3D cell culture
A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments (e.g. a Petri dish), a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.
Early studies in the 80’s, led by Mina Bissell from the Lawrence Berkeley National Laboratory, highlighted the importance of 3D techniques for creating accurate in vitro culturing models. This work focused on the importance of the extracellular matrix and the ability of cultures in artificial 3D matrices to produce physiologically relevant multicellular structures, such as acinar structures in healthy and cancerous breast tissue models. These techniques have been applied to for in vitro disease models used to evaluate cellular responses to pharmaceutical compounds.
Eric Simon, in a 1988 NIH SBIR grant report, showed that electrospinning could be used to produced nano- and submicron-scale polystyrene and polycarbonate fibrous mats (now known as scaffolds) specifically intended for use as in vitro cell substrates. This early use of electrospun fibrous lattices for cell culture and tissue engineering showed that various cell types including Human Foreskin Fibroblasts (HFF), transformed Human Carcinoma (HEp-2), and Mink Lung Epithelium (MLE) would adhere to and proliferate upon the fibers. It was noted that as opposed to the flattened morphology typically seen in 2D culture, cells grown on the electrospun fibers exhibited a more histotypic rounded 3-dimensional morphology generally observed in vivo.
In living tissue, cells exist in 3D microenvironments with intricate cell-cell and cell-matrix interactions and complex transport dynamics for nutrients and cells. Standard 2D, or monolayer, cell cultures are inadequate representations of this environment, which often makes them unreliable predictors of in vivo drug efficacy and toxicity. 3D spheroids more closely resemble in vivo tissue in terms of cellular communication and the development of extracellular matrices. These matrices help the cells to be able to move within their spheroid similar to the way cells would move in living tissue. The spheroids are thus improved models for cell migration, differentiation, survival, and growth. Furthermore, 3D cell cultures provide more accurate depiction of cell polarization, since in 2D, the cells can only be partially polarized. Moreover, cells grown in 3D exhibit different gene expression than those grown in 2D.
The third dimension of cell growth provides more contact space for mechanical inputs and for cell adhesion, which is necessary for integrin ligation, cell contraction and even intracellular signalling. Normal solute diffusion and binding to effector proteins (like growth factors and enzymes) is also reliant on the 3D cellular matrix, so it is critical for the establishment of tissue scale solute concentration gradients 
For the purposes of drug toxicology screening, it is much more useful to test gene expression of in vitro cells grown in 3D than 2D, since the gene expression of the 3D spheroids will more closely resemble gene expression in vivo. Lastly, 3D cell cultures have greater stability and longer lifespans than cell cultures in 2D. This means that they are more suitable for long-term studies and for demonstrating long-term effects of the drug. 3D environments also allow the cells to grow undisturbed. In 2D, the cells must undergo regular trypsinization in order to provide them with sufficient nutrients for normal cell growth. 3D spheroids have been cultured in a lab setting for up to 302 days while still maintaining healthy, non-cancerous growth.
Classification of 3D culture methods
There are a large number of commercially available culturing tools that claim to provide the advantages of 3D cell culture. In general, the platforms can be classified in two types of 3D culturing methods: scaffold techniques and scaffold-free techniques.
Scaffold techniques include the use of solid scaffolds, hydrogels and other materials. In a recent study potentiality of human CD34+ stem cells explored by generating in vitro agarose gel 3D model to understand the bone ossification process.
As the natural extracellular matrix (ECM) is important in the survival, proliferation, differentiation and migration of the cells, different hydrogel matrices mimicking natural ECM structure are considered as potential approaches towards in vivo –like cell culturing. Hydrogels are composed of interconnected pores with high water retention, which enables efficient transport of e.g. nutrients and gases. Several different types of hydrogels from natural and synthetic materials are available for 3D cell culture, including e.g. animal ECM extract hydrogels, protein hydrogels, peptide hydrogels, polymer hydrogels, and wood-based nanocellulose hydrogel.
Scaffold free techniques employ another approach independent from the use scaffold. Scaffold-free methods include e.g. the use of low adhesion plates, hanging drop plates, micropatterned surfaces, and rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting.
