Isolation Of Primary Cells Research Articles

Tendon stem/progenitor cells are promising reparative cell sources for multiple musculoskeletal injuries of concomitant articular cartilage lesions associated with ligament injuries

BackgroundTrauma-related articular cartilage lesions usually occur in conjunction with ligament injuries. Torn ligaments are frequently reconstructed with tendon autograft and has been proven to achieve satisfactory clinical outcomes. However, treatments for the concomitant articular cartilage lesions are still very insufficient. The current study was aimed to evaluate whether stem cells derived from tendon tissue can be considered as an alternative reparative cell source for cartilage repair.MethodsPrimary human tendon stem/progenitor cells (hTSPCs) were isolated from 4 male patients (32 ± 8 years) who underwent ACL reconstruction surgery with autologous semitendinosus and gracilis tendons. The excessive tendon tissue after graft preparation was processed for primary cell isolation with an enzyme digestion protocol. Decellularization cartilage matrix (DCM) was used to provide a chondrogenic microenvironment for hTSPCs. Cell viability, cell morphology on the DCM, as well as their chondrogenic differentiation were evaluated.ResultsDAPI staining and DNA quantitative analysis (61.47 μg per mg dry weight before and 2.64 μg/mg after decellularization) showed that most of the cells in the cartilage lacuna were removed after decellularization process. Whilst, the basic structure of the cartilage tissue was preserved and the main ECM components, collagen type II and sGAG were retained after decellularization, which were revealed by DMMB assay and histology. Live/dead staining and proliferative assay demonstrated that DCM supported attachment, survival and proliferation of hTSPCs with an excellent biocompatibility. Furthermore, gene expression analysis indicated that chondrogenic differentiation of hTSPC was induced by the DCM microenvironment, with upregulation of chondrogenesis-related marker genes, COL 2 and SOX9, without the use of exogenous growth factors.ConclusionDCM supported hTSPCs attachment and proliferation with high biocompatibility. Moreover, TSPCs underwent a distinct chondrogenesis after the induction of a chondrogenic microenvironment provided by DCM. These results indicated that TSPCs are promising reparative cell sources for promoting cartilage repair. Particularly, in the cohort that articular cartilage lesions occur in conjunction with ligament injuries, autologous TSPCs can be isolated from a portion of the tendon autograph harvested for ligaments reconstruction. In future clinical practice, combined ligament reconstruction with TSPCs- based therapy for articular cartilage repair can to be considered to achieve superior repair of these associated injuries, in which autologous TSPCs can be isolated from a portion of the tendon autograph harvested for ligaments reconstruction.

Targeting the AKT/mTOR/p70S6K Pathway for Oligodendrocyte Differentiation and Myelin Regeneration in Neurological Disorders.

The AKT/mTOR/p70S6K pathway has been shown to potentially promote spinal cord injury (SCI) repair in rats. However, its exact mechanism and beyond needs to be further explored. This study aims to explore the AKT/mTOR/p70S6K pathway in oligodendrocyte precursor cell (OPC) differentiation, microglial polarization differentiation, and the role of these in myelin regeneration in vitro. The isolation, induction and characterization of rat primary neuronal stem cells, OPCs and oligodendrocytes were investigated with immunofluorescence and RT-qPCR. Then, the role of AKT/mTOR/p70S6K signaling was explored using western blotting and immunofluorescence, the effect on myelination was examined with OPC-dorsal root ganglion (DRG) neurons co-culture, and the influence of M1/M2 polarization status of microglia on myelin formation was also observed by adding M1/M2 supernatants into OPC-DRG neurons co-culture. Activation of the AKT/mTOR/p70S6K pathway elevated the expression of oligodendrocyte differentiation markers, including MBP, PLP and MOG, which also promoted the colocalization of MBP and NFH in OPC-DRG neurons co-culture. More interestingly, stimulation of the AKT/mTOR/p70S6K pathway facilitated M2 polarization of rat microglia. M2 polarization of microglia enhanced OPC differentiation to oligodendrocytes and myelin formation. Our findings highlight the potential of targeting the AKT/mTOR/p70S6K pathway in promoting oligodendrocyte differentiation and myelin regeneration in neurological disorders such as SCI.

