Microfluidic device combined with MCTs has several advantages in drug screening, such as in vivo-like environment, high analytical throughput, enhanced sensitivity, and facile parallelization through multiplexing. MCTs culture for a long timeIn a drug screening employing MCTs, it is essential to stably supply oxygen and nutrients for a considerable time and remove cellular waste products to maintain a similar environment as?in vivo?conditions. mechanical support and offers cell-to-ECM interaction opportunities [42, 43]. The scaffold can be produced with various biomaterials, including natural and synthetic compositions. Natural polymers, such as gelatin, alginate, collagen, and Matrigel, are preferred because of their biocompatibility and formability [44C48]. Or, the synthetic polymers, such as poly (lactic-co-glycolic) acid (PLGA) or polycaprolactone (PCL), and poly (ethylene glycol) (PEG), are used in AC-5216 (Emapunil) 3D scaffold fabrication. The synthetic polymers provide abundant availability; they can be produced in large uniform quantities and tailored for specific applications [49C52]. In a scaffold-free culture, four major techniques are available for spheroid formation, including agitation-based technique, liquid overlay technique, hanging drop technique, and microfluidic technique. CDK4 In the agitation\based technique, cells aggregate under continuous stirring to prevent the cell from adhering to surfaces [37, 43]. The hanging drop technique uses the surface tension of a cell liquid drop suspended on a glass coverslip and gravity to induce aggregation and accelerate spheroid formation . In the liquid overlay technique, cells are seeded on non-adhesive surfaces to avoid cell attachment . Super-hydrophobic agar or agarose are frequently applied to make non-adherent surfaces . Microfluidics has been widely investigated as a promising technique because it can offer 3D environments, long-term culture, and precise handling of spheroids [54, 55]. Among the above-mentioned techniques, agitation\based technique, hanging drop technique, and liquid overlay technique are easy and cheap to operate with no specialized equipment needed. Conversely, microfluidics offers the scale\up of spheroid formation under precisely controlled conditions, making it suitable for high\throughput screening [20, 43]. Both agitation\based and liquid overlay techniques need optimization to form MCTs with uniform size and morphology, and the hanging drop technique is labor- and time-consuming. Choosing a MCTs formation technique is very important because they are not equivalently working to form spheroids (Table ?(Table2).2). During the same culture period, MCF-7 and MDA-MB-231 spheroids created using agitation\based (nutator) and hanging drop techniques grew larger than those created using the liquid overlay technique . In addition, the MCTs generated using agitation-based and hanging drop techniques revealed higher collagen type AC-5216 (Emapunil) I levels than those created using the liquid overlay technique. In the liquid overlay technique, the degree of MCTs formation depends on medium additives (25% methocel, 25% methocel?+?1% Matrigel or 3.5% Matrigel) . Bladder cancer cells (RT4) can form compact spheroids with both hanging drop and liquid overlay techniques; however, the growth rate of spheroids relative to cell seeding density is better in the liquid overlay technique . Taken together, it seems that the hanging drop technique is more effective than the liquid overlay technique for forming highly compact tumor spheroids in certain cell types. Therefore, it is necessary to establish standardized and reproducible protocols for MCTs formation with comparable size and morphology. Table. 2. MCTs formation depends on culture methods is the limiting volume, and is the specific growth rate. This model predicts an approaching at the asymptotic volume of AC-5216 (Emapunil) Vmax. The MCTs size increases with increasing cell seeding density (Fig.?3b). A positive linear correlation between MCTs size and cell seeding density has been reported for MCTs of several cancer cell types, such as glioma cells (U251, U87) [30, 69], breast adenocarcinoma MCF-7 , and the mesothelioma cell line H2052 . However, the increasing AC-5216 (Emapunil) rates of MCTs size as a function of cell seeding density were all different. Other correlations between MCTs size and cell density AC-5216 (Emapunil) have also been reported. The increasing rate of MCTs size is high at relatively small cell density but gets lower as the density increases, and then, it reaches a plateau [13, 71C74]. For example, the size of a breast cancer spheroid (T47D) was 200C300?m at a cell density of 1 1??106 cells/mL and increased to 250C300?m with a seeding density of 3??106 cells/mL . For a seeding density of 3C10??106 cells/mL, the mean diameter of the spheroid was almost the same, with a minor difference.