Spheroids are a type of three-dimensional cell modeling that better simulate a live cell’s environmental conditions compared to a two-dimensional cell model, specifically with the reactions between cells and the reactions between cells and the matrix. Spheroids are useful in the study of changing physiological characteristics of cells, the difference in the structure of healthy cells and tumor cells, and the changes cells undergo when forming a tumor. Spheroids co-cultured with tumor and healthy cells were used to simulate how cancerous cells interact with normal cells. Spheroids can be grown with a few different methods. One common method is to use low cell adhesion plates, typically a 96 well plate, to mass-produce spheroid cultures, where the aggregates form in the rounded bottom of the cell plates. Spheroids can also be cultured using the hanging drop method, involving forming cell aggregates in drops that hang from the surface of a cell plate. Other methods under investigation include the use of rotating wall vessel bioreactors, which spins and cultures the cells when they are constantly in free fall and forms aggregates in layers 
The bioreactors used for 3D cell cultures are small plastic cylindrical chambers that are specifically engineered for the purpose of growing cells in three dimensions. The bioreactor uses bioactive synthetic materials such as polyethylene terephthalate membranes to surround the spheroid cells in an environment that maintains high levels of nutrients. They are easy to open and close, so that cell spheroids can be removed for testing, yet the chamber is able to maintain 100% humidity throughout. This humidity is important to achieve maximum cell growth and function. The bioreactor chamber is part of a larger device that rotates to ensure equal cell growth in each direction across three dimensions.
MC2 Biotek has developed a bioreactor to incubate proto-tissue that uses gas exchange to maintain high oxygen levels within the cell chamber. This is an improvement over previous bioreactors because the higher oxygen levels help the cell grow and undergo normal cell respiration.
The various cell structures in the human body must be vascularized to receive the nutrients and gas exchange help that they need to survive. Similarly, 3D cell cultures in vitro require certain levels of fluid circulation, which can be problematic for dense, 3D cultures where cells may not all have adequate exposure to nutrients. This is particularly important in hepatocyte cultures because the liver is a highly vascularized organ. One study cultured hepatocytes and vascular cells together on a collagen gel scaffold between microfluidic channels, and compared growth of cells in static and flowing environments, and showed the need for models with tissues and a microvascular network.
Pharmacology and toxicology
A primary purpose of growing 3D cell spheroids in vitro is to test pharmacokinetic and pharmacodynamic effects of drugs in preclinical trials. Toxicology studies have shown 3D cell cultures to be nearly on par with in vivo studies for the purposes of testing toxicity of drug compounds. When comparing LD50 values for 6 common drugs: acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid, the 3D spheroid values correlated directly with those from in vivo studies. Although 2D cell cultures have previously been used to test for toxicity along with in vivo studies, the 3D spheroids are better at testing chronic exposure toxicity because of their longer life spans. The matrix in 3D Spheroids causes cells to maintain actin filaments and is more relevant physiologically in cytoskeletal organization and cell polarity and shape of human cells. The three-dimensional arrangement allows the cultures to provide a model that more accurately resembles human tissue in vivo without utilizing animal test subjects.
Existing 3D methods are not without limitations, including scalability, reproducibility, sensitivity, and compatibility with high-throughput screening (HTS) instruments. Cell-based HTS relies on rapid determination of cellular response to drug interaction, such as dose dependent cell viability, cell-cell/cell-matrix interaction, and/or cell migration, but the available assays are not optimized for 3D cell culturing. Another challenge faced by 3D cell culturing is the limited amount of data and publications that address mechanisms and correlations of drug interaction, cell differentiation, and cell-signalling in these 3D environments. None of the 3D methods have yet replaced 2D culturing on a large scale, including in the drug development process; although the number of 3D cell culturing publications is increasing rapidly, the current limited biochemical characterization of 3D tissue diminishes the adoption of new methods.
There are also problems using spheroids as a model for cancerous tissue. Although beneficial for 3D tissue culture, tumor spheroids have been criticized for being challenging or impossible to “manipulate gradients of soluble molecules in [3D spheroid] constructs, and to characterize cells in these complex gradients”, unlike the paper-supported 3D cell culture for tissue-based bioassays explored by Ratmir et al.
- Fey, Stephen; Wrzesinski, Krzysztof (2013). "5" (PDF). In Boucher, Alexis (ed.). Valproic Acid. Nova Science Publishers, Inc. pp. 141–165. ISBN 978-1-62417-952-5. Archived from the original (PDF) on 2013-12-02. Retrieved 2013-11-25.