A method for isolating and culturing ectopic epithelial and stromal cells to study human adenomyosis.

Although adenomyosis is a common and benign gynecological disease, the specific pathogenesis of this condition is yet to be fully elucidated. It is difficult to culture primary cells of the ectopic endometrial epithelia and stroma from human adenomyosis lesions. Most of the previous of studies on adenomyosis were based on primary eutopic endometrium cells. However, as yet, no efficient protocols have been developed for the isolation, culture or purification of primary ectopic epithelial and stromal cells from human adenomyosis lesions. Therefore, the present study aimed to develop an efficient protocol for the isolation and culture of primary ectopic epithelial and stromal cells from human adenomyosis lesions. In the present study, we aimed to obtain ectopic endometrium tissue from human adenomyosis foci and use a simple and operable type I collagenase digestion method for primary culture. Cells were isolated by sterile cell strainer filtration and flow cytometry was performed to identify, purify, and evaluate the viability of isolated ectopic endometrial cells. Using our method, we successfully isolated and cultured highly purified and active ectopic endometrial epithelial and stromal cells from human adenomyosis foci. Ep-CAM was expressed in ectopic epithelial cells of human adenomyosis with a purity of 93.74% and a viability of 80.58%. In addition, CD10 were robustly expressed by ectopic stromal cells in human adenomyosis. Cellular purity and viability were determined to be 96.37 and 93.49%, respectively. Our method provides a new experimental model for studying the molecular pathogenesis of human adenomyosis.

Development and characterisation of mgTHP-1, a novel in vitro model for neural macrophages with microglial characteristics

ABSTRACTNeuroinflammation is primarily characterised by activation of the brain’s resident macrophages – the microglia. However, other central nervous system (CNS) cells also contribute to this response, including the astrocytes and endothelial cells. In addition, there is infiltration into the CNS of peripherally derived immune cells. Together these cells mediate inflammation by the production of cytokines, chemokines, reactive oxygen species, and secondary messengers, and enacting of the appropriate response to those signals. However, deciphering the specific contributions of each cell type has been challenging. Studying CNS cell biology is often challenging, as the isolation of primary cells is not always feasible, and differentiation towards microglia-like cells is complex. Here, we demonstrate a novel method whereby THP-1 monocytic cells are differentiated into neural macrophage cells with microglia-like cell characteristics. The cells, designated mgTHP-1, show typical morphological and gene expression patterns of resident CNS macrophages and functionally respond to inflammatory stimuli by producing inflammatory cytokines. Furthermore, with the addition of Vicenin-2 (an anti-inflammatory flavonoid) such responses can be reversed. This novel cell model will allow further investigations, and hence insights, into the neuroinflammatory mechanisms associated with CNS diseases.

An In Vitro Bilayer Model of Human Primary Retinal Pigment Epithelial and Choroid Endothelial Cells for Permeability Studies.

The blood-retinal barrier (BRB) present in the posterior chamber of the eye plays a major role in maintaining the proper function and integrity of the retina. Retinal pigment epithelium and choriocapillaris form the outer blood retinal barrier (oBRB), and breakdown of this barrier leads to vision-threatening diseases like macular edema, macular degeneration, and diabetic retinopathy. A simplified cell culture model of oBRB will be of great importance in elucidating the molecular mechanism of the disease progression. This chapter describes methods for primary cell isolation from donor eyes to culture human retinal pigment epithelial cells (hRPE) and choroidal endothelial cells (hCEC) and the protocol for construction of a simplified in vitro model of oBRB on fibronectin-coated Transwell inserts. Further, we explained the permeability study using FITC-dextran conjugated tracers for validating the bilayer model. The permeability experiments ensured that the system could easily be manipulated to recapitulate the pathological condition in vitro. Thus, it would be an optimal system for studying the disease mechanisms related to retinal and choroidal pathologies, for screening small molecules, and for performing drug permeability kinetics. Moreover, fundamental understanding of paracellular and transcellular trafficking of cargo in hRPE and hCEC could also be studied using this model.

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice.

