Three-dimensional (3D) cell culture has taken the life sciences community by storm, as evidenced by the recent surge in publications on this subject. Compared with standard 2D cultures, 3D methods provide a structural niche in which cells more closely replicate their in vitro behavior. For example, cells grown in 3D are in close proximity, as they are in living tissues and organs. They acquire nutrients and eliminate waste through more in vivo-like gradients, and they are susceptible to neighboring cells’ chemical messaging.
The growth substrates utilized in 2D cultures are more rigid, causing cells to lose their optimal physical shape and polarity. Cell growth and proliferation, to the extent that cells are capable, is more in vivo-like in 3D, as is cellular access to the extracellular matrix (ECM) and available growth factors. Where cells in 2D respond rapidly and uniformly to chemical and physical stimuli, the biochemical gradient inherent in 3D cell assembly is slower and more nuanced. Perhaps most relevant to modern biology, 3D-cultured cells often express critical genes more faithfully relative to their physiologic counterparts than do cells in 2D cultures.
Spheroids are simple yet effective 3D cell models that form from adherent cells’ natural tendency to aggregate. Researchers generate spheroids from a broad range of cell types resulting in tumor spheroids, embryoid bodies, and tissue-relevant 3D models such as hepatospheres, neurospheres, and mammospheres.
Spheroids develop metabolic gradients that create heterogeneous cell aggregates exhibiting interactions among and between cells, and with the ECM, that are more life-like. Spheroids thus can mimic typical microenvironments of tissue types in disease states. Spheroids may self-assemble scaffold-free, without the aid of ECMs or other physical supports. Scaffold-free methods include suspension cultures, hanging drop methods, spinner flask cultures, or growth in attachment-resistant cultureware.
Typical scaffold-free applications include the formation of uniform embryoid bodies from induced pluripotent stem cells to study stem cell fate and behavior, tumor biology to test the efficacy of cancer drugs and drug candidates, and immuno-oncology to quantify the cytotoxicity of CAR-T cancer treatments.
Spheroids also form through interactions with hydrogels—natural or synthetic crosslinked polymers or proteins that are used to recapitulate ECM.
Organoids, a somewhat more complex 3D cell culture model, have become increasingly popular in drug discovery and development. Organoids are 3D cultures, usually consisting of several cell types, that recreate some of the functions of intact living organs. Some organoids are produced through 3D printing, a method that allows exquisite control over the cellular composition of these structures. When combined with an appropriate matrix, stem cells or progenitor cells from normal or diseased tissue form what are essentially mini-organ models of kidney, thyroid, liver, brain, etc., and thus support the study of organogenesis, disease modeling, and investigations into personalized therapies.
Organoid applications can also include multiparameter models of tumor progression, invasion, and metastasis. For metastasis to occur tumor cells must cross a biological matrix to reach the circulatory system, a process emulated in scaffold-based organoid models. Similarly, tumor cell survival, apoptosis, and invasion may be modeled in appropriately scaffolded organoids for basic research or investigations into anticancer agents.
The right tools are critical to the consistency of 3D cell cultures, and ultimately to their success.
Corning® Matrigel® matrix, the most widely used natural bioactive ECM for 3D cultures, exhibits the chemical and mechanical properties of living ECM. The matrix creates an ideal ex vivo microenvironment for cell growth and development. Matrigel consists of a reconstituted extract from mouse tumor, and is rich in laminin, collagen IV, entactin, heparin sulfate proteoglycans, and growth factors, making it ideal for cancer and stem cell research.
Corning Collagen Type I is a natural hydrogel component of stromal compartments of dermis, tendon, and bone. Collagen Type I supports life-like 3D growth and differentiation, by providing physiological interactions with receptors that modulate gene expression related to cancer cell invasion, susceptibility to anticancer drugs, cell proliferation, and cell migration. Additionally, the matrix has been used to culture intestinal, primary mammary, and salivary organoids.
Corning Spheroid microplates combine the Corning Ultra-Low Attachment surface with an innovative U-shaped well geometry resulting in the formation of a single spheroid in each well without the addition of an ECM. The plates provide an accessible, highly reproducible platform for generating, culturing, and assaying individual spheroids. Corning Spheroid microplates feature opaque side walls and a gridded-plate bottom to reduce well-to-well crosstalk and background fluorescence/luminescence for drug and toxicity screens. Corning spheroid microplates are available in 96-, 384-, and 1536-well automation-friendly formats and can be combined with matrices and 96 HTS Transwells for added complexity.
The time has never been better to consider 3D cell culture for chemical, toxicological, pharmaceutical, and physical testing of cell models. Having the right tools for the job can spell the difference between failure or inconsistent results, and reproducible data to support mission-critical experiments. With a varied portfolio of reagents, microplates, ECMs and permeable supports, Corning is an ideal partner to advance your research with the 3D cell culture.
1. Simian, Marina & J Bissell, Mina. (2016). Organoids: A historical perspective of thinking in three dimensions. The Journal of Cell Biology. 216. 10.1083/jcb.201610056.
2. Edmondson, R., Broglie, J. J., Adcock, A. F., & Yang, L. (2014). Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay and Drug Development Technologies, 12(4), 207-18.
3. Fang Y and Eglen RM. Three-Dimensional cell cultures in drug discovery and development. SLAS Discovery (2017) 22:456-472
4. Fatehullah, A., S.H. Tan, and N. Barker. 2016. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18:246–254. doi:10.1038/ncb3312
5. Dawson E, Mapili G, Erickson K, Taqvi S, Roy K. Biomaterials for stem cell differentiation. Adv Drug Deliv Rev. (2008) 60(2):215-28.
6. Li X, Nadauld L, Ootani A, et al. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nature Medicine (2014) 20:769-777.
7. Linnemann JR, Miura H, Meixner LK. Quantification of regenerative potential in primary human mammary epithelial cells. Development (2015) 142:3239-3251.
8. Nanduri Lalitha SY, et al. Purification and ex vivo expansion of fully functional salivary glands stem cells. Stem Cell Reports (2014) 3(6):957-964.