Isolation Of Primary Cells Research Articles

Adipose-derived cells surpass muscle-derived cells in primary cell isolation efficacy.

Adipose-derived cells surpass muscle-derived cells in primary cell isolation efficacy.

Deciphering the Impact of Collagenase Treatment Duration on Primary Breast Cancer Cell Proteome: A Comprehensive Study

Primary cell isolation is essential for studying cellular behavior and disease mechanisms, with collagenase-mediated tissue dissociation playing a critical role in the process. However, the impact of collagenase treatment duration on the proteome of primary cells, particularly in breast cancer research, remains underexplored. This study aims to investigate the effects of collagenase II treatment duration on the proteomic profiles of primary breast cancer cells. Breast cancer tissues from patients diagnosed with infiltrating ductal carcinoma were treated with collagenase II for either 1 or 3 hours. Subsequent proteomic analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Identified proteins were subjected to bioinformatic analyses to determine the functional implications of the proteomic changes induced by the different treatment durations. Bioinformatic analyses showed that 1-hour treatment predominantly affected proteins involved in cytoskeletal organization and cell adhesion, with significant enrichment in actin cytoskeleton dynamics and structural molecule activity. In contrast, 3-hour treatment led to significant metabolic reprogramming, with enhanced regulation of pathways involved in energy production, including the TCA cycle and glycolysis. This study reveals for the first time that, collagenase II treatment duration significantly alters the proteomic profile of primary breast cancer cells, with shorter durations affecting structural proteins and longer durations inducing metabolic changes. Optimizing treatment time is crucial for targeted proteomic studies.

Optimization of process parameters for specific pathogen-free chicken embryonic fibroblast cultivation for yellow fever vaccine production

The yellow fever (YF) vaccine is usually produced with egg-based methods, which has limitations, including potential adverse effects and low production yields. Alternatively, producing the vaccine using Vero cells or HEK 293 cells can overcome some of these issues, but these methods are significantly more expensive. In the current study, the YF vaccine candidate 17DD virus was produced in primary chicken embryo fibroblast (CEF) cells. The primary CEF cells isolation from eggs was optimized through a two-step process. In the first step, the important parameters that contribute to the development of the egg embryo, such as egg position, relative humidity (RH), and incubation time are optimized. In second step, primary CEF release parameters namely; trypsin volume and incubation temperature are optimized. Both steps were optimized using statistical methods. Further, the seeding cell density of isolated CEF was also optimized. It was observed that 5 x 104 cells/cm2 gave the highest virus titer of 3.89 million PFU/ml. The 17DD yields achieved in primary CEFs are much higher than egg-based production and it is an economically viable method.

Isolation and identification of primary cells: A comprehensive primary cell culture experiment for graduate students.

Experimental teaching is an important part of postgraduate training in basic and clinical medicine. While primary cell isolation and identification are among the most important research techniques for medical graduate students, most graduate students do not understand and master these techniques before starting their research experience. In particular, many students lack training in this field, and high-quality teaching and learning materials are still very sparse. Here, we designed a practical experiment course for graduate students engaged in research. The target students usually have research projects involving primary cell culture in their future research, making the course highly applicable for the students. The lab exercise focused on the methods of primary cell isolation (including mechanical grinding method, explant culture method and enzymatic digestion method) and identification (including flow cytometry, immunofluorescence, and periodic acid-Schiff (PAS) staining). It aimed to help students master the conceptual, principle, technical, operation, and analytical skills related to primary cell culture and contributed to their foundation for future research. Students generally reflect that they have initially mastered the isolation and identification of primary cell culture as a result of the course. Student feedback also indicates significantly increased confidence in the practical application of primary cell culture in the future. Here, we provide our experience for others who may want to implement similar courses.

