3D Cell Culture Tips To Optimize Your Workflow

How To Guide 1 3D Cell Culture Tips To Optimize Your Workflow Kaja Ritzau-Reid, PhD Three-dimensional (3D) in vitro cell culture provides a window to human disease and development by creating tissue structures that mimic the architecture and functionality of tissues in vivo. This has revolutionized biomedical research, providing a more advanced platform for diagnostics, drug discovery and personalized therapeutics.1,2 The last decade has seen huge development in 3D culture technology, from identifying essential factors in the culture environment for controlling growth and development, to the development of technologies to help automate and streamline the culture process.3 However, 3D cell culture comes with its own unique set of challenges, and ensuring the validity and reliability of your experimental results requires careful planning, optimization and sometimes a lot of patience! This guide will address some of the common pitfalls in 3D culture and provide tips and tricks to help you optimize your 3D culture workflow, from cell selection to culture maintenance and post-culture characterization. 3D cell culture models: A trade-off between complexity and reproducibility 3D cell culture broadly falls into three categories: spheroids, organoids and bioprinted tissues. There are advantages and disadvantages to each of these, depending on your experimental goals. Spheroids are simpler aggregates of cells that are commonly used as tumor models.4 Organoids are complex tissues with diverse cell types that mimic organ development, while bioprinted tissues use engineering-based methods to assemble the cells rather than cells self-assembling.5,6 This means that they are typically more reproducible but offer less complexity than an organoid model. Selecting the right materials Cell source and type The starting point for successful 3D culture is selecting the right cells. These can be derived from different sources. 3D CELL CULTURE TIPS TO OPTIMIZE YOUR WORKFLOW 2 How To Guide • Primary cells are terminally differentiated cells obtained directly from a patient’s tissue. This means that they have the advantage of closely mimicking the physiological state of cells in vivo. However, the availability of cells and different cell types is dependent on donor availability, which can be limited. The differentiated nature of the cells also means that they have a short lifespan in culture, limiting the maturation potential of the 3D culture. • Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are frequently used for creating complex organoid tissues, owing to their ability to self-organize and their unlimited potential for differentiation.1 iPSC technology means that patient-specific cells can be used, opening up the opportunity for patient-specific organoid models.7 The maintenance and handling of these cells can be more specialized and labor-intensive compared to other cell types. • Adult stem cells, such as mesenchymal stem cells can also be used for 3D tissue culture. Adult stem cells are multipotent, so don’t have the same potential as ESCs or iPSCs to differentiate into any cell type. This limits the cell diversity and complexity of the 3D tissue. However, adult stem cells can be easier to source and are easier to culture for those less experienced with stem cells in the lab. The decision about which cell type to use depends on the specific application, including the desired complexity of the 3D culture and the intended clinical or research use. Whichever cell type you choose, it is important to spend time standardizing your cell preparation methods to ensure consistency in your experiments. Extracellular matrix (ECM) and scaffold materials In 3D cell culture, it is important to create a microenvironment for the cells that closely resembles the in vivo environment to guide cell growth. Natural and synthetic hydrogel matrices are used because of their ability to mimic the ECM. • Matrigel is a decellularized murine matrix commonly used for organoid culture, providing biochemical cues for cell growth. However, its heterogeneous protein mix can cause batch-to-batch variability, affecting experimental consistency. For more consistency, consider using Geltrex, a murine basement membrane with a more uniform composition and lower growth factor content. • Individual components of the ECM, including collagen, laminin and glycosaminoglycans, can be used as 3D matrices to grow cells in 3D. It is worth noting that these can lack the biochemical complexity required to grow complex 3D tissues. • Synthetic matrices such as poly(ethylene glycol) (PEG) hydrogels can be engineered with specific stiffnesses