Analytical and computational studies predict negligible risk of cell death from eddy generation off flat surfaces in cell culture flow systems.

Cell-based therapies represent the current frontier of biomedical innovations, with the technologies required underpinning treatments as broad as CAR-T cell therapies, stem cell treatments, genetic therapies and mRNA manufacture. A key bottleneck in the manufacturing process for each of these lies in the expansion of cells within a bioreactor vessel, requiring by far the greatest share of time for what are often time-critical therapies. While various designs, culture feeding and mixing methods are employed in these bioreactors, a common concern among manufacturers and researchers lies in whether shear stresses generated by culture media flow will damage cells and inhibit expansion. This study develops an analytical tool to link macro-scale measures of flow to risk of cell death using relationships with eddy size and dissipation rates, from eddies generated off flat surfaces. This analytical tool was then employed using computational fluid dynamics (CFD) to replicate a range of generic bioreactor geometries and flow conditions. We found that no combination of flow condition or design parameter was predicted by the tool to cause cell death within eddies, indicating negligible risk of cell death due to eddy formation within cell culture systems. While this requires experimental validation, and does not apply when cells are expanded using microcarriers, this tool nonetheless provides reassurance and accessible prediction of bioreactor design parameters that could result in cell death. Finally, our findings show that bioreactor design can be tailored such that the shear stress stimulation of cells can be selectively altered through small changes in flow rate.

Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models.

Three-dimensional (3D) ex vivo cultures allow the study of cancer progression and drug resistance mechanisms. Here, we present a protocol for measuring on-target drug sensitivity in a scaffold-free 3D culture system through quantification of apoptotic tumor cells. We provide detailed steps for sample processing, immunofluorescence staining, semi-high-throughput confocal imaging, and imaged-based quantification of 3D cultures. This protocol is versatile and can be applied in principle to any patient-derived material.

Protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages.

Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity. For complete details on the use and execution of this protocol, please refer to Xu et al.1.

The use of hyaluronic acid in a 3D biomimetic scaffold supports spheroid formation and the culture of cancer stem cells.

Three-dimensional (3D) bioprinting culture models capable of reproducing the pathological architecture of diseases are increasingly advancing. In this study, 3D scaffolds were created using extrusion-based bioprinting method with alginate, gelatin, and hyaluronic acid to investigate the effects of hyaluronic acid on the physical properties of the bioscaffold as well as on the formation of liver cancer spheroids. Conformational analysis, rheological characterization, and swelling-degradation tests were performed to characterize the scaffolds. After generating spheroids from hepatocellular carcinoma cells on the 3D scaffolds, cell viability and proliferation assays were performed. Flow cytometry and immunofluorescence microscopy were used into examine the expression of albumin, CD44, and E-cadherin to demonstrate functional capability and maturation levels of the spheroid-forming cells. The results show that hyaluronic acid in the scaffolds correlates with spheroid formation and provides high survival rates. It is also associated with an increase in CD44 expression and a decrease in E-cadherin, while there is no significant change in the albumin expression in the cells. Overall, the findings demonstrate that hyaluronic acid in a 3D hydrogel scaffold supports spheroid formation and may induce stemness. We present a promising 3D scaffold model for enhancing liver cancer spheroid formation and mimicking solid tumors. This model also has the potential for further studies to examine stem cell properties in 3D models.

Dynamic cell culture modulates colon cancer cell migration in a novel 3D cell culture system.

