Two-dimensional cell cultures

Table 2. Correlation between the extent of cell adhesion and the subsequent cell behavior in a conventional two-dimensional cell culture system [40]...

Although investigations of mechanical strain on a two-dimensional cell culture platform allow the researchers to gain insights on the mechanotransduction of cells, this environment does not represent the physiological conditions of the native cells in a three-dimensional in vivo environment as the presence of several other... 

Micromechanical stimulator devices have been fabricated and used to precisely assess the mechanical strain effects on the two-dimensional ceU cultures. For instance, a high-throughput perfusi(Mi-based micro-bioreactor platform was developed to exert uniform tensile strain in a controUable maimer to the attached cells within the central circular membrane (Fig. 1). This microdevice was capable of applying tensile strain similar to that experienced by the articular chondrocytes during walking in human and was used to demonstrate the effect of tensile strain on the physiology of bovine articular chondrocytes. 

Since the initial development of methods for in vitro cnlture of human keratinocytes (Rheinwald and Green, 1975), two-dimensional monolayer cultures of submerged keratinocytes have been widely utilized for skin-related research. Primary and immortalized fibroblast and melanocyte cultures are also now widely available for in vitro research and toxicology testing applications (Hsu and Herlyn, 1996 Costin and Hearing, 2007). Skin-resident immune cells (e.g., Langerhans cells) are difficult to isolate and maintain in primary culture. However, monocyte-derived dendritic cells and dendritic cells derived from cord blood precursors are well established, as are a variety of immortalized dendritic-like cell lines (Ayehunie et al., 2009 Reuter et al., 2011). 

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. 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

As was stressed by Professor Ubbelohde, in the process of cell recognition not only the lateral diffusion of the binding sites has to be considered, but also the mechanical effects resulting from the local change of surface tension, inducing convection at the cell surface. It is well known, in the cell-to-cell contact inhibition of motion, in tissue culture, that a cell approaches another cell by touching it by means of microvilli and that this process can be affected when adding surfactants to the culture. Now the point is, What is the relative importance of both diffusion and convection Well, in binary surface films, it was observed that the transport process induced by two-dimensional convection is much more rapid than the two-dimensional diffusion. 

Steinberg RA, Coffino P. Two-dimensional gel analysis of cyclic AMP effects in cultured S49 mouse lymphoma cells protein modifications, inductions and repressions. Cell 1979 18 719-733. 

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. 

We allow R to depend on voltage because in a two dimensional medium R must contain terms from the exchange of molecules with the culture medium with which the sheet of cells is in contact. Defining the culture medium to have voltage V then V in R(c, v) is the voltage within a cell minus V, i.e. v(r) is the voltage drop f om the culture medium to the interior of a cell surrounding point r. 

Figure 4. Two-dimensional heteronuclear multiple quantum coherence spectrum of a medium sample taken from a culture of HeLa cells which had been grown for 48 hours in the presence of 2 mM L[2-15N]glutamine. In this type of experiment the, 5N label (FI axis) is detected indirectly via spin-coupled protons (F2 axis). The peaks labeled in the contour plot arise from alanine (Ala), glutamate (Clu), glutamine (Gin), glycine (Gly), aspartate (Asp), and pyrollidone carboxylic acid (Pyr). From Street etal., 1993, with permission.

Other primary cells, like cardiomyocytes or chondrocytes, can rapidly undergo de-differentiation to fibroblast-like cells when plated on a two-dimensional (flat) surface. In this case, three-dimensional matrices can help maintain the original features. BioLevitator by Hamilton Robotics, 3D Insert by 3dbiotek, and 3D cell culture by Invitrogen are only some of the products available to facilitate the correct growth and morphology of particular primary cell types. 

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