Then, the required volume of rat tail collagen type I (3.38 mg/mL, Corning, 354236) was calculated to Paricalcitol reach the final collagen concentration. for the stem-like phenotype through better self-renewal and de-differentiation (Notch? to Notch+ transition). More interestingly, we found that the Notch? de-differentiated cells were more resistant to doxorubicin and cisplatin than the Notch+ cells. Combining the 3D ECM culture and single cell resolution, the presented platform can automatically analyze the individual cell actions of hundreds of cells using a small amount of drug and reagents. Keywords: 3D cell culture, extracellular matrix, ECM, collagen, single cell, cell heterogeneity, differentiation, drug screening Introduction In vitro cell culture has been widely used in cell behavior studies for more than 100 years. However, it is not until the 80s that people started to spotlight the importance of 3D cell culture, especially for understanding the functions of the extracellular matrix (ECM) in tissue physiology and malignancy pathology . To bridge Rabbit polyclonal to WBP2.WW domain-binding protein 2 (WBP2) is a 261 amino acid protein expressed in most tissues.The WW domain is composed of 38 to 40 semi-conserved amino acids and is shared by variousgroups of proteins, including structural, regulatory and signaling proteins. The domain mediatesprotein-protein interactions through the binding of polyproline ligands. WBP2 binds to the WWdomain of Yes-associated protein (YAP), WW domain containing E3 ubiquitin protein ligase 1(AIP5) and WW domain containing E3 ubiquitin protein ligase 2 (AIP2). The gene encoding WBP2is located on human chromosome 17, which comprises over 2.5% of the human genome andencodes over 1,200 genes, some of which are involved in tumor suppression and in the pathogenesisof Li-Fraumeni syndrome, early onset breast cancer and a predisposition to cancers of the ovary,colon, prostate gland and fallopian tubes the different drug responses between standard 2D cell culture and in vivo experiments, more and more malignancy researchers are performing experiments in the more realistic 3D culture environment [2-3]. Compared to the 2D culture, which develops cells on artificial rigid polystyrene (Young’s modulus: 3 GPa) or glass (Young’s modulus: 50-90 GPa), the 3D Paricalcitol culture develops cells in the elastic ECM (Young’s modulus: 50-5000 Pa, depending on the tissue) environment and can better mimic the in vivo environment [4-7]. In addition to their mechanical house, cells are known to sense the biochemical signals from the surrounding ECM using numerous transmission transduction cascades via receptors around the cell membrane . Therefore, it is important to apply 3D ECM culture in microfluidics for re-capitulating the tumor microenvironment. Due to the genomic and epi-genomic instability of tumors, malignancy cells are notorious for their heterogeneity. Among the various sub-populations, malignancy stem-like cells (CSCs), which play crucial functions in malignancy metastasis, therapeutic resistance, and relapse, are important clinical targets [9-12]. As stem-like cells, CSCs are capable of either self-renewal (symmetric division) to generate CSCs or differentiation (asymmetric division) to make differentiated malignancy cells . Considerable evidence suggests that the symmetric division of CSCs is critical for the progression of tumors, whereas skewing toward asymmetric division can lead to tumor suppression [14-15]. Although it is usually believed that this 3D culture environment is usually favorable for stem-like phenotypes, it is not clear Paricalcitol whether this is caused by (1) reduced asymmetric division, (2) increased symmetric division, or (3) better survival of stem-like cells [16-17]. In addition, CSCs are typically more resistant to chemotherapies; however, it is not obvious whether self-renewing CSCs have stronger resistance compared to differentiating CSCs [18-19]. Using the conventional dish-based approach, only the final cell number and gene expression (or Live/Dead) can be counted by fluorescence-activated cell sorting (FACS). The averaged end-point results provide little insight into the cellular heterogeneity of CSCs and not the process of how the populace is usually skewed. To decipher the changes of CSC populations in different conditions and treatments, there is a need for single cell analysis to monitor the fate of each individual cell. Due to the benefits of small sample volumes, precise fluid control, and high-throughput scaling, microfluidic technology has emerged as a state-of-the-art approach for single cell analyses [20-24]. There are Paricalcitol a number of previous reports on microfluidic platforms for 3D cell culture, but many of them use a suspension culture with hydrogel, which cannot emulate the cell-ECM interactions in vivo [25-27]. To incorporate 3D ECM in microfluidics, some experts control the hydrogel matrix using laminar circulation , surface tension Paricalcitol (achieved using micropillars) , and physical confinements [30-31], but these cannot accomplish precise spatial control for performing a single cell assay. Hydrogel droplet formation [32-33] and 3D bioprinting  encapsulating single cells have merits in high-throughput and precise micro-environment control. However, only a limited quantity of bio-materials can be utilized for these technologies, thus making it difficult to study a wide range of different ECMs, which have unique biochemical properties. In addition, the shear pressure induced by inkjet printing can compromise the cell viability. Although filling a hydrogel with cells.