- MERIT Award Recipient: Mina J. Bissell, Ph.D. (n.d.). Retrieved June 16, 2016, from http://www.cancer.gov/research/nci-role/spotlight/merit/Bissell
- Simon, Eric M. (1988). "NIH PHASE I FINAL REPORT: FIBROUS SUBSTRATES FOR CELL CULTURE (R3RR03544A) (PDF Download Available)". ResearchGate. Retrieved 2017-05-22.
- Marx, Vivien (11 April 2013). "A Better Brew" (PDF). Nature. Retrieved July 9, 2013.
- Souza, Glauco (14 March 2010). "Three-dimensional tissue culture based on magnetic cell levitation". Nature Nanotechnology. 5 (4): 291–296. doi:10.1038/nnano.2010.23. PMC 4487889. PMID 20228788.
- Pampaloni, Francesco (October 2007). "The third dimension bridges the gap between cell culture and live tissue". Nature Reviews Molecular Cell Biology. 8 (10): 839–845. doi:10.1038/nrm2236. PMID 17684528.
- Boudreau, Nancy (5 May 2006). "A pericellular collagenase directs the 3-dimensional development of white adipose tissue". Cell. 125 (3): 577–91. doi:10.1016/j.cell.2006.02.050. PMID 16678100.
- Yamada, KM (24 August 2007). "Modeling tissue morphogenesis and cancer in 3D". Cell. 130 (4): 601–10. doi:10.1016/j.cell.2007.08.006. PMID 17719539.
- Friedrich, Seidel (12 February 2009). "Spheroid-based drug screen: considerations and practical approach". Nature Protocols. 4 (3): 309–324. doi:10.1038/nprot.2008.226. PMID 19214182.
- Prestwich, Glenn (15 August 2007). "Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach". Journal of Cellular Biochemistry. 101 (6): 1370–83. doi:10.1002/jcb.21386. PMID 17492655.
- Griffith, Linda; Melody A. Swartz (March 2006). "Capturing complex 3D tissue physiology in vitro". Nature Reviews Molecular Cell Biology. 7 (3): 211–224. doi:10.1038/nrm1858. PMID 16496023.
- Lee, J; Cuddihy MJ; Kotov NA. (14 March 2008). "Three-dimensional cell culture matrices: state of the art". Tissue Engineering Part B: Reviews. 14 (1): 61–86. doi:10.1089/teb.2007.0150. PMID 18454635.
- Haycock JW. (2011). 3D cell culture: a review of current approaches and techniques. Methods Mol. Biol. Methods in Molecular Biology. 695. pp. 1–15. doi:10.1007/978-1-60761-984-0_1. ISBN 978-1-60761-983-3. PMID 21042962.
- Prestwich, G.D. (15 August 2007). "Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach". Journal of Cellular Biochemistry. 101 (6): 1370–83. doi:10.1002/jcb.21386. PMID 17492655.
- Suuronen, E. J.; Sheardown, H.; Newman, K.D.; McLaughlin, C.R. & Griffith, M. (2005). Building in vitro Models of Organs. Building in Vitro Models of Organs. International Review of Cytology. 244. pp. 137–173. doi:10.1016/s0074-7696(05)44004-8. ISBN 9780123646484. PMID 16157180.
- Louekari, K (2004). "Status and prospects of in vitro tests and risk assessment". Altern. Lab. Anim. 32: 431–435.
- Knight, B.; et al. (2000). "Visualizing Muscle Cell Migration in situ". Curr. Biol. 10 (10): 576–585. doi:10.1016/s0960-9822(00)00486-3.
- Roskelley, CD; Desprez PY; Bissell MJ (1994). "Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction". Proc. Natl. Acad. Sci. USA. 91 (26): 12378–12382. doi:10.1073/pnas.91.26.12378. PMC 45441. PMID 7528920.
- Krzysztof Wrzesinski; et al. (2013). "HepG2/C3A spheroids exhibit stable physiological functionality for at least 24 days after recovering from trypsinisation". Toxicol. Res. 2 (3): 163–172. doi:10.1039/C3TX20086H.
- "After trypsinisation, 3D spheroids of C3A hepatocytes need 18 days to re-establish similar levels of key physiological functions to those seen in the liver" (PDF). Archived from the original (PDF) on 2015-04-02. Retrieved 2013-11-25.
