**2. Artificially fabricated 3D scaffolds**

this limiting approach. Growing fibroblasts on flat, rigid surface of the Petri dish or flask results in major differences in adhesion formation and maturation, proliferation, cell signaling, migration and cytoskeletal function, compared to the three-dimensional environment [8-12]. Even changes in stiffness of the two-dimensional (2D) substrate have shown significant difference in fibroblasts response to the environment [13, 14]. Being cultured on 2D surfaces, these non-polar cells are forced to adapt to polar setting, thus significantly changing their response to the environment. One could argue that this culturing method would be ideal for epithelial cells, allowing them to form distinct apical and basal layers. Nevertheless growth in 3D environment allows normal polarization and differentiation of epithelial cells [15] and is a prerequisite for the formation of duct-like structures in vitro [16]. Thereby the emerging differences between conventional, monolayer cell cultures and in vivo cellular behavior led to

The idea of cell culturing in 3D environment is not a novel one though [17], but until recently the importance of the cell environment – its dimensionality, stiffness, elasticity, composition and remodeling during tissue morphogenesis and disease, remained neglected. Number of studies have shown that knockdown of extracellular matrix (ECM) components like fibronec‐ tin, collagen, laminin, aggrecan, etc. lead to lethal phenotypes or severe pathologies during embryonic or post-natal development [18-24]. Furthermore, recent discoveries like the importance of Cdc42 for the appropriate acquisition of apical and basal polarity during morphogenesis or cell specification during tubulogenesis wouldn't have been possible without the use of 3D model systems [25, 26]. Thus the concept of the importance of the surrounding settings emerged and an entirely new scientific direction evolved, focusing on the role of the

In the living organism cells are usually embedded in a complex three-dimensional extracellular matrix that is dynamic in its structure[27] and rarely do have the opportunity to attach to planar, rigid substrates. Reciprocal interactions between cells and the ECM facilitate signaling to and from cells and lead to continuous reorganization of the environment [10]. Investigation of the dynamics of the ECM, its structure in different tissues and cellular response to changes in the mechanical properties of the extracellular matrices have shown that cells are not only able to feel and respond to the environment, but also to cause changes to the environment's mechanical properties [14]. These interactions deliver further signals responsible for cell growth and differentiation, survival, migration and reorganization of the resident tissue [28]. In addition when one thinks of cells in the third dimension it has to be considered that cells are not just randomly incorporated in the ECM but form complex 3D structures, characteristic

The recognition of the importance of dimensionality of cellular environment encouraged the creation of a variety of three-dimensional culturing systems. Essentially they could be divided into two groups – three-dimensional systems made of artificial materials, mimicking the natural components of the matrix and three-dimensional culturing systems based on natural ECM components, or cell-synthesized ECM. This article's focus will be mainly on the later 3D

the increasing number of scientists aiming to provide in vivo-relevant results.

extracellular matrix and its importance in cell biology.

of the specific tissue or organ [29].

222 Regenerative Medicine and Tissue Engineering

culturing models.

Design of artificial 3D environments for cells aims at creating an environment that would mimic the physical properties of the natural extracellular matrix along with the signaling cues it provides for cell development. Such scaffolds should be non-toxic, preferably biodegradable in time and must allow cell attachment and migration as well as diffusion of vital nutrients [30]. Cells should be able to proliferate and differentiate, and subsequently to synthesize and organize their own ECM. In time, eventually, the naturally synthesized and organized extracellular matrix should substitute the artificial scaffold. All those prerequisites mean that the surface should be suitable for cell attachment, have the adequate chemical and mechanical properties to support adhesion, proliferation and differentiation. To our knowledge there are currently several reported methods for fabricating appropriate artificial scaffolds:


All these methods find application in medicinal practice and tissue engineering, but there are still many questions to be answered regarding their safety with long term usage, biodegrad‐ ability, their influence on cellular signaling, proliferation and differentiation, etc. that are beyond the scope of this review.
