29. Jahrestagung der Deutschen Gesellschaft für Biomaterialien

Prof. Dr.

Chair

Fabrication of 3D microstructures within a perfusable vasculature – on – a – chip system using two – photon polymerization

Additive manufacturing (e. g. 3D printing)

Abstract text (incl. figure legends and references) Tissue engineering is an interdisciplinary research area with great potential for modeling human physiology and disease. One of the main challenges in this field is generation of a fine and perfusable vasculature with appropriate mechanical properties and biocompatibility. Fine capillary structures can be formed in vitro by combining angiogenesis with two-photon printing. Perfusion of an engineered vascular network is crucial to provide natural cell culture conditions that maintain optimal cell function. This work aims to design a vasculature-on-a-chip device, which provides a vascular network that can be used, for example, to study angiogenesis under physiological and pathological conditions. We outline the chip fabrication process that combines stereolithography with two-photon printing. Stereolithography is used for printing the main chip part, whereas two-photon technology aims to finalize the chip by printing 3D microstructures inside the chip. We describe the composition and advantages of the synthesized photosensitive material and present a biofunctionalization strategy to support cell culture. We demonstrate the chip with 3D microstructures printed and imaged with two-photon technology and a successful culture of endothelial cells in this chip under perfusion. The creation of perfusable vascular structures would make a significant contribution to the investigation of the influence of various treatments on the mechanism of angiogenesis.

Short Talk

A drop-on-demand bioprint process for the fabrication of vascularized Organ-on-a-Chip cultures

Biofabrication

Abstract text (incl. figure legends and references) Introduction &amp; Objectives Biomimetic Organs-on-a-Chip are key for advanced drug screening and research, but require complex 3D arrangements of multiple cell types representing organ-specific cells, connective tissue, and vasculature. Combined with suitable bioinks, drop-based 3D-bioprinting is a powerful tool to create these models. This work includes an in-depth analysis of various bioinks and cultivation protocols for a print process of a multicellular and vascular tissue model on a microfluidic chip. To demonstrate the feasibility of the approach, HepG2 islets are embedded in a microvascular bed of HUVEC and HDF. Materials &amp; Methods The microfluidic chip was designed in an open lid setup enabling the direct bioprinting into the cultivation chamber. Bioink formulations comprising agarose, collagen, and gelatin were investigated for the fabrication of HepG2 cell islands, while fibrin was selected as vascular bed promoting bioink. The gels were characterized regarding rheology, microstructure and cell behavior. In parallel, a medium formulation that supports growth of all three cell types was identified using proliferation assays. The printability of the selected bioinks was monitored by various imaging technologies both in-flight and post-printing. Finally, the tissue model was printed, cultured under perfusion and vascular network formation monitored. Results &amp; Conclusion Two bioinks – a collagen-gelatin blend and agarose - were identified for the HepG2 islets as suitable considering printability, gel structure and cellular behavior. HUVECs and HDF mixed in 10 mg/ml fibrin at a total cell concentration of 5 Mio/ml resulted in vascular network formation throughout the chip and around the HepG2 islets. By combining both bioinks, we achieved a reproducible, fast and scalable multi-material print process, demonstrating the high versatility of combining microfluidics and 3D-bioprinting and its potential for the future fabrication of vascularized tissue models.

Professor

Magnetic nanoparticles pass a differentiating in vitro blood-placenta barrier

Clinical applications and translation

Cell-material interactions

Abstract text (incl. figure legends and references) Introduction The biomedical application of nanomaterials is growing with a strong need to understand the interaction between nanomaterials and the human body. Since clinical trials on pregnant women are difficult to perform, physiologically appropriate models of the human placenta to study nanoparticle-placenta interactions in vitro are needed. Objectives We aim to gain a better understanding of the interactions of magnetic nanoparticles (MNP), with the blood-placenta barrier. In this study, we investigated how the fusion of cytotrophoblasts to syncytiotrophoblasts affects the involved pericytes and the passage of MNPs through the barrier. Methods The in vitro BPB was established in a microfluidic chip by using the cytotrophoblast cell line BeWo on the apical (maternal) side and human primary placental pericytes on the basolateral (fetal) side. Differentiation of the BeWo cells was induced by a 24h-incubation with 20 µM forskolin. Barrier integrity was verified by sodium-fluorescein (Na-Flu) permeability. The forskolin-induced changes were confirmed by fluorescence microscopy and qPCR. Citrate-coated MNP (hydrodynamic diameter 116 nm, z-potential -35 mV) were applied for 24 h. For quantification of the MNP content magnetic particle spectroscopy (MPS) was performed. Results Na-Flu incubation showed a 5-fold reduction in permeability for cell-covered barriers compared to empty ones. Forskolin caused a 3.6-fold increase of chorion gonadotropin expression. After forskolin and MNP incubation the distribution of MNPs in the apical and basolateral compartment as well as in the cell layer was determined by MPS. The formation of syncytiotrophoblasts did not affect the penetration rate of MNPs. In the basolateral compartment, no significant difference in MNP content was measured before forskolin addition (2.3% ± 2.0%) and after cytotrophoblast fusion (2.4% ± 1.6%). Conclusion We demonstrate that MNPs pass a cytotrophoblastic cell layer as well as a syncytiotrophoblast.

