One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

In this second part of our systematic review on the research area of 3D cell culture in micro-bioreactors we give a detailed description of the published work with regard to the existing micro-bioreactor types and their applications, and highlight important results gathered with the respective systems. As an interesting detail, we found that micro-bioreactors have already been used in SARS-CoV research prior to the SARS-CoV2 pandemic. As our literature research revealed a variety of 3D cell culture configurations in the examined bioreactor systems, we defined in review part one “complexity levels” by means of the corresponding 3D cell culture techniques applied in the systems. The definition of the complexity is thereby based on the knowledge that the spatial distribution of cell-extracellular matrix interactions and the spatial distribution of homologous and heterologous cell–cell contacts play an important role in modulating cell functions. Because at least one of these parameters can be assigned to the 3D cell culture techniques discussed in the present review, we structured the studies according to the complexity levels applied in the MBR systems.

(1) Background: We describe a 4D cell culture platform with which we tried to detect and to characterize migration dynamics of single hematopoietic stem cells in polymer film microcavity arrays integrated into a microtiter plate. (2) Methods: The system was set up with CD34-expressing KG-1a cells as a surrogate for hematopoietic stem cells. We then evaluated the system as an artificial hematopoietic stem cell niche model comprised of a co-culture of human hematopoietic stem cells from cord blood (cord blood CD34⁺ cells, hHSCs) and human mesenchymal stromal cells (hMSCs) from bone marrow over a period of 21 days. We used a software-based cell detection method to count single hematopoietic stem cells (HSCs) in microcavities. (3) Results: It was possible to detect single HSCs and their migration behavior within single microcavities. The HSCs displayed a pronounced migration behavior with one population of CD34-expressing cells located at the bottom of the microcavities and one population located in the middle of the microcavities at day 14. However, at day 21 the two populations seemed to unite again so that no clear distinction between the two was possible anymore. (4) Conclusions: Single cell migration detection was possible but microscopy and flow cytometry delivered non-uniform data sets. Further optimization is currently being developed.

The sodium triple-quantum (TQ) magnetic resonance (MR) signal created by interactions of sodium ions with macromolecules has been demonstrated to be a valuable biomarker for cell viability. The aim of this study was to monitor a cellular response using the sodium TQ signal during inhibition of Na/K-ATPase in living cancer cells (HepG2). The cells were dynamically investigated after exposure to 1 mM ouabain or K⁺-free medium for 60 min using an MR-compatible bioreactor system. An improved TQ time proportional phase incrementation (TQTPPI) pulse sequence with almost four times TQ signal-to-noise ratio (SNR) gain allowed for conducting experiments with 12–14 × 10⁶ cells using a 9.4 T MR scanner. During cell intervention experiments, the sodium TQ signal increased to 138.9 ± 4.1% and 183.4 ± 8.9% for 1 mM ouabain (n = 3) and K⁺-free medium (n = 3), respectively. During reperfusion with normal medium, the sodium TQ signal further increased to 169.2 ± 5.3% for the ouabain experiment, while it recovered to 128.5 ± 6.8% for the K⁺-free experiment. These sodium TQ signal increases agree with an influx of sodium ions during Na/K-ATPase inhibition and hence a reduced cell viability. The improved TQ signal detection combined with this MR-compatible bioreactor system provides a capability to investigate the cellular response of a variety of cells using the sodium TQ MR signal.

Precise control and measurement of the cellular microenvironment, particularly oxygen concentration, are crucial for developing physiologically relevant in vitro models. However, current methods often lack the spatial resolution and throughput needed to investigate complex, oxygen-dependent biological mechanisms in 3D cell cultures. Here, we present an advanced platform based on microcavity arrays featuring integrated, ratiometric oxygen sensors, so-called SensoSpheres. A unique bevel design at the cavity entrance enables the non-invasive, real-time measurement of pericellular oxygen concentration and oxygen gradients. We established protocols for generating spheroids from various cell lines (e.g., HepG2, HeLa) and characterized their metabolic responses under precisely controlled hypoxic, normoxic, and hyperoxic conditions. Using a dose–response assay, we demonstrate the platform’s sensitivity in capturing distinct metabolic shifts in response to acetaminophen and cisplatin. Furthermore, we introduce the Oxygen Consumption Recovery Rate (OCRR) as a novel parameter to quantify cellular resilience after exposure to toxic compounds such as cisplatin and acetaminophen. This high-throughput-compatible platform represents a significant methodological advancement, enabling detailed studies of oxygen-dependent cellular processes, drug toxicity, and metabolic adaptation. Its potential for integration into microfluidic systems paves the way for more sophisticated organ-on-chip models, ultimately improving the predictive power of preclinical research.