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 °C, and gently suspended in culture medium using a 200 µL pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached >90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Culture of primary human meibomian gland cells from surgically excised eyelid tissue

Meibomian gland dysfunction is one of the most common ocular diseases, with therapeutic treatment being primarily palliative due to our incomplete understanding of meibomian gland (MG) pathophysiology. To progress in vitro studies of human MG, this study describes a comprehensive protocol, with detailed troubleshooting, for the successful isolation, cultivation and cryopreservation of primary MG cells using biopsy-size segments of human eyelid tissue that would otherwise be discarded during surgery. MG acini were isolated and used to establish and propagate lipid-producing primary human MG cells. The primary cell viability during culture procedure was maintained through the application of Rho-associated coiled-coil containing protein kinase inhibitor (Y-27632, 10 μM) and collagen I from rat tails. Transcriptomic analysis of differentiated primary human MG cells confirmed cell origin and revealed high-level expression of many lipogenesis-related genes such as stearoyl-CoA desaturase (SCD), ELOVL Fatty Acid Elongase 1 (ELOVL1) and fatty acid synthase (FASN). Primary tarsal plate fibroblasts were also successfully isolated, cultured and cryopreserved. Established primary human MG cells and tarsal plate fibroblasts presented in this study have potential for applications in 3D models and bioengineered tissue that facilitate research in understanding of MG biology and pathophysiology.

The Influences of Cryopreservation Methods on RNA, Protein, Microstructure and Cell Viability of Skeletal Muscle Tissue.

Background: Different experiments require different sample storage methods. The commonly used preservation methods in biobank practice cannot fully meet the multifarious requirements of experimental techniques. Programmable controlled slow freezing (PCSF) can maintain the viability of tissue. In this study, we hypothesized that PCSF-preserved samples have potential advantages in matching subsequent experiments compared with existing methods. Methods: We compared the differences on skeletal muscle tissue RNA integrity, protein integrity, microstructure integrity, and cell viability between four existing cryopreservation methods: liquid nitrogen (LN2) snap-freezing, LN2-cooled isopentane snap-freezing, RNAlater®-based freezing, and PCSF. RNA integrity was evaluated using agarose gel electrophoresis and RNA integrity number. Freezing-related microstructural damage in the muscle tissue was evaluated using ice crystal diameter and muscle fiber cross-sectional area. Protein integrity was evaluated using immunofluorescence staining. Cell viability was evaluated using trypan blue staining after primary muscle cell isolation. Results: PCSF preserved RNA integrity better than LN2 and isopentane, with a statistically significant difference. RNAlater preserved RNA integrity best. PCSF best controlled ice crystal size in myofibers, with a significant difference compared with LN2. The PCSF method best preserved the integrity of protein epitopes according to the mean fluorescence intensity results, with a significant difference. Cell viability was best preserved in the PCSF method compared with the other three methods, with a significant difference. Conclusion: PCSF protected the RNA integrity, microstructural integrity, protein integrity, and cell viability of skeletal muscle tissue. The application of PCSF in biobank practice is recommended as a multi-experiment-compatible cryopreservation method.

Swiss Vascular Biobank: Evaluation of Optimal Extraction Method and Admission Solution for Preserving RNA from Human Vascular Tissue.

Proper biobanking is essential for obtaining reliable data, particularly for next-generation sequencing approaches. Diseased vascular tissues, having extended atherosclerotic pathologies, represent a particular challenge due to low RNA quality. In order to address this issue, we isolated RNA from vascular samples collected in our Swiss Vascular Biobank (SVB); these included abdominal aortic aneurysm (AAA), peripheral arterial disease (PAD), healthy aorta (HA), and muscle samples. We used different methods, investigated various admission solutions, determined RNA integrity numbers (RINs), and performed expression analyses of housekeeping genes (ACTB, GAPDH), ribosomal genes (18S, 28S), and long non-coding RNAs (MALAT1, H19). Our results show that RINs from diseased vascular tissue are low (2-4). If the isolation of primary cells is intended, as in our SVB, a cryoprotective solution is a better option for tissue preservation than RNAlater. Because RNA degradation proceeds randomly, controls with similar RINs are recommended. Otherwise, the data might convey differences in RNA degradation rather than the expressions of the corresponding genes. Moreover, since the 18S and 28S genes in the diseased vascular samples were degraded and corresponded with the low RINs, we believe that DV200, which represents the total RNA's disintegration state, is a better decision-making aid in choosing samples for omics analyses.