Isolation methods and characterization of primary rat neurovascular cells

BackgroundThere is significant interest in isolating cells of the blood-brain barrier (BBB) for use in in vitro screening of therapeutics and analyzing cell specific roles in neurovascular pathology. Primary brain cells play an advantageous role in BBB models; however, isolation procedures often do not produce cells at high enough yields for experiments. In addition, although numerous reports provide primary cell isolation methods, the field is lacking in documentation and detail of expected morphological changes that occur throughout culturing and there are minimal troubleshooting resources. Here, we present simplified, robust, and reproducible methodology for isolating astrocytes, pericytes, and endothelial cells, and demonstrate several morphological benchmarks for each cell type throughout the process and culture timeframe. We also analyze common considerations for developing neurovascular cell isolation procedures and recommend solutions for troubleshooting.ResultsThe presented methodology isolated astrocytes, pericytes, and endothelial cells and enabled cell attachment, maturation, and cell viability. We characterized milestones in cell maturation over 12 days in culture, a common timeline for applications of these cell types in BBB models. Phase contrast microscopy was used to show initial cell plating, attachment, and daily growth of isolated cells. Confocal microscopy images were analyzed to determine the identity of cell types and changes to cell morphology. Nuclear staining was also used to show the viability and proliferation of glial cells at four time points. Astrocyte branches became numerous and complex with increased culture time. Microglia, oligodendrocytes, and neurons were present in mixed glial cultures for 12 days, though the percentage of microglia and neurons expectedly decreased after passaging, with microglia demonstrating a less branched morphology.ConclusionsNeurovascular cells can be isolated through our optimized protocols that minimize cell loss and encourage the adhesion and proliferation of isolated cells. By identifying timepoints of viable glia and neurons within an astrocyte-dominant mixed culture, these cells can be used to evaluate drug targeting, uptake studies, and response to pathological stimulus in the neurovascular unit.

Epigenomic, cistromic, and transcriptomic profiling of primary kidney tubular cells.

Spatiotemporal regulation of gene expression is essential for maintaining cellular homeostasis throughout kidney development and disease progression. Transcription factors (TFs) and epigenetic modifications play pivotal roles in controlling gene expression. Profiling chromatin modifications across the genome, along with the distribution and target regulation by TFs in specific kidney cell types, is crucial for understanding the dynamic changes in gene expression. Here, we presented a comprehensive workflow for epigenomic, cistromic, and transcriptomic analyses of primary kidney tubular cells. Specifically, our methodologies included the isolation of primary kidney tubular epithelial cells, RNA extraction, assay for transposase-accessible chromatin using sequencing, ultra-low-input micrococcal nuclease-based native chromatin immunoprecipitation, cleavage under targets and release using nuclease, and subsequent bioinformatic analysis. This protocol provides a methodological framework for investigating the roles of TFs and epigenetic modifications in kidney development and diseases.

Novel genetically glycoengineered human dendritic cell model reveals regulatory roles of α2,6-linked sialic acids in DC activation of CD4+ T cells and response to TNFα.

Dendritic cells (DCs) are central for the initiation and regulation of appropriate immune responses. While several studies suggest important regulatory roles of sialoglycans in DC biology, our understanding is still inadequate primarily due to a lack of appropriate models. Previous approaches based on enzymatic- or metabolic-glycoengineering and primary cell isolation from genetically modified mice have limitations related to specificity, stability, and species differences. This study addresses these challenges by introducing a workflow to genetically glycoengineer the human DC precursor cell line MUTZ-3, described to differentiate and maturate into fully functional dendritic cells, using CRISPR-Cas9, thereby providing and validating the first isogenic cell model for investigating glycan alteration on human DC differentiation, maturation, and activity. By knocking out (KO) the ST6GAL1 gene, we generated isogenic cells devoid of ST6GAL1-mediated α(2,6)-linked sialylation, allowing for a comprehensive investigation into its impact on DC function. Glycan profiling using lectin binding assay and functional studies revealed that ST6GAL1 KO increased the expression of important antigen presenting and co-stimulatory surface receptors and a specifically increased activation of allogenic human CD4 + T cells. Additionally, ST6GAL1 KO induces significant changes in surface marker expression and cytokine response to TNFα-induced maturation, and it affects migration and the endocytic capacity. These results indicate that genetic glycoengineering of the isogenic MUTZ-3 cellular model offers a valuable tool to study how specific glycan structures influence human DC biology, contributing to our understanding of glycoimmunology.

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Isolation and Culture of Primary Human Mammary Epithelial Cells.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

MiR-29b-3p Affects the Hypertrophy of Ligamentum Flavum in Lumbar Spinal Stenosis and its Mechanism.

