Methods in molecular biology最新文献

Recent findings from studies involving astronauts and animal models indicate that microgravity increases immune cell activity and potentially alters the white and gray matter of the central nervous system (CNS). To further investigate the impact of microgravity on CNS cells, we established cultures of three-dimensional neural organoids containing isogenic microglia, the brain's resident immune cells, and sent them onboard the International Space Station. When using induced pluripotent stem cell (iPSC) lines from individuals affected by neuroinflammatory and neurodegenerative diseases such as multiple sclerosis (MS) and Parkinson's disease (PD), these cultures can provide novel insights into pathogenic pathways that may be exacerbated by microgravity. We have devised a cryovial culture strategy that enables organoids to be maintained through space travel and onboard the International Space Station (ISS) without the need for medium or carbon dioxide exchange. Here, we provide a comprehensive description of all the steps involved: generating various types of neural organoids, establishing long-term cultures, arranging plans for shipment to the Kennedy Space Center (KSC), and ultimately preparing organoids for launch into low-Earth orbit (LEO) and return to Earth for post-flight analyses.

Neuroblastoma, the most prevalent extracranial solid tumor in children, poses therapeutic challenges due to its variable clinical course and propensity for metastasis. Despite advances in treatment strategies like chemotherapy, drug resistance remains a significant concern, highlighting the need for improved models to study tumor behavior and drug responses. This chapter proposes the development of a three-dimensional multicellular model of human neuroblastoma using Matrigel as an ECM analogue. Such models aim to replicate the complexity of the tumor microenvironment, providing valuable insights into tumor progression and drug resistance mechanisms. By recapitulating key features of neuroblastoma within a physiologically relevant context, these models offer a platform for preclinical drug screening and therapeutic development.

Telocytes, distinctive interstitial cells, have recently emerged as crucial components of the stem-cell niche in the intestine. Notably, telocytes are distinguished by their extremely long cellular protrusions extending hundreds of microns from the cell body, forming an interconnected network along the intestinal crypt villus axis. Due to these unique cellular characteristics, there is a need for tailored working protocols to effectively characterize and target telocytes. Here, we outline advanced and progressive protocols for tissue fixation, dissociation, visualization, and the use of tamoxifen-induced transgenic mouse models to specifically target telocytes.

Preparation of highly porous biocompatible and bioresorbable nerve conduit or scaffold by electrospinning based on synthetic polycaprolactone with a molecular weight of 80 kDa (PCL 80 kDa) has significance in the context of regenerative medicine with special emphasis on their application in neurotrauma. PCL conduits/scaffolds serving as a support structure for seeded stem cells show promising regenerative potential to promote functional recovery and tissue regeneration in models of neurotrauma. Here we describe a standard protocol for the production of conduits by electrospinning at high field-forming voltages (24kB) using a 6% solution of PCL 80 kDa in a chloroform/methanol mixture.

Epithelial organoid monoculture is a powerful tool to model stem cell dynamics in vitro. However, extensive efforts have recently revealed various niche players and their significant roles in regulating epithelial stem cells. Among these niche components, fibroblasts have been heavily recognized in the field as a critical niche signal secretor. Thus, understanding the roles of fibroblasts in epithelial dynamics has become increasingly relevant and crucial. This propels the development of approaches to coculture epithelial 3D organoids with fibroblasts to model epithelial-fibroblast crosstalk in vitro. Here, we describe a stepwise coculture method to isolate and culture primary intestinal fibroblasts and epithelial organoids together. Aligned with the recent literature, our coculture protocol allows for primary intestinal fibroblast support of epithelial organoid growth.

Compared with traditional 2D cell culture, 3D cell culture more closely resembles the original state of cells in vivo and enables the establishment of in vivo-like microenvironments and cell-cell interactions, thereby providing valuable cellular materials for numerous studies. The direct establishment of in vitro patient tumor models can enhance drug testing, cancer research, and individualized precision therapy. In this study, we propose a microfluidic chip based on microwell arrays for 3D tumor cell culture. This chip combines nanoscale channels and microwell arrays to precisely control cell distribution and nutrient diffusion, thus closely mimicking the tumor microenvironment. The incorporation of microwell arrays allows for simple and rapid high-throughput preparation of tumor spheroids, while promoting the formation of cell-cell and cell-matrix interactions, ultimately enhancing cell viability and function. Preliminary experiments using tumor cell lines validate the ability of the chip to support 3D tumor growth with enhanced physiological relevance. The microfluidic chip serves as a reliable and scalable platform for studying tumor biology and evaluating therapeutic efficacy and is anticipated to expedite cancer research and drug discovery.

Remodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy-dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use of GelMA hydrogels with defined stiffness represents a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells.