细胞生物学

Primary oligodendrocyte cultures are a crucial driving force for in vitro research on oligodendrocytes (OLs) and myelin. Various methods are available to obtain oligodendrocyte lineage cells, primarily from neonatal rodent brains or human induced pluripotent stem cells (iPSCs). In this protocol, we describe a step-by-step procedure for detaching and cryopreserving primary rat oligodendrocyte progenitor cells (OPCs), followed by the thawing, proliferation, and differentiation of the cryopreserved OPCs. After freezing in a serum-free cryopreservation medium, the OPCs can be preserved at -80 °C for up to two months without notable changes in viability, proliferation, or differentiation into mature OLs. Cryopreserved OPCs can be differentiated into mature OLs with robust myelin processes and the capacity to wrap around neuron-mimicking structures. Combined with the author’s method for primary OL culture, which allows for bulk production of OPCs, OPC cryopreservation may substantially improve the efficiency of in vitro OL research.

Endometritis is a prevalent gynecological condition, often resulting from bacterial infections, which poses significant risks to women’s reproductive health, including recurrent pregnancy loss, spontaneous abortion, and intrauterine adhesions. While conventional in vitro models have provided valuable insights into the pathogenesis of bacterial-induced endometritis, they often fail to replicate the complex cellular architecture and microenvironment of the endometrium due to species-specific differences and variations in the menstrual cycle. In this study, we present a novel organoid-based culture system that establishes a bacterial-induced endometritis model using endometrial organoids derived from primary epithelial cells. This protocol involves culturing endometrial organoids in a Matrigel-based three-dimensional matrix, followed by infection with Escherichia coli at a defined multiplicity of infection (MOI). The model effectively recapitulates key pathological features of bacterial-induced endometritis, including disruption of the epithelial barrier, release of inflammatory cytokines, and cellular damage. By preserving epithelial polarity, this approach offers enhanced physiological relevance, improves host–pathogen interaction studies, and provides a robust platform for evaluating potential therapeutic interventions.

Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells, followed by the migration, differentiation, and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens, such as FGF8. Disruption of this developmental balance can lead to brain malformations, which underlie a range of complex neurodevelopmental disorders, including epilepsy, autism, and intellectual disabilities. Studying the early stages of human brain development, whether under normal or pathological conditions, remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently, human brain organoids have emerged as a powerful in vitro alternative, allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures, where neural progenitors and neurons are grown on flat surfaces, 3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However, 3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore, few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning.

To address these limitations, we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs), our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation, NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors, where they self-organize into neural rosettes and neuroepithelial structures, surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids, leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity, enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas.

The study of choroidal endothelial cells is essential for understanding the pathological mechanisms underlying choroidal neovascularization and other vision-threatening disorders. Traditional methods for isolating and culturing primary endothelial cells often yield mixed populations or require specialized equipment, limiting their widespread use. Here, we present a straightforward protocol for isolating and culturing primary mouse choroidal endothelial cells. This protocol involves enzymatic digestion of choroidal tissue, magnetic-activated cell sorting (MACS) to enrich CD31⁺ endothelial cells, and optimized culture conditions to promote cell proliferation and maintain endothelial phenotype. The protocol is strategic, reproducible, and requires minimal specialized equipment, making it accessible for researchers across various fields. By providing a robust method for obtaining pure choroidal endothelial cell cultures, this protocol facilitates the study of cell-specific behaviors and responses, advancing research into choroidal vascular diseases.

Cancer-associated mesenchymal stem cells (Ca-MSCs), an integral part of the tumor microenvironment, play a major role in modulating tumor progression; they have been reported to progress as well as inhibit various cancers, including cervical cancer. To understand the exact role of Ca-MSCs in tumor modulation, it is necessary to have an optimized protocol for Ca-MSCs isolation. This work demonstrates the isolation and expansion of a primary culture of cervical cancer–associated MSCs (CCa-MSCs) from the biopsy sample of cervical cancer patients using the explant culture technique. The isolated cells were characterized according to International Society for Cellular Therapy (ISCT) guidelines. Morphological analysis revealed that cells were adherent to the plastic surface and possessed spindle-shaped morphology. Flow cytometry analysis of the cells showed high expression (~98%) for MSC-specific cell surface markers (CD90, CD73, and CD105), negative expression (<0.5%) for endothelial cell marker (CD34) and hematopoietic cell marker (CD45), and negligible expression for HLA-DR, as recommended by ISCT. Further, trilineage differentiation potential analysis of the cells showed their osteogenic and chondrogenic potential and adipogenic differentiation. This standardized protocol will assist in the cultivation of CCa-MSCs and the study of their interactions with tumor cells and other components of the tumor microenvironment. This protocol may be utilized in the establishment of Ca-MSCs from other types of cancers as well.

