Isolation of Chondrocytes and Chondroprogenitors using Fibronectin Adhesion and Migratory Assay.

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened chondrogenesis, and limited hypertrophic traits. The enrichment of progenitors is achieved through differential fibronectin adhesion and migration-based explant assays, with Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs) and Migratory Chondroprogenitors (MCPs) demonstrating superior potential compared to chondrocytes. This article delves into the details of isolating resident cartilage-derived cells, namely chondrocytes and chondroprogenitors. While valuable insights from chondrocyte research contribute to our understanding of cartilage repair, ongoing efforts are directed toward the use of chondroprogenitors and exploring their potential as an alternative therapeutic approach. Additionally, this methodology article provides a detailed step-by-step protocol for isolating three specific cell types from cartilage: chondrocytes, FAA-CPs, and MCPs. By following standardized procedures, this protocol facilitates the successful extraction of these cell subtypes. Grounded in extensive research, the article focuses on the intricate techniques utilized in isolating the different subsets and the optimized culture conditions required to expand and maintain their cultures. The methodology encompasses enzymatic isolation of human articular cartilage-derived chondrocytes, differential fibronectin adhesion following sequential enzymatic digestion, and migration-based explant assays to obtain cartilage-resident cells.

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of <12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth. Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 °C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3 (451 µm x 462.84 µm)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice.

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.

Isolation of Primary Mouse Retinal Glial Müller Cells.

The primary supporting cell of the retina is the retinal glial Müller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, Müller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse Müller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, Müller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for Müller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn't be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Isolation of Mouse Pancreatic Endothelial Cells.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage.

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

Advances in diagnostic liquid-based cytology.

Liquid-based cytology (LBC) has changed the landscape of gynaecological cytology. A growing demand exists for LBC in diagnostic cytology, particularly for ancillary testing, such as immunocytochemistry and molecular testing. Ancillary testing solely based on conventional preparation (CP) methods remains challenging. Recently, the increased demand for specialist testing and minimally invasive techniques, such as endoscopic ultrasonography fine-needle aspiration, to obtain cellular samples has led to an increasing demand for ancillary testing on cytology LBC supernatant, slides and cell block (CB). This facilitates the diagnosis and prognosis in cytology samples enabling personalized treatment. An understanding of the history and future prospects of LBC is crucial for its application in routine diagnostics by cytopathologists and cytotechnologists. In this review, we initiated an internet search using the keyword 'liquid-based cytology', and we conducted a literature review to discuss the usefulness of combined diagnosis of LBC and CP, immunocytochemistry and molecular testing and assessed the quality of nucleic acids in diagnostic LBC. High-quality and cell-rich diagnostic LBC surpassed the CP method alone in terms of reliability and versatility of ancillary testing in cytological diagnosis. Conclusively, diagnostic LBC lends itself to various new technologies and is expected to continue evolving with innovations in the future.

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages.

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a 'niche-based approach on stemness' for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a 'fibroblast-like' spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells.

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

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.

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

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.

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

Impression cytology of ocular surface in xeroderma pigmentosum.

PURPOSE: To describe cellular alterations detected by impression cytology of the ocular surface in patients with xeroderma pigmentosum. The secondary objective was to assess the reliability of impression cytology in diagnosing ocular surface squamous neoplasia. METHODS: Patients with xeroderma pigmentosum underwent a single-day complete ophthalmological examination and impression cytology for ocular surface evaluation using 13 mm diameter mixed cellulose esters membrane filters and combined staining with Periodic Acid Schiff, Hematoxylin and Eosin, and Papanicolaou stains followed by microscopic analysis. The cytological findings were correlated with the clinical diagnosis. The impression cytology findings at baseline and one-year follow-up were correlated with the clinical course (no tumor, treated tumor, residual tumor recurrent tumor, new tumor). RESULTS: Of the 42 patients examined, impression cytology was performed in 62 eyes of 34 participants (65% females). The mean age of patients was 29.6 ± 17 years (range 7-62). Fifteen eyes had a clinical diagnosis of ocular surface squamous neoplasia. Impression cytology showed goblet cells (47, 75%), inflammatory cells (12, 19%), keratinization (5, 8%), and squamous metaplasia (30, 48%). Impression cytology was positive for atypical cells in 18 patients (12 with and 6 without ocular surface squamous neoplasia). The sensitivity, specificity, positive predictive value, and negative predictive value of impression cytology (at baseline) for diagnosis of ocular surface squamous neoplasia were 80%, 87%, 67%, and 93%, respectively, using clinical diagnosis of ocular surface squamous neoplasia as the reference standard. CONCLUSION: Impression cytology has a moderate positive predictive value for the diagnosis of ocular surface squamous neoplasia in patients with xeroderma pigmentosum. However, the lack of detection of atypical cells on impression cytology has a high negative predictive value for ocular surface squamous neoplasia. Integration of impression cytology in the long-term management of high-risk patients, such as patients with xeroderma pigmentosum, can avoid unnecessary diagnostic biopsies.