Isolation and Micromass Culturing of Primary Chicken Chondroprogenitor Cells for Cartilage Regeneration.

Much of the skeletal system develops by endochondral ossification, a process that takes place in early fetal life. This makes the early stages of chondrogenesis, i.e., when chondroprogenitor mesenchymal cells differentiate to chondroblasts, challenging to study in vivo. In vitro methods for the study of chondrogenic differentiation have been available for some time. There is currently high interest in developing fine-tuned methodology that would allow chondrogenic cells to rebuild articular cartilage and restore joint functionality. The micromass culture system that relies on embryonic limb bud-derived chondroprogenitor cells is a popular method for the study of the signaling pathways that control the formation and maturation of cartilage. In this protocol, we describe a technique fine-tuned in our laboratory for culturing limb bud-derived mesenchymal cells from early-stage chick embryos in high density (Basic Protocol 1). We also provide a fine-tuned method for high-efficiency transient transfection of cells before plating using electroporation (Basic Protocol 2). In addition, protocols for histochemical detection of cartilage extracellular matrix using dimethyl methylene blue, Alcian blue, and safranin O are also provided (Basic Protocol 3 and Alternate Protocols 1 and 2, respectively). Finally, a step-by-step guide on a cell viability/proliferation assay using MTT reagent is also described (Basic Protocol 4). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Micromass culture of chick embryonic limb bud-derived cells Basic Protocol 2: Transfection of cells with siRNA constructs using electroporation prior to micromass culturing Basic Protocol 3: Qualitative and quantitative assessment of cartilage matrix production using dimethyl methylene blue staining and image analysis Alternate Protocol 1: Qualitative assessment of cartilage matrix production using Alcian blue staining Alternate Protocol 2: Qualitative assessment of cartilage matrix production using safranin O staining Basic Protocol 4: Measurement of mitochondrial activity with the MTT assay.

Dengue Virus Infection of Human Retinal Müller Glial Cells.

Retinopathy is a recently recognized complication of dengue, affecting up to 10% of hospitalized patients. Research on the pathogenesis has focused largely on effects of dengue virus (DENV) at the blood-retinal barrier. Involvement of retinal Müller glial cells has received little attention, although this cell population contributes to the pathology of other intraocular infections. The goal of our work was to establish the susceptibility of Müller cells to infection with DENV and to identify characteristics of the cellular antiviral, inflammatory, and immunomodulatory responses to DENV infection in vitro. Primary human Müller cell isolates and the MIO-M1 human Müller cell line were infected with the laboratory-adapted Mon601 strain and DENV serotype 1 and 2 field isolates, and cell-DENV interactions were investigated by immunolabelling and quantitative real-time polymerase chain reaction. Müller cells were susceptible to DENV infection, but experiments involving primary cell isolates indicated inter-individual variation. Viral infection induced an inflammatory response (including tumour necrosis factor-α, interleukin [IL]-1β, and IL-6) and an immunomodulatory response (including programmed death-ligand [PD-L]1 and PD-L2). The type I interferon response was muted in the Müller cell line compared to primary cell isolates. The highest infectivity and cell responses were observed in the laboratory-adapted strain, and overall, infectivity and cell responses were stronger in DENV2 strains. This work demonstrates that Müller cells mount an antiviral and immune response to DENV infection, and that this response varies across cell isolates and DENV strain. The research provides a direction for future efforts to understand the role of human retinal Müller glial cells in dengue retinopathy.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines. Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Two Methods for the Isolation and Cultivation of Porcine Primary Corneal Cells.

