The cultivation of cells in a favorable artificial environment has become a versatile tool in cellular and molecular biology. Cultured primary cells and continuous cell lines are indispensable in investigations of basic, biomedical, and translation research. However, despite their important role, cell lines are frequently misidentified or contaminated by other cells, bacteria, fungi, yeast, viruses, or chemicals. In addition, handling and manipulating of cells is associated with specific biological and chemical hazards requiring special safeguards such as biosafety cabinets, enclosed containers, and other specialized protective equipment to minimize the risk of exposure to hazardous materials and to guarantee aseptic work conditions. This review provides a brief introduction about the most common problems encountered in cell culture laboratories and some guidelines on preventing or tackling respective problems.

Human Periodontal Ligament Fibroblasts (hPDLF), as part of the periodontal apparatus, modulate inflammation, regeneration and bone remodeling. Interferences are clinically manifested as attachment loss, tooth loosening and root resorption. During orthodontic tooth movement (OTM), remodeling and adaptation of the periodontium is required in order to enable tooth movement. hPDLF involvement in the early phase-OTM compression side was investigated for a 72-h period through a well-studied in vitro model. Changes in the morphology, cell proliferation and cell death were analyzed. Specific markers of the cell cycle were investigated by RT-qPCR and Western blot. The study showed that the morphology of hPDLF changes towards more unstructured, unsorted filaments under mechanical compression. The total cell numbers were significantly reduced with a higher cell death rate over the whole observation period. hPDLF started to recover to pretreatment conditions after 48 h. Furthermore, key molecules involved in the cell cycle were significantly reduced under compressive force at the gene expression and protein levels. These findings revealed important information for a better understanding of the preservation and remodeling processes within the periodontium through Periodontal Ligament Fibroblasts during orthodontic tooth movement. OTM initially decelerates the hPDLF cell cycle and proliferation. After adapting to environmental changes, human Periodontal Ligament Fibroblasts can regain homeostasis of the periodontium, affecting its reorganization.

Extracellular vesicles such as exosomes are small-sized, bilayered extracellular biovesicles generated by almost every cell and released into the surrounding body fluids upon the fusion of multivesicular bodies and the plasma membrane. Based on their origin, they are enriched with a variety of biologically active components including proteins, lipids, nucleic acids, cellular metabolites, and many other constituents. They can either attach or fuse with the membrane of a target cell, or alternatively be taking up via endocytosis by a recipient cell. In particular, milk exosomes have been recently shown to be a fundamental factor supporting infant growth, health, and development. In addition, exosomes derived from different cell types have been shown to possess regenerative, immunomodulatory, and anti-inflammatory properties, suggesting that they are a potential therapeutic tool in modulating the pathogenesis of diverse diseases. Therefore, efficient protocols for the isolation of milk exosomes in a high quantity and purity are the basis for establishing clinical applications. Here, we present an easy-to-follow protocol for exosome isolation from bovine and human milk. Electron microscopic analysis and nanoparticle tracking analysis reveal that the protocols allow the isolation of highly enriched fractions of exosomes. The purified exosomes express the typical exosomal protein markers, CD81 and ALIX.

Estrogen receptor alpha (ERα) is widely expressed in reproductive organs, but also in non-reproductive tissues of females and males. There is evidence that lipocalin 2 (LCN2), which has diverse immunological and metabolic functions, is regulated by ERα in adipose tissue. However, in many other tissues, the impact of ERα on LCN2 expression has not been studied yet. Therefore, we used an Esr1-deficient mouse strain and analyzed LCN2 expression in reproductive (ovary, testes) and non-reproductive tissues (kidney, spleen, liver, lung) of both sexes. Tissues collected from adult wild-type (WT) and Esr1-deficient animals were analyzed by immunohistochemistry, Western blot analysis, and RT-qPCR for Lcn2 expression. In non-reproductive tissues, only minor genotype- or sex-specific differences in LCN2 expression were detected. In contrast, significant differences in LCN2 expression were observed in reproductive tissues. Particularly, there was a strong increase in LCN2 in Esr1-deficient ovaries when compared to WTs. In summary, we found an inverse correlation between the presence of ERα and the expression of LCN2 in testes and ovaries. Our results provide an important basis to better understand LCN2 regulation in the context of hormones and in health and disease.

This study presents a comprehensive genetic characterization of the H9c2 cell line, a widely used model for cardiac myoblast research. We established a short tandem repeat (STR) profile for H9c2 that is useful to confirm the identity and stability of the cell line. Additionally, we prepared H9c2 metaphase chromosomes and performed karyotyping and molecular cytogenetics to further investigate chromosomal characteristics. The genetic analysis showed that H9c2 cells exhibit chromosomal instability, which may impact experimental reproducibility and data interpretation. Next-generation sequencing (NGS) was performed to analyze the transcriptome, revealing gene expression patterns relevant to cardiac biology. Western blot analysis further validated the expression levels of selected cardiac genes identified through NGS. Additionally, Phalloidin staining was used to visualize cytoskeletal organization, highlighting the morphological features of these cardiac myoblasts. Our findings collectively support that H9c2 cells are a reliable model for studying cardiac myoblast biology, despite some genetic alterations identified resembling sarcoma cells. The list of genes identified through NGS analysis, coupled with our comprehensive genetic analysis, will serve as a valuable resource for future studies utilizing this cell line in cardiovascular medicine.