To explore the effect of miR-29b-3p on fibrosis and hypertrophy of ligamentum flavum (LF) in lumbar spinal stenosis (LSS) and its underlying mechanism. Patients with LSS and lumbar disc herniation (LDH) (control) undergoing posterior lumbar laminectomy were included in this study. Human LF samples were obtained for LF cell isolation, RNA, and protein extraction. Histomorphological analysis of LF was performed using hematoxylin-eosin (HE) staining. After isolation, culture, and transfection of primary LF cells, different transfection groups were constructed: NC-mimic, miR-29b-3p-mimic, NC-inhibitor, and miR-29b-3p-inhibitor. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29b-3p in LF and LF cells. Western blot analysis detected the protein expressions of P16 and CyclinD1. ELISA detected the protein expressions of TGF-β1, Smad2, Smad3, TLR4, Type I collagen, and Type III collagen. Finally, LF cell viability was detected using the Cell Counting Kit-8 (CCK8) assay. The thickness of LF was significantly thicker in the LSS group compared to the LDH group (p < 0.05), accompanied by a higher calcification degree, more fibroblasts, and a larger area of collagen fiber proliferation. miR-29b-3p expression was significantly lower in LSS-derived LF tissues and cells than in LDH-derived tissues and cells (both p < 0.05). Compared to the NC-mimic group, the miR-29b-3p-mimic group exhibited significantly higher miR-29b-3p expression, decreased protein expressions of Type I collagen, Type III collagen, TGF-β1, Smad2, Smad3, TLR4, P16, and CyclinD1, and inhibited LF cell proliferation (all p < 0.05). As expected, the miR-29b-3p-inhibitor group displayed contrasting expression patterns (all p < 0.05). Compared to the phosphate buffer saline (PBS) group, the Trimethylamine-N-Oxide (TMAO) group showed significantly increased expressions of TGF-β1, Smad2, Smad3, TLR4, Type I collagen, Type III collagen, P16, and CyclinD1, as well as enhanced LF cell proliferation (all p < 0.05). However, there was no significant difference between the TMAO group and the Ang II group (all p > 0.05). Upregulation of miR-29b-3p expression may play a role in improving LF fibrosis and hypertrophy in LSS by inhibiting P16 expression and suppressing the activation of the TGF-β/Smad signaling pathway. This finding offers new insights into future gene modification therapy for this patient population.

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Observation of the Effects of Different Enzyme Digestion Time and Enzyme Digestion Methods on the Extraction of Primary Renal Tubular Epithelial Cells from Sprague-Dawley Suckling Mice.

Renal tubular epithelial cells are one of the essential functional cells in the kidney. Optimizing the isolation and culture method of primary renal tubular epithelial cells from SD mammary rats provides better experimental materials for renal tubule-related studies, which is essential for studying the pathogenesis of renal diseases, especially diabetic nephropathy and drug screening. SD rat renal tubular epithelial cells were isolated and purified by 2.5-mg/ml collagenase II or 2mg/ml trypsin + 2.5mg/ml collagenase II enzymatic digestion. The isolation and purification were observed at different time points (15min, 30min, 45min, and 60min) to determine the optimal extraction time for the enzymatic digestion method. After comparing the two enzymatic methods, it was determined that the trypsin + collagenase II enzymatic method was more effective. The primary renal tubular epithelial cells extracted by the trypsin + collagenase II digestion method were identified by the marker Cytokeratin 18 of renal tubular epithelial cells at 45min of digestion with high purity. We established a simple, efficient, and reproducible method for isolation and culture of renal tubular epithelial cells in SD mammary gland rats.

Isolation and Culturing of Primary Murine Chondroprogenitor Cells: A Mammalian Model of Chondrogenesis.

