 12: HVMF printed into the biological membrane, cocultured with HPVEC on basolateral side and BeWo cells on apical side. Cells within membrane stain positively for vimentin (Vim), while apical cells showed cytokeratin 7 (CK7) staining (a). Endothelial marker von Willebrand factor (vWF) and CD31 stain positively for cells on basolateral side of biological membrane (b). Scale bars $100\mathrm{\mu }$m.