The progression of cancer cell migration, invasion and subsequent metastasis is the main cause of mortality in cancer patients. Through creating more accurate cancer models, we can achieve more precise results, which will lead to a better understanding of the invasion process. This holds promise for more effective prevention and treatment strategies. Although numerous 2D and 3D cell culture systems have been developed, they poorly reflect the in vivo situation and many questions have remained unanswered. This work describes a novel dynamic 3D cell culture system aimed at advancing our comprehension of cancer cell migration. With the newly designed cultivation chamber, 3D tumor spheroids were cultivated within a collagen I matrix in the presence of fluid flow to study the migration of cancer cells from spheroids in the matrix. Using light sheet microscopy and histology, we demonstrated that the morphology of spheroids is influenced by dynamic culture and that, in contrast to static culture, spheroids in dynamic culture are characterized by the absence of a large necrotic core. Additionally, this influence extends to an increase in the size of migration area, coupled with an increase in expression of some genes related to epithelial-mesenchymal transition (EMT). The results here highlight the importance of dynamic culture in cancer research. Although the dynamic 3D cell culture system in this study was used to investigate migration of one cell type into a matrix, it has the potential to be further developed and used for more complex models consisting of different cell types or to analyze other steps of metastasis development such as transendothelial migration or extravasation.

Chitinase 3-Like 1 and C-X-C motif chemokine ligand 5 proteins and the hair cycle.

Dermal papilla cells (DPCs) exhibit self-recovery ability, which may be involved in hair growth. Therefore, we tested whether DPCs subjected to temporary growth-inhibiting stress (testosterone, 17ß-estradiol, mitomycin C, or undernutrition) treatments exhibit self-recovery behavior that can activate hair follicle growth, and examined the changes in cell proliferation capacity and gene expression. Related proteins were identified and their relationships with the hair cycle was examined using a mouse model. Recovery-period DPCs (i.e., from day 3 after loading) were subjected to microarray analysis to detect genetic variations common to each stress treatment. Co-culture of recovery-period DPCs and outer root sheath cells (ORSCs) confirmed the promotion of ORSC proliferation, suggesting that the activation of hair follicle growth is promoted via signal transduction. Chitinase 3-like 1 (CHI3L1) and C-X-C motif chemokine 5 (CXCL5) exhibited ORSC proliferation-promoting effects. Measurement of protein content in the skin during each phase of the hair cycle in mice revealed that CHI3L1 and CXCL5 secretion increased immediately after anagen transition. In a hair-loss mouse model treated with testosterone or 17ß-estradiol, CHI3L1 and CXCL5 secretion was lower in treated telogen skin than in untreated skin. Our results suggest that CHI3L1 and CXCL5 secreted by recovery-state DPCs promote hair growth.

Completely noninvasive multi-analyte monitoring system for cell culture processes.

Although online monitoring of dissolved O2, pH, and dissolved CO2 is critical in bioprocesses, nearly all existing technologies require some level of direct contact with the cell culture environment, posing risks of contamination. This study addresses the need for an accurate, and completely noninvasive technique for simultaneous measurement of these analytes. A "non-contact" technique for simultaneous monitoring of dissolved O2, pH, and dissolved CO2 was developed. Instead of direct contact with the culture media, the measurements were made through permeable membranes via either a sampling port in the culture vessel wall or a flow cell. The efficacy of the "non-contact" technique was validated in Escherichia coli (E.coli), Chinese hamster ovary (CHO) culture processes, and dynamic environments created by sparging gases in cell culture medium. The measurements obtained through the developed techniques were comparable to those obtained through control methods. The noninvasive monitoring system can offer accurate, and contamination-minimized monitoring of critical process parameters including dissolved O2, pH, and dissolved CO2. These advancements will enhance the control and optimization of cell culture processes, promising improved cell culture performance.

Primary human mesenchymal stem cell culture under xeno-free conditions using surface-modified cellulose nanofiber scaffolds.