Telecytology - remote rapid on-site evaluation for ultrasound-guided head and neck fine needle aspiration utilising a clinical imaging assistant with an extended practice role.

OBJECTIVE: Rapid On-Site Evaluation (ROSE) of fine needle aspirations (FNA) is widely accepted as best practice, resulting in better outcomes and delivery of care for patients. However, it is not always practical for cytology laboratories to release staff. To increase the availability of ROSE, this study aimed to robustly test the effectiveness of Telecytology ROSE (TCROSE) utilising a clinical imaging assistant (CIA) to prepare the samples and operate the microscope. METHODS: The study was divided into 3 phases. Phase 1, equipment testing, validation and in-house training for the CIA and the Consultant Biomedical Scientist (CBMS) performing TCROSE. Phase 2, Verifying TCROSE on the same site as the cytology laboratory and phase 3, TCROSE utilising a clinic at a peripheral site away from the cytology laboratory. RESULTS: 78/80 (97% sensitivity, 95% accuracy) of TCROSE cases matched the final report for assessment of adequacy and sufficient sampling, demonstrating 94% reliability with a 95% confidence value. An appropriately trained CIA effectively prepared the samples and operated the microscope for remote interpretation. The samples were triaged effectively, and biopsy requests were appropriate to reduce the need for repeat procedures and delays in treatment. This approach received positive feedback from patients. CONCLUSION: TCROSE utilising a CIA provides a highly effective alternative to conventional ROSE, minimising the resources required from cytopathology services and improving patient care and access to best practice. This study supports the validity of trained CIAs for a more involved role in the ultrasound-guided FNA service.

Cell block practices in European cytopathology laboratories.

BACKGROUND: There are numerous methods and procedures described for the preparation of cell blocks (CBs) from cytological samples. The objective of this study was to determine current practices and issues with CBs in European laboratories. METHODS: A link to an online survey, with 11 questions about CB practices, was distributed to cytology laboratories via participants of United Kingdom National External Quality Assurance Service for Cellular Pathology Techniques and national representatives in the European Federation of Cytology Societies. RESULTS: A total of 402 laboratories responded completely (337/402, 84%) or partially (65/402, 16%) to the survey by February 4, 2022. The most common CB practice is embedding cell pellets using plasma and thrombin (23.3%), agar (17.1%), Shandon/Epredia Cytoblock (11.4%), HistoGel (7.9%), and Cellient (3.5%). Other methods such as CytoFoam, albumin, gelatin, Cytomatrix, and collodion bags are rarely used (1.0%, 0.7%, 0.7%, 0.3%, and 0.2%, respectively). CBs are also prepared from naturally occurring clots or tissue fragments (29.5%) and cells scraped from unstained or prestained smears (4.4%). The most frequent issues with the CBs in a daily cytology practice are low cellularity (248/402, 62%) and dispersed cells (89/402, 22%), regardless of the CBs preparation method or how the samples for embedding were selected. CONCLUSIONS: There is a great variability in CB practices in European laboratories with low cellular CBs as the main issue. Additional studies are mandatory to evaluate and improve performance and cellular yield of CBs.