Enzymatic Tissue Digestion Research Articles

10× single-cell sequencing revealed cellular composition heterogeneity in cardiac myxoma with malignant glandular properties

Cardiac myxoma is the most common primary cardiac tumor in adults. The histogenesis and cellular composition of myxoma are still unclear. This study aims to reveal the role of myxoma cell components and their gene expression in tumor development. We obtained single living cells by enzymatic digestion of tissues from 4 cases of surgically resected cardiac myxoma. Of course, there was 1 case of glandular myxoma and 3 cases of nonglandular myxoma. Then, 10× single-cell sequencing was performed. We identified 12 types and 11 types of cell populations in glandular myxoma and nonglandular myxoma, respectively. Heterogeneous epithelial cells are the main components of glandular myxoma. The similarities and differences in T cells in both glandular and nonglandular myxoma were analyzed by KEGG and GO. The most important finding was that there was active communication between T cells and epithelial cells. These results clarify the possible tissue occurrence and heterogeneity of cardiac myxoma and provide a theoretical basis and guidance for clinical diagnosis and treatment.

Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Isolation of Human Adipose-Derived Stem Cells.

Human adipose-derived stromal/stem cells (hASCs) are a promising source of adult stem cells used in numerous applications in regenerative medicine. We present the protocols from our laboratory for isolating and expanding hASCs. The isolation of hASCs involves the enzymatic digestion of adipose tissue and subsequent culturing of the isolated cells.

Human papillomavirus infection affects the immune microenvironment and antigen presentation in penile cancer.

Penile squamous cell carcinoma (PSCC) is a largely neglected condition, predominantly affecting underdeveloped regions, and is associated with risk factors such as low socioeconomic status, phimosis, and human papillomavirus (HPV) infection. Unlike other urogenital cancers, its pathophysiology and therapeutic targets remain poorly understood, particularly regarding the immune response to the tumor microenvironment. This study aims to investigate immune cell infiltration profiles, dendritic cell maturation, and lymphocyte apoptosis in both HPV-positive and HPV-negative PSCC. Clinical and histopathological data, along with peripheral blood and tumor tissue samples, were collected from 30 patients (66.6% were HPV-positive and 33.3% HPV-negative), with an additional 19 healthy donors serving as controls. Tumor-infiltrating immune cells were analyzed following enzymatic digestion of tumor tissue, enabling detailed phenotypic characterization. A simulated tumor microenvironment was created using supernatants derived from primary cultures of HPV-positive PSCC tumors. Peripheral blood mononuclear cells were isolated and differentiated into dendritic cells (Mo-DCs) for further phenotyping and lymphoproliferation assays. Lymphocytes from healthy donors and patients were exposed to tumor culture supernatants to evaluate apoptosis induced by the tumor microenvironment. Results showed that HPV-positive tumors exhibited lower T lymphocyte frequencies compared to HPV-negative tumors. Additionally, patients infected with high-risk HPV demonstrated reduced maturation rates of Mo-DCs and decreased expression of co-stimulatory molecules on these cells compared to healthy donors. Furthermore, Mo-DCs from hrHPV-positive patients showed impaired lymphoproliferation capacity relative to controls, while HPV-negative patients exhibited a trend towards reduced lymphoproliferative ability. Regarding the simulated tumor microenvironment, lymphocytes from healthy donors underwent apoptosis, contrasting with patients' lymphocytes, which showed increased viability when cultured with tumor supernatants. These results underscore the impact of HPV infection on T lymphocyte infiltration, Mo-DC maturation, and lymphocyte survival in PSCC, offering critical insights for advancing our understanding of the tumor microenvironment and guiding the development of immunotherapy strategies.

Systemically delivered adipose stromal vascular fraction mitigates radiation-induced gastrointestinal syndrome by immunomodulating the inflammatory response through a CD11b+ cell-dependent mechanism