Stem cell culture often requires various animal-derived components such as serum and collagen. This limits its practical use. Therefore, xeno-free (xenogeneic component-free) culture systems are receiving increased attention. Herein, we propose xeno-free, plant-derived cellulose nanofibers (CNFs) with different surface chemistry: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized CNFs (TOCNFs) with carboxy groups and surface-sulfated CNFs (S-CNFs) for the proliferation of human mesenchymal stem cells (hMSCs) under various serum conditions. We cultured bone marrow-derived hMSCs on CNF scaffolds with various fiber lengths and functional group contents. Original CNFs were bioinert materials that did not contribute to cell adhesion. In contrast, the surface-modified CNFs facilitated the proliferation of immortalized hMSCs under normal and low-serum conditions. The TOCNFs (COONa: 1.47 mmol g-1; length: 0.53 µm), the S-CNFs (OSO3Na: 0.64 mmol g-1; 0.61 µm), and a combination of the two (1:1 by weight) enabled immortalized hMSCs to maintain their multipotency, even under serum-free conditions. Primary cultured hMSCs proliferated well on the TOCNF/S-CNF scaffolds in a completely serum-free medium, comparable to animal-derived type I collagen, although few hMSCs adhered to the standard polystyrene substrate. Our strategy of using surface-modified CNFs will inform the development of xeno-free culture systems to avoid the use of animal-derived materials for both cell culture media and scaffolds.

Bionano Interactions of Organosilica Nanoparticles with Myeloid Derived Immune Cells.

Investigating the interactions between nanomaterials and the cells they are likely to encounter in vivo is a critical aspect of designing nanomedicines for imaging and therapeutic applications. Immune cells such as dendritic cells, macrophages, and myeloid derived suppressor cells have a frontline role in the identification and removal of foreign materials from the body, with interactions shown to be heavily dependent on variables such as nanoparticle size, charge, and surface chemistry. Interactions such as cellular association or uptake of nanoparticles can lead to diminished functionality or rapid clearance from the body, making it critical to consider these interactions when designing and synthesizing nanomaterials for biomedical applications ranging from drug delivery to imaging and biosensing. We investigated the interactions between PEGylated organosilica nanoparticles and naturally endocytic immune cells grown from stem cells in murine bone marrow. Specifically, we varied the particle size from 60 nm up to 1000 nm and investigated the effects of size on immune cell association, activation, and maturation with these critical gatekeeper cells. These results will help inform future design parameters for in vitro and in vivo biomedical applications utilizing organosilica nanoparticles.

Integrative Proteomics-Metabolomics of In Vitro Degeneration of Cardiovascular Cell Lines.

Long-term cell culture is an important biological approach but is also characterized by degeneration in cellular morphology, proliferation rate, and function. To explore this phenomenon in a systematic way, we conducted an integrative proteomics-metabolomics measurement of two cardiovascular cell lines of AC16 and HUVECs. The 18th culturing passages, i.e., G18, showed as the turning points by cell metabolism profiles, in which the metabolomic changes demonstrated the dysfunction of energy, amino acid, and ribonucleotide metabolism metabolic pathways. Although active protein networks showed mitochondria abundance AC16 and oxidative/nitrative sensitive HUVECs indicated the different degeneration patterns, the G18 and G30 proteomics evidenced the senescence by processes of signal transduction, signaling by interleukins, programmed cell death, cellular responses to stimuli, cell cycle, mRNA splicing, and translation. Some crucial proteins (RPS8, HNRNPR, SOD2, LMNB1, PSMA1, DECR1, GOT2, OGDH, PNP, CBS, ATIC, and IMPDH2) and metabolites (L-glutamic acid, guanine, citric acid, guanosine, guanosine diphosphate, glucose 6-phosphate, and adenosine) that contributed to the dysregulation of cellular homeostasis are identified by using the integrative proteomic-metabolomic analysis, which highlighted the increased cellular instability. These findings illuminate some vital molecular processes when culturing serial passages, which contribute holistic viewpoints of in vitro biology with emphasis on the replicative senescence of cardiovascular cells.

Human research islet cell culture outcomes at the Alberta Diabetes Institute IsletCore.

