Spheroid-on-chip – Combining the benefits of dynamic cell culture with the complexity of spheroids

In recent years, the integration of spheroids and organoids with microfluidic chip technology has become a growing trend in biomedical research. This combination, often referred to as “spheroid-on-chip” or “organoid-on-chip”, provides a dynamic, but also highly controlled environment that more accurately mimics the complexity of in vivo tissue when compared to traditional culture methods. As the field advances, spheroid-on-chip systems are increasingly recognized for their potential to revolutionize drug testing, disease modeling, and personalized medicine.

What Are Spheroids?

Spheroids are three-dimensional (3D) cell aggregates that closely resemble the architecture and function of natural tissues. In contrast to 2D-cultures, spheroids enable realistic cell-cell and cell-matrix interactions. These interactions are critical for studying cellular behavior in a context that mirrors in vivo conditions (Moshksayan 2018 et al.).

Spheroids are compact, spherical structures formed by cells that have been suspended in a culture medium and allowed to aggregate. This aggregation occurs through cell-cell interactions, primarily mediated by integrin-ECM binding and cadherin expression. The resulting structure can consist of multiple cell types, such as epithelial, mesenchymal, and endothelial cells, that establish a microenvironment akin to that found in tissues (Fang 2023 et al.). Spheroids can be generated from cell lines, single cells, or even tissue-derived cells obtained via biopsy.

A key characteristic of spheroids is their ability to develop distinct layers that reflect the spatial organization of cells in solid tumors or other tissues. These layers include an outer proliferating zone, an intermediate quiescent zone, and a central necrotic core, particularly in larger spheroids (Pinto 2020 et al.). This stratification is essential for mimicking the gradients of oxygen, nutrients, and metabolites that are characteristic of in vivo tissues (Biju 2023 et al.).

Assembly of (Tumor) Spheroids

When considering the assembly of tumor spheroids, the ability to recreate the structural and functional heterogeneity of tumors in vitro is of paramount importance. In traditional 2D cultures, cells often fail to exhibit the same behavior as they would in a 3D environment, leading to less predictive results in drug screening and disease modeling. Spheroids, on the other hand, offer several advantages over 2D cultures, including the establishment of proliferation gradients that result in a layered cell architecture (Moshksayan 2018 et al.). This structure not only better represents the in vivo tumor environment but also influences how cells within the spheroid respond to treatments.

Spheroid formation can be divided into two stages: the initial formation of loose cell aggregates and the subsequent compaction into a well-rounded spheroid. The first stage involves the binding of cells to the extracellular matrix (ECM) via integrins, which helps to constrain the cells within a confined space, such as round-bottom wells. The second stage is characterized by the accumulation and interaction of cadherins, which are crucial for the formation of tight, compact spheroids (Fang 2023 et al.).

We won’t go into great detail on spheroid formation here but would like to guide you to this review article by Moshksayan et al. to learn more about this topic.

Spheroid-on-Chip vs. Non-Microfluidic Methods

While traditional methods of spheroid formation, such as hanging drop cultures or microwell plates, have been instrumental in the development of 3D cell culture, they come with significant limitations (Tevlek 2023 et al.). The static nature of these culture environments can lead to the rapid depletion of oxygen and nutrients, as well as the accumulation of waste products, which adversely affect spheroid viability and the reliability of experimental results.

In contrast, microfluidic chips offer a more sophisticated approach to spheroid culture. These chips allow for precise control over the microenvironment, including the regulation of chemical gradients, pressure, and shear stress (Zhang 2023 et al.). This level of control is crucial for mimicking the dynamic conditions of in vivo tissues. For instance, continuous perfusion of culture medium in microfluidic systems ensures a steady supply of oxygen and nutrients while removing waste products, which enhances spheroid viability and function (Pinto 2020 et al.). In another approach by Dynamic42, the spheroids are not perfused directly but indirectly via a vasculature. Similar to direct perfusion, fresh nutrients are continuously provided, waste removed, an an oxygen gradient is created, but spheroids are left without shear stress, which is usually physiological.

Interestingly, the dynamic culture conditions provided by microfluidic systems have been shown to increase the resistance of cancer spheroids to drug treatments, a phenomenon that closely mirrors the drug resistance observed in tumors in vivo (Ruppen 2014 et al.). This makes spheroid-on-chip platforms invaluable for studying the mechanisms of drug resistance and for identifying more effective therapeutic strategies.

Applications of Spheroid-on-Chip

The applications of spheroid-on-chip technology are vast and span multiple areas of biomedical research. One of the most promising applications is in the study of cancer metastasis, which is the leading cause of cancer-related deaths (Collins 2021 et al.). Traditional in vivo metastasis models, such as murine assays, have limitations in terms of ethical concerns, cost, and translatability to human biology (Jackson 2017 et al.). Spheroid-on-chip systems offer an alternative by providing a controlled environment to study the impact of continuous flow and mechanical cues on metastatic potential in vitro. Collins 2021 et al. were able to detect secretion of pro-metastatic factors (cytokines vascular endothelial growth factor and interleukin 6) using physiological flow conditions in vitro. Consequently, these platforms allow for the identification of novel metastasis biomarkers and the evaluation of molecular targets that are not feasible in conventional models.

Another significant application is in cardiotoxicity testing using vascularized cardiac spheroids-on-a-chip. This approach enables the modeling of systemic drug delivery and the assessment of therapeutic toxicity in a more physiologically relevant context (Di Cio 2024 et al.). By integrating vascular networks within the spheroid-on-chip platform, researchers can better predict how drugs will behave in the human body, potentially reducing the likelihood of adverse effects in clinical trials.

Conclusion

The integration of spheroids with microfluidic chips represents a significant advancement in the field of in vitro modeling. Spheroid-on-chip systems provide a dynamic and highly controllable microenvironment that better recapitulates the complexity of living tissues compared to traditional methods. As this technology continues to evolve, it holds great promise for improving the accuracy of drug testing, enhancing our understanding of disease mechanisms, and paving the way for personalized medicine.

Would you like to establish spheroid-on-chip in your lab? Our DynamicOrgan System in combination with our Biochip BC003 features 2 times 25 microcavities for spheroid cultivation. You can learn more in this flyer detailing our work in spheroid-on-chip for cancer research.

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