In ophthalmic research, there is a strong need for in vitro corneal cell models. Here, we describe different protocols for the cultivation of primary corneal cells that were isolated from porcine eyes. This primary cell culture can be used to test new therapeutic options for corneal diseases, such as dry eye disease, traumatic injuries, or corneal infections, and to study limbal epithelial stem cell (LESC) expansion. Two different isolation methods were performed: the outgrowth and the collagenase method. To perform the outgrowth protocol, small explants of the corneal limbus were generated and incubated in culture flasks in an incubator for 4-5 weeks. Regarding the collagenase method, to extract corneal cells, porcine corneas were removed, cut into small pieces, and incubated with collagenase. After incubation and centrifugation, the cells were seeded in 6- or 12-well plates and incubated in an incubator for 2-3 weeks. The differences between corneal cell cultivation with fetal bovine serum (FBS) and without it are also discussed. Therefore, the main advantages of the outgrowth method are that it requires fewer porcine eyes, and it takes less time to be performed compared to the collagenase method. On the other hand, with the collagenase method, mature cells are obtained earlier, at about 2 to 3 weeks.

The effect of 1,25-dihydroxyvitamin D3 on the Wnt signaling pathway in bovine intestinal epithelial cells is mediated by the DKK2 (dickkopf2) Wnt antagonist

The Wnt/β-catenin signaling pathway is aberrantly activated in most colorectal cancers. High-dose 1,25(OH)2D3 has anticancer effect by regulating Wnt signal pathway. However, it is not clear whether high-dose of 1,25(OH)2D3 have an effect on normal cells. The aim of the present study was to investigate the mechanism of high-dose 1,25(OH)2D3 on the Wnt signaling pathway in bovine intestinal epithelial cells. The potential mechanism of action was investigated after knockdown and overexpression of the Wnt pathway inhibitor, DKK2, in intestinal epithelial cells by observing the effects of 1,25(OH)2D3 on proliferation, apoptosis, pluripotency and the expression of genes related to the Wnt/β-catenin signaling pathway. In the present study, we introduced the method of isolation and culture of primary bovine intestinal epithelial cells. After cells were treated with 50 ng/mL 1,25(OH)2D3 or DMSO for 48 h, total RNA was extracted, and six differentially expressed genes, including SERPINF1, SFRP2, SFRP4, FZD2, WISP1 and DKK2 were identified by transcriptome sequencing, which were related to Wnt signaling pathway. To further explore the mechanism of 1,25(OH)2D3 on the Wnt/β-catenin signaling pathway, we constructed knockdown and overexpression plasmids of DKK2. After transfecting these plasmids into bovine intestinal epithelial cells, we measured the expression of DKK2 mRNA and protein through GFP expression, qRT-PCR and western blot analyses to verify the transfection efficiency. In addition, the CCK-8 assay was used to detect the cell proliferation rate after transfection. Subsequently, the transfected cells were treated with 1,25(OH)2D3 for 48 h, and the proliferation- (Ki67 and PCNA), apoptosis- (Bcl-2, p53, casp3 and casp8), pluripotency- (Bmi-1, Lrig1, KRT19 and TUFT1) and Wnt/ β-catenin signaling pathway- related genes (LGR5, DKK2, VDR, β- Catenin, SFRP2, WISP1 and FZD2) were detected by qRT-PCR and western blot analyses. Our results showed that the expression trend of some genes in bovine intestinal epithelial cells under high-dose 1,25(OH)2D3 was consistent with the sequencing results, including SFRP2 (P < 0.001), SFRP4 (P < 0.05), FZD2 (P < 0.01), WISP1 (P < 0.001) and DKK2 (P < 0.001). In addition, knockdown of DKK2 inhibited cell proliferation (P < 0.01), but DKK2 overexpression promoted cell proliferation (P < 0.01). Compared to the control group, 1,25(OH)2D3 promoted the expression of Wnt/β-catenin signaling pathway-related proteins in bovine intestinal epithelium, thus maintaining intestinal homeostasis in normal intestinal epithelium. In addition, knockdown and overexpression of DKK2 indicated that 1,25(OH)2D3 weakened the inhibitory effect of DKK2 on the Wnt/β-catenin signaling pathway. Together, these results suggest that high-dose 1,25(OH)2D3 has no killing effect on normal intestinal epithelial cells and regulates Wnt/β-catenin signaling pathway through DKK2.