and to contain bioactive components.8 • Fibrous and porous scaffolds can be used to mimic the native ECM microenvironment effectively, providing structural support for cell growth and migration. Scaffolds can either be custom designed in the laboratory or purchased commercially. Cell crown culture inserts can be useful for immobilizing scaffold samples in well plates of different sizes. Survival of the fittest: Culturing cells in 3D Cell seeding Successfully seeding your 3D cell culture is critical to ensure that your model is reproducible and reliable. Here are some tips to help ensure that you are using a reliable seeding method: • When direct seeding in a culture dish or scaffold, gently mix the suspension before seeding to ensure that you distribute the cells evenly. 3D CELL CULTURE TIPS TO OPTIMIZE YOUR WORKFLOW 3 How To Guide • Use an appropriate seeding density. If the cells are too sparse, they will fail to aggregate properly. If the cell density is too high, this can cause cell clumping or necrosis in the center. You can optimize seeding density by starting with a lower density and gradually increasing until you see proper aggregation or integration into a scaffold. • For spheroid and organoid generation, using low cell attachment plates ensure that the cells aggregate and don’t stick to the bottom of the dish. • Consider using commercially available plates such as spheroid microplates, or low attachment U bottom 96 well plates for high-throughput spheroid generation. These are often compatible with plate readers and high-content analysis platforms and are a good option for screening or high-throughput experiments. Culture medium optimization Choosing the right culture medium directly impacts cell growth and differentiation. Unlike two-dimensional (2D) cell culture, 3D cell culture require more specialized media formulations that may differ at various stages of growth. Growth factors and supplements are also often required to maintain cell growth and differentiation. The specific cocktail of media and supplements will vary according to the type of 3D culture, and optimizing culture media by adjusting growth factors and supplements is a key step to creating a reliable 3D culture workflow. Monitoring and controlling the microenvironment It is important to optimize the culture environment to support both growth and waste. Here are some key points to consider: • Regularly exchange media to prevent the buildup of waste products. • For organoid or spheroid cultures that are growing in suspension, orbital shakers can be used to agitate the cultures continuously while incubating to ensure even nutrient distribution. • Incorporating bioreactor systems in 3D culture can improve scalability and reproducibility. Bioreactors facilitate the continuous flow of nutrients to cells and circulate the culture media to ensure even distribution of nutrients. Automated media exchange continually removes waste products like lactic acid. • Maintaining a stable pH is vital and using pH buffers in the media can help to maintain stability for long-term cultures. • Tools can be set up for real-time monitoring of culture conditions such as temperature, pH and oxygen. Specialized tools can be integrated into the culture, or form part of a bioreactor system. Consider using monitoring tools for long-term cultures.9 Validating and characterizing your 3D cell cultures It is important to validate your 3D models to produce reproducible results that represent the in vivo tissue. Consider the following tips to help validate your 3D culture and plan your characterization methods during culture and post-fixation. Analysis and characterization of live 3D cultures • Use brightfield imaging techniques to characterize your 3D cultures morphologically. • Fluorescent dyes can be used to label specific cell populations. For example, a live/dead stain can reveal cell necrosis in an organoid model. 3D CELL CULTURE TIPS TO OPTIMIZE YOUR WORKFLOW 4 How To Guide • Time lapse microscopy can be useful to monitor morphology and real-time cellular behaviors. Used in conjunction with fluorescent dyes, this can provide valuable insights into processes such as cell migration and proliferation. • A variety of commercially available molecular probes and assays are available to monitor cell health, proliferation, differentiation and apoptosis. Post-fixation analysis and characterization The tissue density of 3D cell cultures can make them more challenging to characterize than 2D cultures, requiring specialized techniques. Here are some techniques to help you plan how you will characterize your 3D cultures: • For tissues that are thicker than 100 µm, consider cryosectioning