Gellan gum based inflammatory adipose tissue model

Tissue regeneration/regenerated medicine

Abstract text (incl. figure legends and references) Obesity is a complex condition associated with an inflammatory state of the adipose tissue, involving immune cell infiltration and the release of pro-inflammatory cytokines. Therefore, reliable in vitro models resembling inflamed adipose tissue are required as a tool to investigate adipose tissue inflammation and associated diseases. In this study, we set up an inflamed co-culture of the human monocytic cell line MM6 and human primary mature adipocytes encapsulated in gellan gum (GG) cultured in a defined medium. As a linear microbial exopolysaccharide, GG possesses many favorable features such as biocompatibility, biodegradability, and a non-toxic nature. Further favorable properties are its high transparency, thermoresponsive features, flexible mechanical properties, ease of manufacturing and crosslinking, stability under physiological conditions, and low price. Gellan gum itself was neither toxic nor monocyte activating. Successful pro-inflammatory activation of the co-culture by stimulation with phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) was proven by clumping assay of the MM6 and quantification of released cytokines IL1b, IL6 and TNFa. Live/dead staining and visualization of intracellular lipids revealed no impact of activation substances on adipocyte viability and integrity. Further, we transferred the co-culture into perfusion culture to simulate more physiological conditions. Viability and intracellular lipid content were high and morphological (actin, perilipin A) analyses showed similar results for both culture methods. Inflammatory stimuli induced morphological changes independently of the culture condition. For PMA and LPS activation, MM6 exhibited membrane protrusions, and ACs showed decreased perilipin A integrity. In summary, we successfully developed an inflamed human adipose tissue model in a completely defined environment, which can be activated in static and perfusion cultures.

Geometry is a key factor in defining the transcriptional background of bone marrow metastatic neuroblastoma cells in a multicellular 3D in vitro model

Abstract text (incl. figure legends and references) Introduction: Metastasis remains one of the leading causes of cancer death. Metastases are frequent in pediatric neuroblastoma (NB), a developing tumor of the sympathetic nervous system. Of particular concern are invasions of neoplastic cells to the bone marrow and bone, which are strongly correlated with tumor aggressiveness. Objectives: In this study, we investigated how the geometry, chemistry, and cellular complexity of a 3D in vitro NB bone metastasis model made of β-tricalcium phosphate (β-TCP) scaffolds with interconnected microchannels affect tumor cell behavior. Material and Methods: The interaction between mesenchymal stromal cells (MSC), endothelial cells (EC), and NB cells and the extracellular matrix was investigated using two-photon microscopy. The release of cytokines was determined by ELISA. Extensive computational analyses were performed to determine how geometry affects the transcriptional background of NB cells. The in-silico data were validated using an immunofluorescence approach. Results: We confirmed that the tumor microenvironment is dynamically shaped by stromal cells, promoting the growth of NB cells in the metastatic niche. Compared with their 2D counterparts, 3D β-TCP structures produce proinflammatory and immunomodulatory cytokines and define the intracellular localization of Connexin 43. Moreover, our data confirmed that 3D growth conditions were crucial for the type of tumor cell response to conventional chemotherapeutic agents. Finally, we verified the crucial contribution of geometry in defining the in vivo resembling transcriptional landscape of tumor cells by activating a subset of genes critical for tumor progression. Conclusions: Our results demonstrate that geometry is an important modulator of cell-cell interaction and of molecular signaling pathways active in metastatic NB cells. The 3D conditions of the metastatic niche support the maintenance of stem-like properties in the tumor cell compartment.

Short Talks 7
Biofabrication / Organ-on-a-Chip

Appointment

Chair

Topics

Programme

Short Talks 7Biofabrication / Organ-on-a-Chip

Appointment

Chair

Topics

Programme

Short Talks 7
Biofabrication / Organ-on-a-Chip