3D culture

Birgersdotter A, Sandberg R, Ernberg I (2005) Gene expression perturbation in vitro—z growing case for three-dimensional (3D) culture systems. Semin Cancer Biol 15 405-412... 

Cell proliferation GI s of the test agent should be determined in preliminary studies using assays compatible with 3D cultures (e.g., CellTiter Glo luminescent assay or WST-1 assay) so that appropriate concentrations can be selected. 

Figure 5. Immunohistochemical staining of phosphorylated EGFR in A431 epidermoid cell lines. Tissues from A431 xenograft bearing SCID mouse (A) and 3D-cultured A431 cells in collagen gel (B) were formalin-fixed, paraffin-embedded, and used for method validation.

In industrial (e.g. recombinant protein) and medical (e.g. bioartificial organ) fields 3D cultures can be used to improve the surface area/volume ratio compared with 2D cultures, which is a useful feature where cells are used as the machinery for biological production. Such approaches promote high cell yield and increased production of cellular or recombinant proteins. 

Diverse techniques for 3D culture are now available and here we will primarily consider ... 

The microcontainer technique is a recently developed method for 3D cultures (Weibezahn et al, 1994). In this patented system cells grow on the vertical walls until a multicellular layer is formed. The containers are then transferred into a vessel and perfused continuously. A controlled supply of growth media of different compositions on each side of the layer allows the culture of highly differentiating ceU types and hence the establishment of tissue-like models. The full range of applications for this technique remains to be determined and it may well provide some useful in vitro tissue models in the future. 

Current hepatocyte in vitro system, including 3D cultures and microtissues, do not yet form a lobular zonation. 

Hepatocytes cultivated between two layers of soft gel collagen represent the most frequently used hepatocyte in vitro system. They establish an apical pole between the cells which contains bile canaliculi (Fig. 3). The hepatocyte membrane facing the collagen gel corresponds to the basolateral side. Therefore, hepatocyte sandwich cultures represent the easiest to handle 3D culture system, although only one sheet of hepatocytes is represented. The hepatocyte phenotype in sandwich culture is characterized by (1) maintenance of susceptibility to apoptosis, (2) a delayed decrease of drug-metabolizing activities compared to monolayer cultures, (3) establishment and maintenance of bile canaliculi, and (4) a resting cell state where stimulation by HGF and EGF induces almost no proliferation events. As previously mentioned, this cultivation system effectively prevents the spontaneous activation of ERK and Akt which occurs in 2D systems [13]. Consistent with the effects of small chemical inhibitors in 2D cultures, expression of a constitutively active form of Ras in sandwich-cultured hepatocytes induces features of EMT and stress fibers. In contrast to Ras, expression of constitutive active Akt in hepatocytes induces an antiapoptotic phenotype and does not cause EMT [13]. 

Three-dimensional brain cell cultures can been obtained from different sources such as cell lines [38], primary cells [39], and stem cells [40]. Depending of the cells used the 3D culturing techniques can vary, e.g., stem cells are commonly forming 3D structures spontaneously, while cell lines and primary cells often need support by for example extracellular matrix, hanging drop, or rotation-mediated culture techniques. 

D Cultures and Primary hepatocytes from human liver remain the gold standard for... 

Human primary hepatocytes remain as the best cell model for human liver, and the recent advances in 3D culture techniques have solved some of the problems with rapid decline in metabolic competence. However, the limited availability of human liver remains as a botdeneck. Therefore there is a clear need for more reliable and consistent source of metabolically competent hepatocytes. Previously in this chapter we have discussed the pros and cons of the HepaRG cell line, which currently represents the best... 

A phenomenon called dielectrophoresis has been used to pattern a variety of cell types on 2D substratesand more recently in 3D culture constructs. Unlike electrophoresis where charged species move in an applied electric field due to Coulombic forces (F = qE), dielectrophoresis capitalizes on the ability of a cell to become polarized when placed in an electric field. Dielectrophoresis is most often used in conjunction with dtemating current (AC) electric fields since AC fields eliminate electrophoretic movement, and have less physiological impact on cells than direct current (DC) fields. When a cell is placed in an AC field, the magnitude and polarity of the induced dipole depend on the frequency of the applied field and the conductivities of the cell and the surrounding medium, described by the equation... 

Since dielectrophoresis relies on the geometry of the electrodes to create the nonuniform electric fields, the method has been limited to the creation of 2D cell patterns within 3D culture constructs (Figure 32.5c). True 3D cell patterning would require more complex electrode geometries than those employed to date. Although dielectrophoresis has been used to levitate cells and could be used for 3D patterning, the method has not been utilized solely or in combination with any other methods to pattern cells in all three dimensions within a 3D culture construct. 

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. ... 

Choi BG, Park MH, Cho S-H, Joo MK, Oh HJ, Kim EH, Paik K, Han DK, Jeong B (2011) Thermal gelling polyalanine-poloxamine-polyalanine aqueous solution for chondrocytes 3D culture initial concentration effect. Soft Matter 7 456-462... 

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