## 2 3D Culture of Cancer Cells or Stem Cells  
Cancer cells are the most common cell types that have been investigated by 3D culture systems, and obvious change of gene expression as well as intracellular signaling in these 3D-cultured cancer cells have been shown in the literature, as compared with those cultured on tissue culture polystyrene (TCPS) plates. The overall change of transcriptome has been analyzed by bioinformatic approaches in several types of cancers. For glioblastoma multiforme (GBM) cells, the change of expression levels of \(\sim\)10 thousand genes has been demonstrated after culture in 3D poly (lactic acid) (PLA) scaffolds [4]. The whole gene expression profiles of lung carcinoma and squamous cell carcinoma cultured in Matrigel were also analyzed. The expression levels of genes related to cell adhesion and immune response were changed when the culture environment of cancer cells was changed from 2D to 3D, suggesting the alternation of tumorigenicity of these 3D-cultured cancer cells [5]. Regarding the cell growth, the proliferation rate of ALDH+ (aldehyde dehydrogenase, a classical marker for cancer stem cells) breast cancer stem cells was amplified (\(\sim\)2-fold after 3 days of culture) by PLA porous scaffolds vs. those cultured on 2D plastic plates [6]. Spheroid-forming lung and liver cancer cells generated on biomaterial-based flat substrates or cellulosic scaffolds have increased cancer stemness, cell mobility, and chemoresistance [7; 8]. Stem cell transplantation is the most promising strategy in regenerative medicine to achieve the functional recovery of impaired tissues or organs. With regard to stem cell culture, the stemness maintenance and differentiation capacities are of critical importance. Several positive effects have been demonstrated for 3D-cultured stem cells. For example, human embryonic stem cells (hESCs) have been successfully amplified in the form of suspended 3D spheroids, and these massively produced hESCs maintain their pluripotent properties to differentiate into the cells belonging to all three germ layers [9]. With the appropriate induction, floating-cultured ESCs self-organize into several different 3D organoids such as neural retina, liver buds, and inner ear [10], whereas it is inapproachable for the traditional 2D culture system. Mesenchymal stem cells (MSCs) cultured in 3D space are intensively examined as well, though MSCs are conventionally defined as plastics-adherent. As compared to 2D culture, several benefits to MSCs have been identified when they are grown in a 3D environment. The benefits include the promotion of self-renewal and differentiation activities as well as up-regulation of paracrine secretion [11]. In addition to these positive effects, the enhancement of therapeutic potential of 3D-cultured MSCs was proven in cardiac and neural disorders [12]. Neural stem cells (NSCs) have been proposed to treat the neurodegenerative diseases, and the clinical trials have already started [13]. The proliferation and differentiation of NSCs were both observed in the scaffold-based or scaffold-free 3D culture system [14; 15]. Furthermore, the differentiation potential of adherent and spheroid-forming (by non-adherent plates) neural progenitors was compared in a recent study. Researchers found that the number of neural progenitors with higher differentiation potential was greater in the spheroid-forming group, as compared to the 2D-cultured neural progenitors. Meanwhile, the differentiated neurons derived from 3D neural spheroids also displayed the longer neurites (\(\sim\)1.5-fold increase) after induction [16]. These evidences indicate the advantages of culturing neural progenitors in a 3D spheroid. The accumulated evidence indicates that the properties of cancer cells and stem cells are significantly distinct after culturing in 2D vs. 3D spaces. Due to the biomimetic features, the 3D-cultured cells have been tested in tissue regeneration, drug screening, and other medical applications [17; 18].