Three-dimensional Cell Culture Systems

Animal cells cultured in two-dimensional (2D) monolayers in traditional glass or plastic tissue culture flasks have been used successfully for many purposes in research and industrial production. However, such cultures may lose key phenotypic characteristics (e.g. virus susceptibility, morphology, surface markers/receptors) after repeated passage. In vivo the presence of three-dimensional (3D) cellular structures is critical to the correct development, function and stability of cells, tissues and organs. The characteristics that the researcher or technologist wishes to utilize are often a feature of the tissue and not individual cells, e.g. a functional bladder epithelium or crypt structures of the gut. In this section we describe some of the approaches that can be used to simulate certain features of the in vivo environment in an attempt to promote natural gene expression and tissue function in cultured cells. The described technologies address these features from two aspects  

Endogenous features (i.e. within 3D cell structures) autocrine and paracrine factors mass transfer characteristics establishment of appropriate cell-cell and cell-matrix contacts cell signalling pathways and pressure and tensile forces. 

Exogenous features (i.e. in culture medium) hormones nutrition, including conditioned media pO, pH pCO, and temperature. 

Many of these specialized functions of cells are lost or expressed at low levels when they are growing in monolayers and this is due, in part, to the lack of appropriate cell-cell or cell-matrix interactions. Cultivation in a 3D system can promote or improve characteristic cell functions such as hormone secretion, production of extracellular matrix components and expression of differentiation markers. 

The resulting cellular structures can be used as models for investigating development, drug metabolism, toxicity, biotransformations, pathogenesis and microorganism replication. 

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Lee EJ, Baek M, Gusev Y, Brackett DJ, Nuovo GJ, Schmitlgen TD (2008) Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumms. RNA 14(1) 3542 Lee JB, Sonb SH, Park MC, Kima TH, Kima MG, Yoo SD, Kim S (2014) A novel in vitro permeability assay using three-dimensional cell culture system. J Biotechnol. doi 10.1016/j. jbiotec.2014.12.019... 

Various tissue constructs have been reassembled from isolated constituents, including resident cell types whose numbers have been amplified or modified in culture. A three-dimensional co-culture system for human skin keratinocytes layered upon a synthetic mesh infiltrated with dermal fibroblasts, when floated to allow contact of the uppermost keratinocytes with air, exhibits stratification and cornification remarkably similar to in vivo squamous epithelia. This reconstructed epithelial model has been recommended as an in vitro replacement for dermal corrosivity testing. It has been anticipated that this and a similar noncomified model will have application in dermal and ocular irritation testing, but thus far validation studies have yielded mixed results. Reconstructed tissues can also provide context for basic toxicological research on aberrant cellular interactions with cellular and acellular constituents, as illustrated by invasion of cancerous epithelial cells into underlying dermis of a skin equivalent model. 

The mechanical properties of the capsule must be carefully considered. Not only does the capsule need sufficient strength to resist mechanical failure and rupture but the elasticity of cell substrates have been shown to influence cell behavior. The mechanism by which substrate elasticity influences differentiation of multipotent cells is not clear. A number of recent studies have analyzed this in both two- and three-dimensional (3D) culture systems, - Integrin engagement is thought to be required to transmit the elasticity of the capsule material to the cells inside. Matrix elasticity has been shown to influence the lineage commitment of naive, mesenchymal stem cells. Another important consideration is how the mechanical properties of a capsule impact the transmission of external mechanical forces to the cells. Many cells respond to mechanical loads and this can be necessary for maintenance of phenotype or to guide differentiation. ... 

Microchambers can be added into the micromechanical stimulators, which allow the incorporation and support of a three-dimensional cell culture. Similar to the rest of the micromechanical stimulators, the systems make use of the deformation of the elastomer membrane to provide either the compressive or tensile strains to the tissue constructs. The systems can be used to monitor and investigate the effect of mechanical loading on the development of a 3D tissue construct. 

Torisawa, Y.-S., Shiku, H., Yasukawa, T., Nishizawa, M., Matsue, T. Three-dimensional micro-culture system with a silicon-based cell array device for multi-channel drug sensitivity test. Sens Actuators B Chem 2005,108, 654-659. 

A more complex system, such as aggregating brain cell cultures, has the advantage of providing a three-dimensional cell system containing all cell types and allowing cell-cell interactions and permits testing of multiple endpoints in... 

D- FCCS Three-dimensional microfluidic cell culture system... 

Nanocapsules covered with phospholipids can be functionalized by attaching or inserting substances with particular properties, like monoclonal antibodies which can be used for targeting. Monoclonal antibodies produced in mammalian cell culture systems are becoming increasingly important as auxiliaries for the treatment of human diseases. Antibodies [65] attached to a phospholipid layer that cover radioactive nanocapsules can be transported by blood to a specific cell for binding to a tumor marker. Functionalization can also be used for self-assembly to obtain organized two- and three- dimensional stmctures [66]. 

By combining microdialysis with nanoESI-MS, low-volume, low-concentration releases of small proteins in a three-dimensional neural cell culture system could be detected [78]. While microdialysis removes interferents, it also has an effect on the temporal resolution of the device ( 1 min). Alternatively, a microdialysis probe can be coupled with paper-spray ionization MS which enables the monitoring of glucose in a cell culture medium [79]. 

Finally, the studies summarized above address the spatial effect of the ECM on the extent of cell distortion and tension in a two-dimensional ceU culture system. With few exceptions, the mechanical interaction between a ceU and the ECM occurs around the whole cell surface in a true physiological setting. While there is not yet a direct measurement method to correlate tractions and the spatial distribution of ECM ligands in a three-dimensional system, it is likely that the correlation is similar to that in a planar culture system. 

To mimic the biological microenvironment of cells, a variety of three-dimensional (3D) biomaterials have been used as substitutes for the ECM. Unlike conventional two-dimensional (2D) culture systems where cells generally grow and proliferate as a horizontal monolayer, 3D scaffolds provide a physical support matrix thus increasing cell-cell and cell-substrate interactions (Martin et al. 1998 Tan et al. 2001). Therefore, bioengineered 3D culture systems have become a promising experimental approach for the differentiation of both adult and ES cells (Martin et al. 1997 Solchaga et al. 1999 Dawson et al. 2008). Table 35.1 summarizes biomaterials used for the expansion and differentiation of hematopoietic cells that are discussed in detail below. 

Alves PM, Moreira JL, Rodrigues JM, Aunins JG, Carrondo MJT (1996), Two dimensional versus three dimensional cultures systems effects on growth and productivity of BHK cells, Biotechnol. Bioeng. 52 429-432. 

Liu M, Xu J, Souza P, Tanswell B, Tanswell AK, Post M. The effect of mechanical strain on fetal rat lung cell proliferation Comparison of two- and three-dimensional culture systems. In Vitro Cell Dev Biol Anim. 1995 31 858-866. 

Another area that has seen considerable advances is that of the physical environment for cell growth. Glassware has almost entirely disappeared from most tissue culture laboratories, to be replaced by an enormous, and expensive, range of plasticware. This has enabled the scientist to derive more sophisticated culture vessels and has led to the manufacture of three-dimensional culture systems and microcarriers. An added benefit has been the reliability and reproducibility of culture systems and, as a result, the avoidance of variability and contamination... 

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