Centrosomes are vital eukaryotic organelles involved in regulating cell adhesion, polarity, mobility, and microtubule (MT) spindle assembly during mitosis. Composed of two centrioles surrounded by the pericentriolar material (PCM), centrosomes serve as the primary microtubule-organizing centers (MTOCs) in proliferating cells. The PCM is crucial for MT nucleation and centriole biogenesis. Centrosome numbers are tightly regulated, typically duplicating once per cell cycle, during the S phase. Deregulation of centrosome components can lead to severe diseases. While traditionally viewed as stable structures, centrosomes can be inactivated or disappear in differentiating cells, such as epithelial cells, muscle cells, neurons, and oocytes. Despite advances in understanding centrosome biogenesis and function, the mechanisms maintaining mature centrosomes or centrioles, as well as the pathways regulating their inactivation or elimination, remain less explored. Studying centrosome maintenance is challenging as it requires the uncoupling of centrosome biogenesis from maintenance. Tools for acute spatial-temporal manipulation are often unavailable, and manipulating multiple components in vivo is complex and time-consuming. This study presents a protocol that decouples centrosome biogenesis from maintenance, allowing the study of critical factors and pathways involved in the maintenance of the integrity of these important cellular structures.

Three-dimensional cell models, such as spheroids, represent a more physiological arrangement in which cells can grow, allowing them to develop cell–cell interactions in all dimensions. The most common methods for growing spheroids are scaffold-based, typically using either extracellular matrix or hydrogels as a physical support for the cellular assembly. One key problem with this approach is that the spheroids that are produced can be highly variable in size and shape. The protocol presented here allows for the systematic production of uniform spheroids in a short time frame by utilising a micropatterned plate. We show that spheroids can be used to investigate fundamental research questions, such as how the endomembrane system is organised in cells. Our protocol can be used in a manual or automated manner, potentially allowing scaling up for screening applications. Furthermore, without the complication of removing the spheroids from the extracellular matrix or hydrogel, as would be required in scaffold-based systems, spheroids can easily be used in other downstream applications.

Since the establishment of the iSLK-BAC16 cell culture system, iSLK-BAC16 cells and their derivatives have been widely used for Kaposi’s sarcoma-associated herpesvirus (KSHV) studies. However, iSLK-BAC16 cells can be difficult to work with, in part due to the lack of standardized protocols and conflicting troubleshooting suggestions. Here, we describe the protocol for general iSLK-BAC16 cell culture and reactivation, which induces lytic KSHV replication and virion production. This protocol achieves robust levels of KSHV reactivation in our hands and can be readily used for studies of KSHV lytic infection mechanisms.

Continuous and balanced bone remodeling is essential for maintaining mechanical integrity, mineral homeostasis, and hematopoiesis. Dysregulated bone metabolism develops pathological conditions, such as osteoporosis and bone metastasis. Functional and analytical recapitulation of bone remodeling in vitro is critical for advancing our understanding of bone mineral metabolism, disease mechanisms, and drug development. However, conventional models fail to replicate the essential complexity of the bone extracellular matrix (ECM) and the dynamic interplay between bone-forming osteoblasts and bone-resorbing osteoclasts. Recently, we developed an osteoid-mimicking demineralized bone paper (DBP) by thin-sectioning demineralized bovine compact bone matrix. DBP supports osteoblastic mineral deposition and the subsequent transition to bone-lining cells. When co-cultured with bone marrow mononuclear cells under biochemical stimulation, osteoblasts shift their regulatory secretion profiles and effectively induce osteoclastogenesis. The semi-transparent nature of DBP, combined with primary osteogenic cells retrieved from DsRed and eGFP reporter mice, enables longitudinal fluorescent monitoring of these multicellular processes and quantitative analysis. In this protocol, we describe the methods for DBP generation, reconstituting mineralized bone tissue complexity with osteoblasts, and recapitulating the bone remodeling cycle through bone marrow monocytes co-culture under biochemical stimulation, offering a useful platform for the related and broader research community.

Dendritic cells (DC) are sentinel cells of the immune system that process and present antigens to activate T cells, thus serving to bridge the innate and adaptive immune systems. DCs are particularly efficient at cross-presentation whereby exogenously acquired antigens are processed and presented in context with MHCI molecules to activate CD8⁺ T cells. Assaying antigen presentation by DCs is a critical parameter in assessing immune functionality. However, the low abundance of bona fide DCs within the lymphoid compartments limits the utility of such assays. An alternative approach employing the culturing of bone marrow cells in the presence of factors needed for DC lineage commitment can result in the differentiation of bone marrow dendritic cells (BMDCs). This protocol details the process of in vitro generation of BMDCs and demonstrates their subsequent utility in antigen presentation assays. The protocol described can be adapted to various conditions and antigens.