2D culture

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. 

The traditional way to cultivate primary hepatocytes is on culture dishes coated with stiff and dry collagen that has been isolated from rat tails. This technique is also named 2D culture. 2D cultures are known to represent the in vitro system which most rapidly dedifferentiates. However, it should be considered that dedifferentiation shows major interspecies differences. While hepatocytes of rats and mice rapidly dedifferentiate showing features of epithelial... 

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]. 

Fig. 1 Examples of 2D and 3D cell culturing. (A) Illustration of 2D flat cell culturing platform and 3D cell culturing. (B) Phase contrast micrographs of 2D monolayer embryoid body cells at day 5 (scale bar 200 pm) and (C) embryoid body cells in 3D scaffold at day 5 (scale bar 500 pm). Arrows show cells migrated from an aggregate. The migrated cells became cordhke and multilayer structures, which were not existed in 2D cultures (Adapted from [4]). (D) Phase contrast micrographs of SKOV-3 cells grown in 2D and (E) in 3D environment (Adapted from [5]). Scale bar 100pm.

Another 3D model had been developed with immortalized human renal cortical cells embedded into a mix of Matrigel and rat tail collagen (DesRochers et al., 2013). These cells displayed some features of PTC but performed also functions that are not typical for PTC. For instance, the cells were responsive to antidiuretic hormone, whereas they did not respond to parathyroid hormone. The effects of cisplatin, gentamicin, and doxorubicin were tested by measuring cell viability, LDH release, and increase of KIM-1 and NGAL. In compatison to 2D cultures, the 3D model did not clearly improve the results. Also here positive responses to doxorubicin were observed, and in this case the 3D model was more sensitive than 2D cultures to this usually non-PT-damaging drag. 

The shape, growth and response ability of cells to cytokine in a 3D collagen gel eulture differs significantly Irom those in a 2D culture (ineluding a 2D culture on a collagen gel) [8], The characteristics of a fibroblast in a 3D collagen gel culture are hsted in Table 1 [11]. 

Formation of 3D cellular structures that are more complex than would naturally occur in the absence of tissue-engineered conditions (i.e., beyond 2D culture and self-forming spheroids)... 

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