BackgroundStromal vascular fraction (SVF) treatment promoted the regeneration of the intestinal epithelium, limiting lethality in a mouse model of radiation-induced gastrointestinal syndrome (GIS). The SVF has a heterogeneous cell composition; the effects between SVF and the host intestinal immunity are still unknown. The specific role of the different cells contained in the SVF needs to be clarified. Monocytes–macrophages have a crucial role in repair and monocyte recruitment and activation are orchestrated by the chemokine receptors CX3CR1 and CCR2.MethodsMice exposed to abdominal radiation (18 Gy) received a single intravenous injection of SVF (2.5 × 106 cells), obtained by enzymatic digestion of inguinal fat tissue, on the day of irradiation. Intestinal immunity and regeneration were evaluated by flow cytometry, RT-PCR and histological analyses.ResultsUsing flow cytometry, we showed that SVF treatment modulated intestinal monocyte differentiation at 7 days post-irradiation by very early increasing the CD11b+Ly6C+CCR2+ population in the intestine ileal mucosa and accelerating the phenotype modification to acquire CX3CR1 in order to finally restore the F4/80+CX3CR1+ macrophage population. In CX3CR1-depleted mice, SVF treatment fails to mature the Ly6C−MCHII+CX3CR1+ population, leading to a macrophage population deficit associated with proinflammatory environment maintenance and defective intestinal repair; this impaired SVF efficiency on survival. Consistent with a CD11b+ being involved in SVF-induced intestinal repair, we showed that SVF-depleted CD11b+ treatment impaired F4/80+CX3CR1+macrophage pool restoration and caused loss of anti-inflammatory properties, abrogating stem cell compartment repair and survival.ConclusionsThese data showed that SVF treatment mitigates the GIS-involving immunomodulatory effect. Cooperation between the monocyte in SVF and the host monocyte defining the therapeutic properties of the SVF is necessary to guarantee the effective action of the SVF on the GIS.

Generation of Connective Tissue-Free Microvascular Fragment Isolates from Subcutaneous Fat Tissue of Obese Mice

BACKGROUND:Microvascular fragment (MVF) isolates are generated by short-term enzymatic digestion of adipose tissue and contain numerous vessel segments for the vascularization of tissue defects. Recent findings indicate that the functionality of these isolates is determined by the quality of the fat source. Therefore, we compared MVF isolates from subcutaneous adipose tissue of obese and lean mice.METHODS:MVF isolates were generated from subcutaneous adipose tissue of donor mice, which received a high fat or control diet for 12 weeks. The isolates were analyzed in vitro and in vivo.RESULTS:Feeding of mice with a high fat diet induced obesity with adipocyte hypertrophy, resulting in a significantly lower collagen fraction and microvessel density within the subcutaneous fat depots when compared to lean controls. Accordingly, MVF isolates from obese mice also contained a reduced number of MVF per mL adipose tissue. However, these MVF tended to be longer and, in contrast to MVF from lean mice, were not contaminated with collagen fibers. Hence, they could be freely seeded onto collagen-glycosaminoglycan scaffolds, whereas MVF from lean controls were trapped in between large amounts of collagen fibers that clogged the pores of the scaffolds. In line with these results, scaffolds seeded with MVF isolates from obese mice exhibited a significantly improved in vivo vascularization after implantation into full-thickness skin defects.CONCLUSION:Subcutaneous adipose tissue from obese mice facilitates the generation of connective tissue-free MVF isolates. Translated to clinical conditions, these findings suggest that particularly obese patients may benefit from MVF-based vascularization strategies.

An enzyme-free method for isolating testicular macrophages from rodent models

Macrophages are the major type of immune cell in the testis of both humans and rodents. Testicular macrophages (TMs) play critical roles in maintaining the testicular microenvironment, such as Leydig cell-dependent hormone production, spermatogenesis, and immune balance. A substantial number of studies have used rodent models to investigate the functions of TMs with various methods and harvest macrophages from the testis. Studies have demonstrated that enzyme digestion, an essential part of these methods, can improve the number and purity of TMs while unavoidably altering the immunoprofile of macrophages, which is detrimental for further study in terms of immune investigation. Here, we modified the existing method of microglia isolation and set up a novel method without the enzyme digestion step to isolate TMs. According to the characteristics of testicular tissue looseness and the physical and biological characteristics of macrophages, by combining mechanical separation, gradient centrifugation, and immuno-magnetic bead selection, we can effectively avoid the enzymatic digestion of testis tissue and maintain the immune characteristics of macrophages. Additionally, we verified the purity of TM with flow cytometry (FC) at approximately 91–95%, and the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was lower than that isolated with enzyme digestion. In contrast to the traditional method, this novel protocol can assist those who have no convenient access to fluorescence-activated cell sorting (FACS) to isolate a sufficient number of TMs and, most importantly, avoid altering the immunoprofile of TMs without enzyme digestion.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

Body fluid-derived stem cells - an untapped stem cell source in genitourinary regeneration.

