Cell-repellent polyampholyte for conformal coating on microstructures.

Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50-150 °C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.

Small molecule LATS kinase inhibitors block the Hippo signaling pathway and promote cell growth under 3D culture conditions.

Although 3D cell culture models are considered to reflect the physiological microenvironment and exhibit high concordance with in vivo conditions, one disadvantage has been that cell proliferation is slower in 3D culture as compared to 2D culture. However, the signaling differences that lead to this slower proliferation are unclear. Here, we conducted a cell-based high-throughput screening study and identified novel small molecules that promote cell proliferation, particularly under 3D conditions. We found that one of these molecules, designated GA-017, increases the number and size of spheroids of various cell-types in both scaffold-based and scaffold-independent cultures. In addition, GA-017 also enhances the ex vivo formation of mouse intestinal organoids. Importantly, we demonstrate that GA-017 inhibits the serine/threonine protein kinases large tumor suppressor kinase 1/2, which phosphorylate Yes-associated protein and transcriptional coactivator with PDZ-binding motif , key effectors of the growth- and proliferation-regulating Hippo signaling pathway. We showed that GA-017 facilitates the growth of spheroids and organoids by stabilizing and translocating Yes-associated protein and transcriptional coactivator with PDZ-binding motif into the cell nucleus. Another chemical analog of GA-017 obtained in this screening also exhibited similar activities and functions. We conclude that experiments with these small molecule large tumor suppressor kinase inhibitors will contribute to further development of efficient 3D culture systems for the ex vivo expansion of spheroids and organoids.

A liquid culture cancer spheroid model reveals low PI3K/Akt pathway activity and low adhesiveness to the extracellular matrix.

Three-dimensional (3D) cultures of cancer cells in liquid without extracellular matrix (ECM) offer in vitro models for metastasising conditions such as those in vessels and effusion. However, liquid culturing is often hindered by cell adhesiveness, which causes large cell clumps. We previously described a liquid culture material, LA717, which prevents nonclonal cell adhesion and subsequent clumping, thus allowing clonal growth of spheroids in an anchorage-independent manner. Here, we examined such liquid culture cancer spheroids for the acquisition of apical-basal polarity, sensitivity to an Akt inhibitor (anticancer drug MK-2206) and interaction with ECM. The spheroids present apical plasma membrane on the surface, which originated from the failure of polarisation at the single-cell stage and subsequent defects in phosphorylated ezrin accumulation at the cell boundary during the first cleavage, failing internal lumen formation. At the multicellular stage, liquid culture spheroids presented bleb-like protrusion on the surface, which was enhanced by the activation of the PI3K/Akt pathway and reduced by PI3K/Akt inhibitors. Liquid culture spheroids exhibited slow proliferation speed and low endogenous pAkt levels compared with gel-cultured spheroids and 2D-cultured cells, explaining the susceptibility to the Akt-inhibiting anticancer drug. Subcutaneous xenografting and in vitro analysis demonstrated low viability and adhesive property of liquid culture spheroids to ECM, while migratory and invasive capacities were comparable with gel-cultured spheroids. These features agree with the low efficacy of circulating tumour spheroids in the settling step of metastasis. This study demonstrates the feature of anchorage-independent spheroids and validates liquid cultures as a useful method in cancer spheroid research.

Novel 3D Liquid Cell Culture Method for Anchorage-independent Cell Growth, Cell Imaging and Automated Drug Screening.

Cells grown in three-dimensional (3D) cultures are more likely to have native cell-cell and cell-matrix interactions than in 2D cultures that impose mechanical constraints to cells. However, most 3D cultures utilise gel matrix which, while serving as a scaffold, limits application due to its solid and opaque nature and inconsistency in cell exposure to exogenous signals. In 3D culture without gel matrix, cells tend to adhere to each other and form clumps with necrotic zone at the centre, making them unsuitable for analyses. Here we report that addition of low-molecular-weight agar named LA717 to culture media allows cells to grow as dispersed clonal spheroids in 3D. LA717 maintains cells dispersed and settled to the bottom of the medium while keeping the medium clear with little additional viscosity, making it suitable for microscopic observation. Importantly, cancer spheroids formed in LA717-containing medium show higher sensitivity to anti-cancer drugs such as Trametinib and MK-2206 that are not as effective in 2D. Because of the small and consistent size of spheroids, cell viability and drug toxicity are readily detectable in automated imaging analysis. These results demonstrate that LA717 offers a novel 3D culture system with great in vivo reflection and practicality.