Hematopoietic stem cell transplantation is successfully applied since the late 1950s; however, its efficacy still needs to be increased. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an improved ex vivo culture system that supports proliferation and maintains HSC pluripotency would override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro, we optimized the HSC medium composition with a panel of cytokines and valproic acid and used an artificial 3D bone marrow-like scaffold made of polydimethylsiloxane (PDMS). This 3D scaffold offered a suitable platform to amplify human HSCs in vitro and, simultaneously, to support their viability, multipotency and ability for self-renewal. Silicon oxide-covering of PDMS structures further improved amplification of CD34+ cells, although the conservation of naïve HSCs was better on non-covered 3D PDMS. Finally, we found that HSC cultivated on non-covered 3D PDMS generated most pluripotent colonies within colony forming unit assays. In conclusion, by combining biological and biotechnological approaches, we optimized in vitro HSCs culture conditions, resulting in improved amplification, multipotency maintenance and vitality of HSCs.

We present a novel lung aerosol exposure system named MALIES (modular air–liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids^®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO₄ aerosol. Cytotoxic effects of BaSO₄- and TiO₂-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air–liquid interface. Experiments with concentrations of up to 5.93 × 10⁵ (BaSO₄) and 1.49 × 10⁶ (TiO₂) particles/cm³, resulting in deposited masses of up to 26.6 and 74.0 µg/cm² were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids^® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO₄-NP in a deposited mass of up to 26.6 µg/cm² resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO₂-NP in a deposited mass of up to 74.0 µg/cm² did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO₄ and TiO₂ in in vitro experiments.

Recent trends in 3D cell culturing has placed organotypic tissue models at another level. Now, not only is the microenvironment at the cynosure of this research, but rather, microscopic geometrical parameters are also decisive for mimicking a tissue model. Over the years, technologies such as micromachining, 3D printing, and hydrogels are making the foundation of this field. However, mimicking the topography of a particular tissue-relevant substrate can be achieved relatively simply with so-called template or morphology transfer techniques. Over the last 15 years, in one such research venture, we have been investigating a micro thermoforming technique as a facile tool for generating bioinspired topographies. We call them MatriGrid^®s. In this research account, we summarize our learning outcome from this technique in terms of the influence of 3D micro morphologies on different cell cultures that we have tested in our laboratory. An integral part of this research is the evolution of unavoidable aspects such as possible label-free sensing and fluidic automatization. The development in the research field is also documented in this account.

Low temperature cofired ceramics (LTCC) provide a technology for the 3-dimensional integration of sensor arrays into bioreactors covering dimensions of several hundred micrometers. Since optical control in such assemblies is not possible, the in situ detection of cell adhesion on impedance electrodes with high spatial resolution would deliver crucial information. A current limitation is the increasing impedance of microelectrodes with decreasing diameter. This study evaluates the suitability of thick film gold electrodes, pristine and coated with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT), for the detection of cell adhesion on the electrode surface. The impedance as criterion for cell attachment is measured with a recording system for electroactive cells with the aim of improving usability. Two cell cultures with different adhesion characteristic are used for adhesion assessment on planar test chips. The impedance increase measured on individual PEDOT coated electrodes due to tight contact of cells reaches a factor of 6.8 in cultures of well-adherent HepG2 cells. Less adhered NG108-15 cells produce a maximum impedance increase by a factor of 2.6. Since the electrode impedance is significantly reduced by PEDOT coating, a reduction of the electrode diameter to values below 100 µm and spatially resolved detection is possible. The results encourage further studies using PEDOT coated thick film electrodes as bio-electronic-interfaces. We presume that such miniaturized electrodes are suitable for 3-dimensional recordings in electroactive cell cultures, providing information of local cell adhesion at the same time.

Real-time monitoring of bioanalytes in organotypic cell cultivation devices is a major research challenge in establishing stand-alone diagnostic systems. Presently, no general technical facility is available that offers a plug-in system for bioanalytics in diversely available organotypic culture models. Therefore, each analytical device has to be tuned according to the microfluidic and interface environment of the 3D in vitro system. Herein, we report the design and function of a 3D automated culture and analysis device (3D-ACAD) which actively perfuses a custom-made 3D microbioreactor, samples the culture medium and simultaneously performs capillary-based flow ELISA. A microstructured MatriGrid^® has been explored as a 3D scaffold for culturing HepaRG cells, with albumin investigated as a bioanalytical marker using flow ELISA. We investigated the effect of acetaminophen (APAP) on the albumin secretion of HepaRG cells over 96 h and compared this with the albumin secretion of 2D monolayer HepaRG cultures. Automated on-line monitoring of albumin secretion in the 3D in vitro mode revealed that the application of hepatotoxic drug-like APAP results in decreased albumin secretion. Furthermore, a higher sensitivity of the HepaRG cell culture in the automated 3D-ACAD system to APAP was observed compared to HepaRG cells cultivated as a monolayer. The results support the use of the 3D-ACAD model as a stand-alone device, working in real time and capable of analyzing the condition of the cell culture by measuring a functional analyte. Information obtained from our system is compared with conventional cell culture and plate ELISA, the results of which are presented herein.

3D‐microcontact printing (3D‐µCP) technique combines the advantages of microcontact printing and microthermoforming for the fabrication of functional biomaterials with complexity closer to real tissue. Despite its unmet advantages in terms of complexity and processability in a single step, this technique is limited to the front side of the substrate. Considering the advantage of inherent topography patterns on the reverse side of the substrate, an additional degree of patterning can be envisioned. However, selective patterning on the reverse side is challenging due to the fragility of the cell culture on the front side. Herein, a technically simple scraping technique is presented in combination with 3D‐µCP to generate 3D cell patterns on both the front and the reverse side of a micro thermoformed substrate. The technical advancement of 3D‐µCP with the scraping technique offers a complex, multi‐layered co‐culture system that can be used to study cell‐to‐cell crosstalk at the microlevel with topographies similar to real tissue. Direct seeding of the third cell type on the scaffold expands cell type complexity, resulting in a tri‐culture model. As a proof of concept, a triculture of EA.Hy 926 endothelial cells, HepG2 hepatoblastoma cells, and NIH3T3 fibroblasts are generated, mimicking the tumor microenvironment in the liver.