Embryonic limb bud-derived micromass cultures are valuable tools for investigating cartilage development, tissue engineering, and therapeutic strategies for cartilage-related disorders. This collection of fine-tuned protocols used in our laboratories outlines step-by-step procedures for the isolation, expansion, and differentiation of primary mouse limb bud cells into chondrogenic micromass cultures. Key aspects covered in these protocols include synchronized fertilization of mice (Basic Protocol 1), tissue dissection, cell isolation, micromass formation, and culture optimization parameters, such as cell density and medium composition (Basic Protocol 2). We describe techniques for characterizing the chondrogenic differentiation process by histological analysis (Basic Protocol 3). The protocols also address common challenges encountered during the process and provide troubleshooting strategies. This fine-tuned comprehensive protocol serves as a valuable resource for scientists working in the fields of developmental biology, cartilage tissue engineering, and regenerative medicine, offering an updated methodology for the study of efficient chondrogenic differentiation and cartilage tissue regeneration. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synchronized fertilization of mice Basic Protocol 2: Micromass culture of murine embryonic limb bud-derived cells Basic Protocol 3: Qualitative assessment of cartilage matrix production using Alcian blue staining.

Interferon-β Overexpression in Adipose Tissue-Derived Stem Cells Induces HepG2 and Macrophage Cell Death in Liver Tumor Organoids via Induction of TNF-Related Apoptosis-Inducing Ligand Expression.

Liver tumor organoids derived from liver tumor tissues and pluripotent stem cells are used for liver tumor research but have several challenges in primary cell isolation and stem cell differentiation. Here, we investigated the potential of HepG2-based liver tumor organoids for screening anticancer drugs by evaluating their responsiveness to IFN-β produced by mesenchymal stem cells (MSCs). Liver tumor organoids were prepared in three days on Matrigel using HepG2, primary liver sinusoidal epithelial cells (LSECs), LX-2 human hepatic stellate cells, and THP-1-derived macrophages at a ratio of 4:4:1:1, with 105 total cells. Hepatocyte-related and M2 macrophage-associated genes increased in liver tumor organoids. IFN-β treatment decreased the viability of liver tumor organoids and increased M1 macrophage marker expression (i.e., TNF-α and iNOS) and TRAIL. TRAIL expression was increased in all four cell types exposed to IFN-β, but cell death was only observed in HepG2 cells and macrophages. Further, MSCs overexpressing IFN-β (ASC-IFN-β) also expressed TRAIL, contributing to the reduced viability of liver tumor organoids. In summary, IFN-β or ASC-IFN-β can induce TRAIL-dependent HepG2 and macrophage cell death in HepG2-based liver tumor organoids, highlighting these liver tumor organoids as suitable for anticancer drug screening and mechanistic studies.

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae.

The maternal decidua is a transient and dynamic tissue that functions as an immunoprivileged matrix related to nutritional and endocrine processes. The function of decidual cells is key to the success of embryo implantation and the maintenance of pregnancy with a positive maternal-fetal outcome. Therefore, establishing a method to optimize the isolation of primary decidual cells is essential. Our protocol described here provides a good yield of decidual cells in an optimized time.

The Y498T499-SARS-CoV-2 spike (S) protein interacts poorly with rat ACE2 and does not affect the rat lung.

The rat is a useful laboratory model for respiratory diseases. SARS-CoV-2 proteins, such as the spike (S) protein, can induce inflammation. This study has investigated the ability of the Q498Y, P499T (QP-YT) amino acid change, described in the S-protein of the mouse-adapted laboratory SARS-CoV-2 MA strain, to interact with rat angiotensin converting enzyme-2 (ACE2) and stimulate responses in rat lungs. A real-time S-ACE2 quantitative fusion assay shows that ancestral and L452R S-proteins fuse with human but not rat ACE2 expressed on HEK293 (human embryonic kidney-293) cells. The QP-YT S-protein retains the ability to fuse with human ACE2 and increases the binding to rat ACE2. Although lower lung of the rat contains both ACE2 and TMPRSS2 (transmembrane serine protease 2) target cells, intratracheal delivery of ancestral or QP-YT S-protein pseudotyped lentivirus did not induce measurable respiratory changes, inflammatory infiltration or innate mRNA responses. Isolation of primary cells from rat alveoli demonstrated the presence of cells expressing ACE2 and TMPRSS2. Infection of these cells, however, with ancestral or QP-YT S-protein pseudotyped lentivirus was not observed, and the QP-YT S-protein pseudotyped lentivirus poorly infected HEK293 cells expressing rat ACE2. Analysis of the amino acid changes across the S-ACE2 interface highlights not only the Y498 interaction with H353 as a likely facilitator of binding to rat ACE2 but also other amino acids that could improve this interaction. Thus, rat lungs contain cells expressing receptors for SARS-CoV-2, and the QP-YT S-protein variant can bind to rat ACE2, but this does not result in infection or stimulate responses in the lung. Further, amino acid changes in S-protein may enhance this interaction to improve the utility of the rat model for defining the role of the S-protein in driving lung inflammation.