- Srikanth, Lokanathan; Sunitha, Manne Mudhu; Kumar, Pasupuleti Santhosh; Chandrasekhar, Chodimella; Vengamma, Bhuma; Sarma, Potukuchi Venkata Gurunadha Krishna (2016). "Gel based in vitro 3D model exploring the osteocytic potentiality of human CD34+ stem cells". Molecular Biology Reports. 43 (11): 1233–1242. doi:10.1007/s11033-016-4053-4. ISSN 1573-4978. PMID 27497820.
- M.W. Tibbitt, K.S. Anseth Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture Biotechnol Bioeng. 2009 Jul 1;103(4):655-63.
- H. Geckil et al. Engineering hydrogels as extracellular matrix mimics Nanomedicine (Lond). 2010 Apr; 5(3): 469–484.
- Fennema, Eelco, et al. “Spheroid Culture as a Tool for Creating 3D Complex Tissues.” Trends in Biotechnology, vol. 31, ser. 02, Feb. 2013. 02. doi: 10.1016/j.tibtech.2012.12.003 PMID 23336996
- Jiang Y, Pjesivac-Grbovic J, Cantrell C, Freyer JP. A multiscale model for avascular tumor growth. Biophys J 2005;89(6):3884–3894. doi: 10.1529/biophysj.105.060640 pmid:16199495
- Guttilla, I.K., Phoenix, K.N., Hong, X. et al. Breast Cancer Res Treat (2012) 132: 75. doi:10.1007/s10549-011-1534-y PMID 21553120
- Kunz-Schughart, Leoni A, et al. “A Heterologous 3-D Coculture Model of Breast Tumor Cells and Fibroblasts to Study Tumor-Associated Fibroblast Differentiation.” Experimental cell research, 2001 doi:10.1006/excr.2001.5210 PMID 11339826
- Fennema, Eelco, et al. “Spheroid Culture as a Tool for Creating 3D Complex Tissues.” Trends in Biotechnology, vol. 31, ser. 02, Feb. 2013. 02. doi: 10.1016/j.tibtech.2012.12.003PMID 23336996
- Xu, Xian, et al. “Three-Dimensional in Vitro Tumor Models for Cancer Research and Drug Evaluation.” Biotechnology Advances, 10 Aug. 2014. doi: 10.1016/j.biotechadv.2014.07.009 PMID 25116894
- Du, Yanan (January 2008). "Synthetic sandwich culture of 3D hepatocyte monolayer". Biomaterials. 29 (3): 290–301. doi:10.1016/j.biomaterials.2007.09.016. PMID 17964646.
- Derda, Ratmir; et al. (2009). "Paper-Supported 3D Cell Culture for Tissue-Based Bioassays". Proceedings of the National Academy of Sciences of the United States of America. 106 (44): 18457–18462. doi:10.1073/pnas.0910666106. PMC 2773961. PMID 19846768.
- Fey, Stephen J. "WO2012022351". European Patent Register.
- Chung, Seok; Kamm, Roger D.; Sudo, Ryo; et al. (July 2009). "Transport-Mediated Angiogenesis in 3D Epithelial Coculture". The Journal of the Federation of American Societies for Experimental Biology (FASEB). 23 (7): 2155–2164. doi:10.1096/fj.08-122820. PMC 2718841. PMID 19246488.
- Fey, Stephen J. (June 2012). "Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line". Toxicological Sciences. 127 (2): 403–11. doi:10.1093/toxsci/kfs122. PMC 3355318. PMID 22454432.
- Messner, S.; Agarkova, I.; Moritz, W.; Kelm, J. M. (August 2012). "Multi-cell type human liver microtissues for hepatotoxicity testing". Archives of Toxicology. 87 (1): 209–213. doi:10.1007/s00204-012-0968-2. PMC 3535351. PMID 23143619.
- Jensen, Janne; Hyllner, Johan; Björquist, Petter (2009). "Human Embryonic Stem Cell Technologies and Drug Discovery". Journal of Cellular Physiology. 219 (3): 513–19. doi:10.1002/jcp.21732. PMID 19277978.
- Ratmir, D; et al. (2009). "Paper-Supported 3D Cell Culture for Tissue-Based Bioassays". Proceedings of the National Academy of Sciences of the United States of America. 106 (44): 18457–18462. doi:10.1073/pnas.0910666106. PMC 2773961. PMID 19846768.