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Understanding the cell cycle at the single-cell level is crucial for cellular biology and cancer research. While current methods using fluorescent markers have improved the study of adherent cells, non-adherent cells remain challenging. In this study, we addressed this gap by combining a specialized surface to enhance cell attachment, the FUCCI(CA)2 sensor, an automated image analysis pipeline, and a custom machine learning algorithm. This approach enabled precise measurement of cell cycle phase durations in non-adherent cells. This method was validated in acute myeloid leukemia cell lines NB4 and Kasumi-1, which have unique cell cycle characteristics, and we tested the impact of cell cycle-modulating drugs on NB4 cells. Our cell cycle analysis system, which is also compatible with adherent cells, is fully automated and freely available, providing detailed insights from hundreds of cells under various conditions. This report presents a valuable tool for advancing cancer research and drug development by enabling comprehensive, automated cell cycle analysis in both adherent and non-adherent cells. Cells regularly grow and divide through a process called the cell cycle. It includes rest periods where no growth or division occurs. When the cells are ready to divide, they duplicate their DNA, so each new cell gets a complete set of instructions. Finally, the cell splits into two new cells through a process called cytokinesis. This whole process can take hours or days to complete, depending on the cell type. Many things can go wrong during these processes, impairing healing or causing tumor formation. Learning more about these processes could help scientists better understand healing and diseases like cancer. Emerging imaging and data analysis tools allow scientists to observe cell-growth processes and watch errors as they occur. But, doing so requires sophisticated equipment and can be time and labor-intensive. Especially, if scientists are trying to track the cell cycle in a large number of cells. It can also be challenging to track free-moving cells, like blood or immune cells. New tools and techniques are needed to help scientists overcome these challenges. Hayatigolkhatmi, Soriani, Soda et al. developed a method in which a sticky surface is used to grow blood cancer cells that allows them to observe the cell cycle in large numbers of the cells at the same time. In the experiments, blood cancer cells were grown on a nano-material-coated surface that kept the usually free-floating cells still. The team compared gene expression in the cells before and after raising them on the surface to confirm that confining the cells did not alter their gene expression or disrupt their normal life cycle. Then, the researchers developed machine learning software that monitors the cell cycle in hundreds of individual cells, quantifies cell cycle phases and analyzes data with minimal human intervention. Usually, it would take a scientist 40-50 hours to oversee the cell cycle in a single experimental condition. This time was reduced to approximately 2 hours for a complete experiment using their pipeline. Finally, they validated their tools by monitoring different types of cancer cells under various treatment conditions. The tools developed by Hayatigolkhatmi, Soriani, Soda et al. provide researchers with a fast, easy and cost-effective tool for studying the cell cycle. It could help scientists study early development and how cells differentiate, grow or age. It could also be helpful for scientists studying cancer and how to treat it or scientists studying the healing process.

Neuroblastoma (NB) is the most common extracranial solid tumour in children. NB is highly heterogeneous and is comprised of a mixture of neuroblastic cancer cells and stromal cells. We previously reported that N‐type cells (neuroblastic cells) and S‐type cells (substrate‐adherent cells) in the SK‐N‐SH cell line shared almost identical genetic backgrounds. Sublines of N‐ and S‐type cells were isolated from an early passage (P35) of SK‐N‐SH. Sequencing analysis revealed that all sublines harboured the anaplastic lymphoma kinase (ALK) F1174L mutation, indicating that they were tumour derived. Surprisingly, over 74% resembled S‐type cells. In coculture experiments, S‐type cells protected N‐type cells from apoptosis induced by the oncogenic ALK inhibitor TAE684. Western blotting analyses showed that ALK, protein kinase A (AKT) and STAT3 signalling were stimulated in the cocultures. Furthermore, the conditioned medium from S‐type cells activated these downstream signalling molecules in the N‐type cells. The activation of STAT3 in the N‐type cells was ALK‐independent, while AKT was regulated by the ALK activation status. To identify the responsible soluble factors, we used a combination of transcriptomic and proteomic analysis and found that plasminogen activator inhibitor 1, secreted protein acidic and cysteine rich, periostin and galectin‐1 were potential mediators of STAT3 signalling. The addition of recombinant proteins to the tumour cells treated with the ALK inhibitor partially enhanced cell viability. Overall, the tumour‐derived S‐type cells prevented apoptosis in the N‐type cells via ALK‐independent STAT3 activation triggered by secreted factors. The inhibition of these factors in combination with ALK inhibition could provide a new direction for targeted therapies to treat high‐risk NB. Neuroblastoma S‐type cells promote survival of N‐type tumor cells through cell‐to‐cell contact, which is associated with upregulation of p‐STAT3 in N‐type cells. Independently, S‐type cells secrete trophic factors that promote the survival of N‐type cells. Trophic factors could act either by stimulating p‐STAT3, or by upregulating p‐AKT and p‐ERK1/2 through an p‐ALK‐dependent pathway. The inhibition of these factors in combination with ALK inhibition could provide a new strategy for treatment of high‐risk neuroblastoma.