your sample to obtain better immunostaining and imaging quality. This can also be useful if you want to perform multiple different stains on the same sample. • If you want to keep your tissue sample intact, consider using clearing techniques to make the tissue transparent for whole-tissue immunostaining and imaging. There are several different clearing methods available such as CLARITY and 2,2’-thiodiethanol (TDE), each suited to different applications.10 • For structural analysis of tissues, electron microscopy can reveal nanometer-resolution cellular details, either on whole or sliced tissue samples. • Gene expression analysis can be performed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) or RNA sequencing. Single cell RNA sequencing is another powerful tool that provides the expression profile of individual genes. This can be costly and bioinformatics services will typically also be required, so plan your experiment carefully. Quality control and troubleshooting Effective documentation of your cell seeding conditions, culture parameters and experimental results are essential to keep track of your data and optimize your workflow. Integrating digital tools for data consistency and traceability can help you to keep organized. Here are some key quality control measures and troubleshooting tips to consider when planning your experiment: • Before starting, identify critical quality control points in your workflow. • Routinely check your cells for contamination by performing mycoplasma testing. • Monitor the viability of your cells in 2D before using them for 3D culture using assays like 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) or live/dead staining. • Before seeding your cells for 3D culture, make sure that your cells are what you think they are! Cells can easily get mixed up during storage or transport and can also change their genetic identity during passaging. If you are using ESCs or iPSCs, it is a good idea to genotype your cells every 10–15 passages. • Monitor cell morphology and behavior using brightfield microscopy. Refer to the “Analysis and characterization of live 3D cultures” section above for more techniques to monitor cell behavior during culture. Key takeaways Culturing cells in 3D typically takes longer, is less reproducible and is more complex to characterize than 2D cultures. But this shouldn’t put you off! Instead, it should highlight how important it is to start with the right cells for your application, be confident with your culture techniques and don’t rush. Taking the time to optimize your workflow will ultimately save you time (and money) in the long run. 3D CELL CULTURE TIPS TO OPTIMIZE YOUR WORKFLOW 5 How To Guide Sponsored by: References 1. Zhao Z, Chen X, Dowbaj AM, et al. Organoids. Nat Rev Methods Prim 2022 21. 2022;2(1):1-21. doi:10.1038/s43586-022- 00174-y 2. Fang G, Chen YC, Lu H, Jin D. Advances in spheroids and organoids on a chip. Adv Funct Mater. 2023;33(19):2215043. doi:10.1002/adfm.202215043 3. Wang Y, Jeon H. 3D cell cultures toward quantitative high-throughput drug screening. Trends Pharmacol Sci. 2022;43(7):569-581. doi:10.1016/j.tips.2022.03.014 4. Gunti S, Hoke ATK, Vu KP, London NR. Organoid and spheroid tumor models: Techniques and applications. Cancers (Basel). 2021;13(4):874. doi:10.3390/CANCERS13040874 5. Vanaei S, Parizi MS, Vanaei S, Salemizadehparizi F, Vanaei HR. An overview on materials and techniques in 3D bioprinting toward biomedical application. Eng Regen. 2021;2:1-18. doi:10.1016/J.ENGREG.2020.12.001 6. Yin X, Mead BE, Safaee H, Langer R, Karp JM, Levy O. Engineering stem cell organoids. Cell Stem Cell. 2016;18(1):25-38. doi:10.1016/j.stem.2015.12.005 7. Kim J, Koo BK, Knoblich JA. Human organoids: Model systems for human biology and medicine. Nat Rev Mol Cell Biol. 2020;21(10):571-584. doi:10.1038/s41580-020-0259-3 8. Maji S, Lee H. Engineering hydrogels for the development of three-dimensional in vitro models. Int J Mol Sci. 2022;23(5):2662. doi:10.3390/IJMS23052662 9. Yousafzai MS, Hammer JA. Using biosensors to study organoids, spheroids and organs-on-a-chip: A mechanobiology perspective. Biosensors. 2023;13(10):905. doi:10.3390/BIOS13100905 10. Susaki EA, Takasato M. Perspective: Extending the utility of three-dimensional organoids by tissue clearing technologies. Front Cell Dev Biol. 2021;9:679226. doi:10.3389/FCELL.2021.679226 About the author: Kaja attained a BSc in genetics, MRes in neurotechnology and a PhD in bioengineering at Imperial College London. During her PhD she worked on bioengineering organoids and spent part of her time working in an organoid group in Vienna, Austria. She is currently working as a freelance science writer.