Patient-derived glioma stem cells

Trends in 3D Cell Culture: Organoids and Spheroids

Amanda Linkous, PhD, discusses new scientific and technological advancements in 3D cell culture in this Q&A with contributing editor Tanuja Koppal, PhD

by Tanuja Koppal, PhD
Amanda Linkous, PhD

Amanda Linkous, PhD, research associate professor, Department of Biochemistry and scientific center manager, NCI Center for Systems Biology of Small Cell Lung Cancer at Vanderbilt University, talks to contributing editor Tanuja Koppal, PhD, about new scientific and technological advancements in 3D cell culture. She discusses some of the emerging applications for the use of 3D organoids and spheroids that have been made possible due to these innovations.

Q: Can you offer some details about your work and expertise?

A: I completed my postdoctoral training in the Neuro-Oncology Branch at the National Cancer Institute (NCI). It was during this time that I developed a passion for studying cancer stem cell biology and the molecular signaling that promotes glioblastoma (GBM) tumor progression. Over the years, I continued to pursue this devastating disease as my primary research focus. As the former director of the Starr Foundation Cerebral Organoid Translational Core at Weill Cornell Medicine, I established a novel, ex vivo 3D system to study the interactions and molecular crosstalk between brain tumor cells and a miniature model of the human brain. We named this cerebral organoid glioma model the “GLICO” model and published our findings in Cell Reports.

Q: What is the difference between 3D spheroids and organoids?

Patient-derived glioma stem cells (green) invading and proliferating within a miniature replica of the human brain.
Amanda Linkous

A: Spheroids and organoids are both multi-cellular 3D structures. Spheroids, however, are cell aggregates typically composed of cancer cells cultured under scaffold-free, non-adherent conditions. The complexity of spheroid cultures is limited; these cultures are also difficult to maintain long-term due to factors such as hypoxia, necrosis, and loss of key genetic features. Organoids, termed “mini-organs” by many, are comprised of organ-specific cell types (from stem cells or progenitor cells) that exhibit lineage-specific differentiation and self-assembly. A scaffolding matrix such as Matrigel is often used to support the architecture of the developing microstructure. The level of self-organization that occurs in organoids is quite remarkable and highly similar to normal organ development in vivo.

Q: Can you discuss some key technical and experimental challenges in culturing and using 3D cellular models?

A: There is a bit of an art-form to working with 3D cell models. In the case of cerebral organoids, each stage of the differentiation process is dependent on timing. If one administers the appropriate growth factor or biological cue, but at the inappropriate time, then there can be wide variation in the resulting cell types that are generated. Therefore, it is critical to monitor the cultures daily and learn to follow the morphological clues that the cultures are giving you. In addition, depending on the size of the organoid, it is easy to shear or damage an organoid when attempting to transfer or physically manipulate the sample. Thus, as with any model system, one must carefully consider the limitations of 3D cellular models when designing experiments.

Q: You mentioned sample size and the importance of timing, but have you encountered other issues when designing experiments with 3D cellular models?

A: Absolutely! As I mentioned, organoids are frequently referred to as “mini-organs” and often resemble small pieces of tissue. This tissue can be incredibly dense, so imaging analysis with standard fluorescence or confocal microscopy can be quite difficult, if not impossible. We were very interested in imaging tumor volume within our mini-brains, so we utilized multi-photon microscopy and even light sheet microscopy for some experiments. The imaging resolution was fantastic; however, these imaging modalities presented challenges of their own. Multi-photon microscopy required the live sample to be immobilized for long periods of time. Since our 3D models required gentle agitation within an incubator in order to remain viable, there was a lot of trial and error to determine the best way to effectively image the tumor and return the organoid to a shaking culture environment, without affecting the viability of the sample itself. Alternatively, light sheet microscopy allowed imaging on fixed 3D samples, but the preparation and labeling of the sample could take weeks.

Another experimental challenge that we faced included isolation of tumor cells from the normal 3D mini-brain. We tried multiple reagents and methods of tissue dissociation, but we finally designed the optimal protocol for our specific needs. With any 3D model, researchers must consider what biological questions they want to ask, and what readout or analysis metric makes the most sense.

Q: What are some of the emerging applications for 3D cell models and how well do they represent in vivo processes and results?

A: 3D cell models have become increasingly more sophisticated in recent years and are used to study a multitude of disease states including neurogenerative diseases, cancer, cardiac disease, cystic fibrosis, and even drug addiction. Moreover, the utilization of 3D models in regenerative medicine is advancing rapidly. One of the reasons that 3D models are so heavily sought after is that they recapitulate the biology and pathophysiology of many in vivo processes. Coupled with their scalability for high-throughput drug screening, 3D models such as organoids offer an unprecedented way to approach personalized medicine. For example, our GLICO model enables the generation of hundreds of patient-specific miniature brain tumors in a way that is not currently possible for any in vivo glioblastoma model.

Q: What advice would you give to lab managers who are looking to get started in 3D cell culture work?

A: 3D cell culture is expensive, but it is extremely important to work with high-quality reagents and never take shortcuts, no matter how tempting it might be. The initial building phase of a 3D culture program is also incredibly time consuming. The amount of training time required to achieve 3D culture competency for new personnel can range from six months to one year. If you struggle with patience, you might want to re-evaluate your decision to enter the world of 3D model systems. If you are willing to make the commitment, however, you will find it to be one of the coolest and most rewarding platforms for studying developmental and/or disease biology.

Amanda Linkous previously served as the director of the Starr Foundation Cerebral Organoid Translational Core at Weill Cornell Medicine (New York, NY). She completed her postdoctoral training in the Neuro-Oncology Branch at the National Cancer Institute (Bethesda, MD). She has extensive expertise in cancer stem cell biology and the molecular signaling that promotes tumor progression. She established a novel, ex vivo 3D system to study the interactions and molecular cross-talk between tumor cells and a miniature model of the human brain—a finding that was featured on CNN Pioneers and in a special edition of Science