Microcarriers with a high surface-to-volume ratio within bioreactor systems have been used for several decades to develop scalable and cost-effective processes for cultivating adherent cells to produce therapeutic cells, vaccines, and proteins. However, the agitation equipment in bioreactors, which is essential for suspending microcarriers and maintaining homogeneous culture conditions, generates high shear forces that adversely affect the growth of adherent cells. Fluidized bed reactors (FBRs), which are capable of suspending particles solely with liquid flow without an impeller, could provide a solution for these issues. Typical FBRs with cylindrical columns encounter difficulties in controlling the fluidized bed, due to a narrow flow rate range for fluidization and changes in the characteristics of microcarriers as cells grow, despite the advantages of low-shear forces. This leads to the failure of microcarrier fluidization and reduced efficiency of the microcarrier-based cell culture. In this study, we demonstrate a newly developed funnel-type fluidized bed reactor (FFBR) system with enhanced culture stability and evaluated its performance by culturing mouse fibroblasts (L929) and human mesenchymal stem cells. The FFBR exhibits stable operation over the entire culture period, unlike the FBRs with cylindrical columns. The cell density per culture area achieved with the FFBR was comparable to that obtained using the two-dimensional plastic culture system for both cell types. The FFBR is a useful tool for the expansion of adherent cells due to its stability and scalability, making it suitable for research, development, and manufacturing for therapeutic applications.

The growth and activity of adherent cells can be enabled or enhanced through attachment to a solid surface. For food and beverage production processes, these solid supports should be food-grade, low-cost, and biocompatible with the cell of interest. Solid supports that are edible can be a part of the final product, thus simplifying downstream operations in the production of fermented beverages and lab grown meat. We provide proof of concept that edible filamentous fungal pellets can function as a solid support by assessing the attachment and growth of two model cell types: yeast, and myoblast cells. The filamentous fungus Aspergillus oryzae was cultured to produce pellets with 0.9 mm diameter. These fugal pellets were inactivated by heat or chemical methods and characterized physicochemically. Chemically inactivated pellets had the lowest dry mass and were the most hydrophobic. Scanning electron microscope images showed that both yeast and myoblast cells naturally adhered to the fungal pellets. Over 48 h of incubation, immobilized yeast increased five-fold on active pellets and six-fold on heat-inactivated pellets. Myoblast cells proliferated best on heat-treated pellets, where viable cell activity increased almost two-fold, whereas on chemically inactivated pellets myoblasts did not increase in the cell mass. These results support the use of filamentous fungi as a novel cell immobilization biomaterial for food technology applications.

Human placenta‐derived adherent cells (PDAC cells) are a culture expanded, undifferentiated mesenchymal‐like population derived from full‐term placental tissue, with immunomodulatory and anti‐inflammatory properties. PDA‐001 (cenplacel‐L), an intravenous formulation of PDAC cells, is in clinical development for the treatment of autoimmune and inflammatory diseases. To elucidate the mechanisms underlying the immunoregulatory properties of PDAC cells, we investigated their effects on immune cell populations, including T cells and dendritic cells (DC) in vitro and in vivo. PDAC cells suppressed T‐cell proliferation in an OT‐II T‐cell adoptive transfer model, reduced the severity of myelin oligodendrocyte glycoprotein peptide‐induced experimental autoimmune encephalomyelitis and ameliorated inflammation in a delayed type hypersensitivity response model. In vitro, PDAC cells suppressed T‐cell proliferation and inhibited Th1 and Th17 differentiation. Analysis of tissues derived from PDAC cell‐treated animals revealed diminished CD86 expression on splenic DC, suggesting that they can also modulate DC populations. Furthermore, PDAC cells modulate the differentiation and maturation of mouse bone marrow‐derived DC. Similarly, human DC differentiated from CD14+ monocytes in the presence of PDAC cells acquired a tolerogenic phenotype. These tolerogenic DC failed to induce allogeneic T‐cell proliferation and differentiation toward Th1, but skewed T‐cell differentiation toward Th2. Inhibition of cyclo‐oxygenase‐2 activity resulted in a significant, but not complete, abrogation of PDAC cells’ effects on DC phenotype and function, implying a role for prostaglandin E2 in PDAC‐mediated immunomodulation. This study identifies modulation of DC differentiation toward immune tolerance as a key mechanism underlying the immunomodulatory activities of PDAC cells. Immunity: Seeking out secrets of placental protection The same mechanisms that protect the fetus from the mother's immune system could help keep autoimmune and inflammatory diseases at bay. A growing body of evidence suggests that placental cells promote maternal tolerance for fetal tissues that might otherwise be perceived as ‘foreign’. Bitao Liang and colleagues at Celgene Cellular Therapeutics in Warren, New Jersey, USA, examined the role of cultured human ‘placenta‐derived adherent cells’ (PDAC® cells), with a view toward developing these cells as therapeutic tools. Cell culture and animal model experiments both demonstrated that PDAC® cells can help restrict proliferation and activation of T‐cell subsets associated with certain immune disorders. The authors found that this occurs through direct effects on T cells and also through factors secreted by PDAC® cells. These factors promote development of particular immune cells that help limit the overall immune response.

Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.

This work presents a pipette tip gap closure migration assay prototype tool (semi-adherent relative upsurge—s-ARU—method) to study cell migration or wound healing in semi-adherent cell lines, such as lymph node carcinoma of the prostate (LNCaP). Basically, it consists of a 6-well cover plate modification, where pipette tips with the filter are shortened and fixed vertically to the inner surface of the cover plate, with their heights adjusted to touch the bottom of the well center. This provides a barrier for the inoculated cells to grow on, creating a cell-free gap. Such a uniform gap formed can be used to study migration assay for both adherent as well as semi-adherent cells. After performing time studies, effective measurement of gap area can be carried out conveniently through image analysis software. Here, the prototype was tested for LNCaP cells, treated with testosterone and flutamide as well as with bacteriophages T4 and M13. A scratch assay using PC3 adherent cells was also performed for comparison. It was observed that s-ARU method is suitable for studying LNCaP cells migration assay, as observed from our results with testosterone, flutamide, and bacteriophages (T4 and M13). Our method is a low-cost handmade prototype, which can be an alternative to the other migration assay protocol(s) for both adherent and semi-adherent cell cultures in oncological research along with other biological research applications.

Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.

Background Umbilical cord blood (UCB) not only contains hematopoietic stem cells (HSCs), but also non-hematopoietic stem cells (NHSCs) that are able to differentiate into a number of distinct cell types. Based on studies published to date, the frequency of NHSCs in UCB is believed to be very low. However, the isolation of these cells is primarily based on their adhesion to tissue culture plastic surfaces. Methods and results In the current study, we demonstrate that this approach overlooks some of the extremely immature NHSCs because they lack the ability to adhere to plastic. Using a native extracellular matrix (ECM), produced by bone marrow (BM) stromal cells, the majority of the UCB-NHSCs attached within 4 h. The colony-forming unit fibroblast frequency of these cells was 1.5 × 10 4 /10 8 mononuclear cells, which is at least 4000-fold greater than previously reported for UCB-NHSCs. The phenotype of these cells was fibroblast-like and different from those obtained by plastic adhesion; they formed embryonic body-like clusters that were OCT4-positive and expressed other human embryonic stem cell-related markers. Importantly, when implanted subcutaneously for 8 weeks into immunocompromised mice, these ECM-adherent and expanded NHSCs generated three germ layer-derived human tissues including muscle, fat, blood vessel, bone, gland, and nerve. Moreover, injection of these cells into muscle damaged by cryoinjury significantly accelerated muscle regeneration. Conclusions These results indicate that UCB may be a virtually unlimited source of NHSCs when combined with isolation and expansion on ECM. NHSCs may be a practical alternative to embryonic stem cells for a number of therapeutic applications.

Analyzing the electrical double layer (EDL) in electrical impedance spectroscopy (EIS) measurement at low frequencies remains a challenging task for sensing purposes. In this work, we propose two approaches to deal with the EDL in measuring impedance for particles and non-adherent cells in an electrolytic suspension. The first approach is a simple procedure to compute a normalized electrical impedance spectrum named dispersed medium index (DMi). The second is the EIS modeling through an equivalent electric circuit based on the so-called effective capacitance (Cef), which unifies the EDL phenomena. Firstly, as an experiment under controlled conditions, we examine polymer particles of 6, 15, and 48 μm in diameter suspended in a 0.9% sodium chloride solution. Subsequently, we used K-562 cells and leukocytes suspended in a culture medium (RPMI-1640 supplemented) for a biological assay. As the main result, the DMi is a function of the particle concentration. In addition, it shows a tendency with the particle size; regardless, it is limited to a volume fraction of 0.03 × 10−4 to 58 × 10−4. The DMi is not significantly different between K-562 cells and leukocytes for most concentrations. On the other hand, the Cef exhibits high applicability to retrieve a function that describes the concentration for each particle size, the K-562 cells, and leukocytes. The Cef also shows a tendency with the particle size without limitation within the range tested, and it allows distinction between the K-562 and leukocytes in the 25 cells/µL to 400 cells/µL range. We achieved a simple method for determining an Cef by unifying the parameters of an equivalent electrical circuit from data obtained with a conventional potentiostat. This simple approach is affordable for characterizing the population of non-adherent cells suspended in a cell culture medium.