Human retinal endothelial cells express functional interleukin-6 receptor

BackgroundInterleukin (IL)-6 is an inflammatory cytokine present in the eye during non-infectious uveitis, where it contributes to the progression of inflammation. There are two major IL-6 signaling pathways: classic signaling and trans-signaling. Classic signaling requires cellular expression of the IL-6 receptor (IL-6R), which exists in membrane-bound (mIL-6R) and soluble (sIL-6R) forms. Prevailing dogma is that vascular endothelial cells do not produce IL-6R, relying on trans-signaling during inflammation. However, the literature is inconsistent, including with respect to human retinal endothelial cells.FindingsWe examined IL-6R transcript and protein expression in multiple primary human retinal endothelial cell isolates, and assessed the effect of IL-6 on the transcellular electrical resistance of monolayers. Using reverse transcription-polymerase chain reaction, IL-6R, mIL-6R and sIL-6R transcripts were amplified in 6 primary human retinal endothelial isolates. Flow cytometry on 5 primary human retinal endothelial cell isolates under non-permeabilizing conditions and following permeabilization demonstrated intracellular stores of IL-6R and the presence of mIL-6R. When measured in real-time, transcellular electrical resistance of an expanded human retinal endothelial cell isolate, also shown to express IL-6R, decreased significantly on treatment with recombinant IL-6 in comparison to non-treated cells across 5 independent experiments.ConclusionsOur findings indicate that human retinal endothelial cells produce IL-6R transcript and functional IL-6R protein. The potential for classic signaling in human retinal endothelial cells has implications for the development of therapeutics targeted against IL-6-mediated pathology in non-infectious uveitis.

RUNX1 promotes liver fibrosis progression through regulating TGF-β signalling.

Liver fibrosis is caused by chronic liver injury. There are limited treatments for it, and the pathogenesis is unclear. Therefore, there is an urgent need to explore the pathogenesis of liver fibrosis, and to try to identify new potential therapeutic targets. For this study we used the carbon tetrachloride abdominal injection induced liver fibrosis animal model in mice. Primary hepatic stellate cell isolation was performed by a density-gradient separation method, and this was followed by immunofluorescence stain analyses. Signal pathway analysis was performed by dual-luciferase reporter assay and western blotting. Our results showed that RUNX1 was upregulated in cirrhotic liver tissues compared with normal liver tissues. Besides, overexpression of RUNX1 caused more severe liver fibrosis lesions than control group under CCl4 -induced conditions. Moreover, α-SMA expression in the RUNX1 overexpression group was significantly higher than in the control group. Interestingly, we found that RUNX1 could promote the activation of TGF-β/Smads in a dual-luciferase reporter assay. Thus we demonstrated that RUNX1 could be considered as a new regulator of hepatic fibrosis by activating TGF-β/Smads signalling. Based on this, we concluded that RUNX1 may be developed as a new therapeutic target in the treatment of liver fibrosis in the future. In addition, this study also provides a new insight about the aetiology of liver fibrosis.

Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures.

The low number of neural progenitor cells (NPCs) present in the adult and aged primate brains represents a challenge for generating high-yield and viable in vitro cultures of primary brain cells. Here we report a step-by-step approach for the fast and reproducible isolation of high-yield and viable primary brain cells, including mature neurons, immature cells and NPCs, from adult and aged macaques. We describe the anesthesia, transcardial perfusion and brain tissue preparation; the subsequent microdissection of the regions of interest and their enzymatic dissociation, leading to the separation of single cells. The cell isolation steps of our protocol can also be used for routine cell culturing, in particular for NPC expansion and differentiation, suitable for studies of hippocampal neurogenesis in the adult macaque brain. The purified primary brain cells are largely free from myelin debris and erythrocytes, paving the way for multiple downstream applications in vitro and in vivo. When combined with single-cell profiling techniques, this approach allows an unbiased and comprehensive mapping of cell states in the adult and aged macaque brain, which is needed to advance our understanding of human cognitive and neurological diseases. The neural cell isolation protocol requires 4 h and a team of four to six users with expertize in primary brain cell isolation to avoid tissue hypoxia during the time-sensitive steps of the procedure.