Human islets from deceased organ donors have made important contributions to our understanding of pancreatic endocrine function and continue to be an important resource for research studies aimed at understanding, treating, and preventing diabetes. Understanding the impacts of isolation and culture upon the yield of human islets for research is important for planning research studies and islet distribution to distant laboratories. Here, we examine islet isolation and cell culture outcomes at the Alberta Diabetes Institute (ADI) IsletCore (n = 197). Research-focused isolations typically have a lower yield of islet equivalents (IEQ), with a median of 252,876 IEQ, but a higher purity (median 85%) than clinically focused isolations before culture. The median recovery of IEQs after culture was 75%, suggesting some loss. This was associated with a shift toward smaller islet particles, indicating possible islet fragmentation, and occurred within 24 h with no further loss after longer periods of culture (up to 136 h). No overall change in stimulation index as a measure of islet function was seen with culture time. These findings were replicated in a representative cohort of clinical islet preparations from the Clinical Islet Transplant Program at the University of Alberta. Thus, loss of islets occurs within 24 h of isolation, and there is no further impact of extended culture prior to islet distribution for research.

A Comprehensive Protocol for the Collection, Differentiation, Cryopreservation, and Resuscitation of Primary Murine Bone Marrow Derived Macrophages (BMDM).

BACKGROUND: The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the "immortal" nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model. METHODS: Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. RESULTS: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. CONCLUSION: Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability.

The superiority of innovative spiral-interdigital microelectrode pattern in increasing the sensitivity of tracing synchronization via serum starvation in cellular metabolism.

In order to investigate the changes in the properties of the cell culture solution in the effect of cell synchronization via cell starvation (for 12, 24, and 36 h), a new spiral-interdigital pattern of microelectrode as a biosensor has been proposed. Then, to test its superiority, the results of this spiral-interdigital pattern with the results of the commercial pattern have been compared. The cells were selected from breast cancer standard lines (MDA-MB-231). Changes in CV peaks of the secretions were recorded by the spiral-interdigital pattern, in which increasing the interactive surface with homogenous electric paths had been considered by simulation before fabrication. The results of the simulation and experimental procedures showed a meaningful correlation. The occurrence of CV oxidative peaks at about 0.1-0.4 V and reductive peaks at approximately 0 V in the spiral-interdigital biosensor in the starved MDA-MB-231 cell line has been observed. The starvation situation resembles one that does not cause meaningful cell apoptosis or necrosis, and this method is only used to make the cells synchronized. Also, no peak is observed in normal cell growth conditions. In addition, by using the commercial design of the electrodes, no peak is observed in any of the conditions of normal and synchronized growth of the cells. Therefore, it seems that the observed peaks are caused by the agents that are secreted in the cell culture solution in a synchronized situation. Moreover, the design of the new spiral-interdigital electrode can significantly increase the sensitivity of the sensor to receive these peaks due to more space and a uniform electric field.

Protocol for establishing inducible CRISPRd system for blocking transcription factor-binding sites in human pluripotent stem cells.

Transcription factor (TF) gene knockout or knockdown experiments provide comprehensive downstream effects on gene regulation. However, distinguishing primary direct effects from secondary effects remains challenging. To assess the direct effect of TF binding events, we present a protocol for establishing a doxycycline (Dox)-inducible CRISPRd system in human pluripotent stem cells (hPSCs). We describe the steps for establishing CRISPRd host hPSCs, designing and preparing single-guide RNA (sgRNA) expression lentivirus vectors, generating CRISPRd hPSCs transduced with sgRNAs, and analyzing CRISPRd TF-block effects by chromatin immunoprecipitation (ChIP)-qPCR. For complete details on the use and execution of this protocol, please refer to Matsui et al.1.

A simulated microgravity-oriented AIE probe-ECM hydrogel-integrated chip for cell culture and superoxide anion radical detection.