Isolation and Culture of Mouse Primary Microglia and Oligodendrocyte Precursor Cells.

Microglia and oligodendrocyte precursor cells (OPCs) are critical glia subsets in the central nervous system (CNS) and are actively engaged in a body of diseases, such as stroke, Alzheimer's disease, multiple sclerosis, etc. Microglia and OPC serve as compelling tools for the study of CNS diseases as well as the repair and damage of myelin sheath in vitro. In this protocol, we summarized a method which is capable of using the same batch of new-born mice to isolate and culture microglia and OPCs. It integrates the characteristics of practicality, convenience, and efficiency, providing a convenient, easy, and reliable technique for research.

Isolation and Enrichment of Major Primary Neuroglial Cells from Neonatal Mouse Brain.

The central nervous system (CNS) relies on the complex interaction of neuroglial cells to carry out vital physiological functions. To comprehensively understand the structural and functional interplay between these neuroglial cells, it is essential to establish an appropriate in vitro system that can be utilized for thorough investigation. Traditional protocols for establishing primary neuronal and mixed glial cultures from prenatal mice or neural stem cells require sacrificing pregnant mice and have the drawback of yielding only specific types of cells. Our current protocol overcomes these drawbacks by utilizing the brain from day-0 pups to isolate CNS resident neuroglial cells including astrocytes, microglia, oligodendrocytes [oligodendrocyte precursor cells (OPCs) and differentiated oligodendrocytes], and meningeal fibroblasts, as well as hippocampal neurons, avoiding sacrificing pregnant mice, which makes this procedure efficient and cost effective. Furthermore, through this protocol, we aim to provide step-by-step instructions for isolating and establishing different primary neuroglial cells and their characterization using cell-specific markers. This study presents an opportunity to isolate, culture, and establish all major CNS resident cells individually. These cells can be utilized in various cell-based and biochemical assays to comprehensively investigate the cell-specific roles and behaviors of brain resident cells in a reductionist approach. Key features • Efficient isolation of major neuroglial cells like meningeal fibroblasts, neurons, astrocytes, oligodendrocytes, and microglia from a single day-0 neonatal mouse pup's brain. • Circumvents the sacrifice of pregnant female mice. • Acts as a bridging experimental method between secondary cell lines and in vivo systems. • Isolated cells can be used for performing various cell-based and biochemical assays.

Canine Adult Adipose Tissue-Derived Multipotent Stromal Cell Isolation, Characterization, and Differentiation.

Adult mesenchymalstromal/stem cells (MSCs) are a standard component of de novo tissue generation to treat and study injury, disease, and degeneration. Canine patients constitute a major component of veterinary practice, and dogs share numerous pathologic conditions with humans. The relative abundance of adipose-derived stromal/stem cells (ASCs) in various canine adipose tissue depots is well described. Refined isolation, characterization, and differentiation techniques contribute to the collective knowledge of ASC phenotypes and subpopulations for specific tissue targets. Continued efforts to advance the knowledge of canine ASC behavior in vivo are critical to harnessing the full potential of primary cell isolates. This chapter contains a description of techniques to isolate, characterize, and differentiate canine ASCs.

Single Cell Isolation from Surgically Resected Tissue Via Mechanical Dissociation Using TissueGrinder.

Primary cells form the basis of modern-day in vitro research analysis tools. Many conventional procedures for generating single-cell suspensions from solid tissue are neither robust nor reproducible. Here we describe primary cells isolation from surgically resected tumor tissue via enzyme-free mechanical dissociation using TissueGrinder, a novel semi-automated benchtop device. The isolated cells can be used for any downstream biochemical or cell-based analytic assay.