Establishment, Long‐Term Maintenance, and Characterization of Primary Liver Cells from Astyanax mexicanus

The tetra fish species Astyanax mexicanus comprises two morphotypes: cavefish that live in caves and surface fish that inhabit rivers and lakes. Because cavefish have adapted to the nutrient-poor conditions in their habitat whereas the surface fish populations can be used as a proxy for the ancestral condition, this species has become a powerful model system for understanding genetic variation underlying metabolic adaptation. The liver plays a critical role in glucose and fat metabolism in the body and hence is an important tissue for studying altered metabolism in health and disease. Cavefish morphs of A. mexicanus have been shown to develop fatty livers and exhibit massive differences in gene expression and chromatin architecture. Primary cell lines from various tissues have become invaluable tools for biochemical, toxicology, and cell biology experiments, as well as genetic and genomic analyses. To enhance the utility of the model system by enabling an expanded set of biochemical and in vitro experiments, we developed protocols for the isolation and maintenance of primary liver cells from A. mexicanus surface fish and cavefish. We also describe methods that can be used for primary cell characterization, including cloning, characterization of cell growth pattern, and lentivirus transduction. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary culture of liver cells Support Protocol 1: Maintenance of A. mexicanus primary liver cells Support Protocol 2: Banking of A. mexicanus primary liver cells Support Protocol 3: Recovery of A. mexicanus primary liver cells Support Protocol 4: Primary liver cell cloning Support Protocol 5: Characterization of A. mexicanus primary liver cell growth pattern Basic Protocol 2: Lentiviral transduction of A. mexicanus primary liver cells.

NF-κB Activator 1 downregulation in macrophages activates STAT3 to promote adenoma-adenocarcinoma transition and immunosuppression in colorectal cancer

BackgroundAdenoma-adenocarcinoma transition is a key feature of colorectal cancer (CRC) occurrence and is closely regulated by tumor-associated macrophages (TAMs) and CD8+ T cells. Here, we investigated the effect of the NF-κB activator 1 (Act1) downregulation of macrophages in the adenoma-adenocarcinoma transition.MethodsThis study used spontaneous adenoma-developing ApcMin/+, macrophage-specific Act1-knockdown (anti-Act1), and ApcMin/+; anti-Act1 (AA) mice. Histological analysis was performed on CRC tissues of patients and mice. CRC patients’ data retrieved from the TCGA dataset were analyzed. Primary cell isolation, co-culture system, RNA-seq, and fluorescence-activated cell sorting (FACS) were used.ResultsBy TCGA and TISIDB analysis, the downregulation of Act1 expression in tumor tissues of CRC patients negatively correlated with accumulated CD68+ macrophages in the tumor. Relative expression of EMT markers in the tumor enriched ACT1lowCD68+ macrophages of CRC patients. AA mice showed adenoma-adenocarcinoma transition, TAMs recruitment, and CD8+ T cell infiltration in the tumor. Macrophages depletion in AA mice reversed adenocarcinoma, reduced tumor amounts, and suppressed CD8+ T cell infiltration. Besides, macrophage depletion or anti-CD8a effectively inhibited metastatic nodules in the lung metastasis mouse model of anti-Act1 mice. CRC cells induced activation of IL-6/STAT3 and IFN-γ/NF-κB signaling and the expressions of CXCL9/10, IL-6, and PD-L1 in anti-Act1 macrophages. Anti-Act1 macrophages facilitated epithelial-mesenchymal-transition and CRC cells’ migration via CXCL9/10-CXCR3-axis. Furthermore, anti-Act1 macrophages promoted exhaustive PD1+ Tim3+ CD8+ T cell formation. Anti-PD-L1 treatment repressed adenoma-adenocarcinoma transition in AA mice. Silencing STAT3 in anti-Act1 macrophages reduced CXCL9/10 and PD-L1 expression and correspondingly inhibited epithelial-mesenchymal-transition and CRC cells’ migration.ConclusionsAct1 downregulation in macrophages activates STAT3 that promotes adenoma-adenocarcinoma transition via CXCL9/10-CXCR3-axis in CRC cells and PD-1/PD-L1-axis in CD8+ T cells.