Human space activities have been continuously increasing. Astronauts experiencing spaceflight are faced with health problems caused by special space environments such as microgravity, and the investigation of cell injury is fundamental. The development of a platform capable of cell culture and injury detection is the prerequisite for the investigation. Constructing a platform suitable for special conditions in space life science research is the key issue. The ground-based investigation is an indispensable part of the research. Accordingly, a simulated microgravity (SMG)-oriented integrated chip platform capable of 3D cell culture and in situ visual detection of superoxide anion radical (O2•-) is developed. SMG can cause oxidative stress in human cells, and O2•- is one of the signaling molecules. Thus, a O2•--responsive aggregation-induced emission (AIE) probe is designed, which shows high selectivity and sensitivity to O2•-. Moreover, the probe exhibits abilities of long-term and wash-free staining to cells due to the AIE behavior, which is precious for space cell imaging. Meanwhile, a chip with a high-aspect-ratio chamber for adequate medium storage for the lack of the perfusion system during the SMG experiment and a cell culture chamber which can integrate the extracellular matrix (ECM) hydrogel for the bioinspired 3D cell culture is fabricated. In addition, a porous membrane is introduced between the chambers to prevent the hydrogel from separating during the SMG experiment. The afforded AIE probe-ECM hydrogel-integrated chip can achieve 3D culturing of U87-MG cells and in situ fluorescent detection of endogenous O2•- in the cells after long-term staining under SMG. The chip provides a powerful and potential platform for ground-based investigation in space life science and biomedical research.

Quantifying Dendritic Arbors In Vitro and In Vivo in Rodent Models.

Dendritic arborization is a critical determinant of neuronal connectivity. The structure of a neuron's dendritic arbor determines the number of synaptic inputs a neuron can receive and how it processes synaptic input from other neurons. Here, we describe methods for visualizing and quantifying the dendritic arbor in primary cell cultures and in the intact rodent brain. These techniques can be used to answer significant scientific questions, such as the effects of disease processes, drugs, growth factors, and diverse environmental stressors on dendritogenesis in both in vitro and in vivo rodent models.

Isolation and Culture of Adult Rat Dorsal Root Ganglion Neurons for the In Vitro Analysis of Peripheral Nerve Degeneration and Regeneration.

Isolation and culture of dorsal root ganglion (DRG) neurons from adult animals is a useful experimental system for evaluating neural plasticity after axonal injury, as well as the neurological dysfunction resulting from aging and various types of disease. In this chapter, we will introduce a detailed method for the culture of mature rat DRG neurons. About 30-40 ganglia are dissected from a rat and mechanically and enzymatically digested. Subsequently, density gradient centrifugation of the digested tissue using 30% Percoll efficiently eliminates myelin debris and non-neuronal cells, to afford neuronal cells with a high yield and purity.

Three-dimensional cell culture using CD9-positive cells isolated from marginal cell layer of intermediate lobe of rats sustains in vivo-like primary niche environment.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

A lentiviral toolkit to monitor airway epithelial cell differentiation using bioluminescence.

Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data is limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the TP63, MUC5AC and FOXJ1 genes to quantitatively assess basal cell, mucosecretory cell and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ('bronchosphere') cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC) cell cultures.

Role of 6mA in the Regulation of Metabolic Biosynthesis in Sorghum Callus.

Plant cell culture technology helps to obtain natural plant-derived metabolites. The callus of sorghum, a prominent cereal crop, possesses various metabolites with potential health benefits. However, the epigenetic mechanism regulating metabolic biosynthetic capabilities in sorghum remains unknown. Therefore, we conducted N6-methyladenine (6mA) methylome analysis using transcriptome profiling and metabolome analysis to investigate the role of 6mA alterations in two calluses having different biosynthetic capacities, which were derived from immature sorghum embryos. Our findings indicate that the 6mA upregulation within gene bodies is crucial in transcriptional activity potentially mediated by the DNA demethylase SbALKBH1. Furthermore, 6mA was significantly enriched in genes involved in the biosynthesis of flavonoids and isoflavonoids. This could serve as a novel source of bioactive compounds for human health. Thus, 6mA could play an essential role in flavonoid biosynthesis in the sorghum callus.