Somatic stem cells have been obtained from solid organs and tissues, including the bone marrow, placenta, corneal stroma, periosteum, adipose tissue, dental pulp and skeletal muscle. These solid tissue-derived stem cells are often used for tissue repair, disease modelling and new drug development. In the past two decades, stem cells have also been identified in various body fluids, including urine, peripheral blood, umbilical cord blood, amniotic fluid, synovial fluid, breastmilk and menstrual blood. These body fluid-derived stem cells (BFSCs) have stemness properties comparable to those of other adult stem cells and, similarly to tissue-derived stem cells, show cell surface markers, multi-differentiation potential and immunomodulatory effects. However, BFSCs are more easily accessible through non-invasive or minimally invasive approaches than solid tissue-derived stem cells and can be isolated without enzymatic tissue digestion. Additionally, BFSCs have shown good versatility in repairing genitourinary abnormalities in preclinical models through direct differentiation or paracrine mechanisms such as pro-angiogenic, anti-apoptotic, antifibrotic, anti-oxidant and anti-inflammatory effects. However, optimization of protocols is needed to improve the efficacy and safety of BFSC therapy before therapeutic translation.

Gut Microbiota Conditioning Promotes Anti-Tumor Immunity and Responsiveness to Immunological Checkpoint Blockade in Colorectal Cancer

Abstract Background Colorectal cancer (CRC) infiltration by T lymphocytes is associated with prolonged survival. However, immune infiltration spontaneously occurs in <20% of tumors and no therapies able to promote it are currently available. CRC tissue is populated by gut bacteria translocated across the neoplastic epithelium. We previously showed that direct contact between tumor cells and bacteria induces overexpression of genes encoding immune cell-recruiting chemokines in tumor cells, and we identified a panel of bacterial species associated with enhanced chemokine expression, high T cell infiltration and favorable prognosis. Aims In this project we tested the ability of identified bacterial species to boost T-cell infiltration and promote tumor regression in orthotopic CRC experimental models. Methods Luciferase-expressing mismatch repair (MMR)-proficient or -deficient murine CRC cells from established cell lines were injected intra-cecum in syngeneic mice. Tumor growth was monitored by bioluminescence. Bacterial species of interest, defined as Operational Taxonomic Units (OTUs), were cultured under anaerobic conditions, and, following tumor development, were administered to tumor-bearing mice by oral gavage. In specific experiments, bacteria administration was combined with immunological checkpoint blockade (ICB), based on injection of anti-PD-1 antibodies. Immune cell infiltration was evaluated by flow cytometry, following tumor harvesting and enzymatic tissue digestion. Results In MMR-deficient tumors, known to be immunogenic and responsive to immunotherapy, administration of the bacterial species OTU8, was sufficient to reduce tumor growth. In MMR-proficient tumors, scarcely infiltrated by immune cells and poorly responsive to ICB, OTU8, did not per se affect tumor growth, but enhanced infiltration by cytotoxic CD8+ T cells, ultimately leading, in combination with anti-PD-1 antibodies, to tumor growth inhibition. Conclusions We demonstrated that administration of defined bacteria promotes endogenous immune responses against CRC. Gut microbiota conditioning might represent an innovative therapeutic strategy to enhance responsiveness to ICB and to broaden its indication beyond MMR-deficient tumors.

Comparison of Osteogenic Capacity and Osteoinduction of Adipose Tissue-Derived Cell Populations.

Stromal vascular fraction (SVF) is the primary isolate obtained after enzymatic digestion of adipose tissue that contains various cell types. Its successful application for cell-based construct preparation in an intra-operative setting for clinical bone augmentation and regeneration has been previously reported. However, the performance of SVF-based constructs compared with traditional ex vivo expanded adipose tissue-derived mesenchymal stromal cells (ATMSCs) remains unclear and direct comparative analyses are scarce. Consequently, we here aimed at comparing the in vitro osteogenic differentiation capacity of donor-matched SVF versus ATMSCs as well as their osteoinductive capacity. Human adipose tissue from nine different donors was used to isolate SVF, which was further purified via plastic-adherence to obtain donor-matched ATMSCs. Both cell populations were immunophenotypically characterized for mesenchymal stromal cell, endothelial, and hematopoietic markers after isolation and immunocytochemical staining was used to identify different cell types during prolonged cell culture. Based on normalization using plastic-adherence fraction determination, SVF and ATMSCs were seeded and cultured in osteogenic differentiation medium for 28 days. Further, SVF and ATMSCs were seeded onto devitalized bovine bone granules and subcutaneously implanted into nude mice. After 42 days of implantation, granules were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) to assess ectopic bone formation. The ATMSCs were shown to be a homogenous cell population during cell culture, whereas SVF cultures consisted of multiple cell types. All donor-matched comparisons showed either accelerated or stronger mineralization for SVF cultures in vitro. However, neither SVF nor ATMSCs loaded on devitalized bone granules induced ectopic bone formation on subcutaneous implantation, as opposed to control granules loaded with bone morphogenetic protein-2 (BMP-2), which triggered ectopic bone formation with 100% incidence. Despite the observed lack of osteoinduction, our findings provide important in vitro evidence on the osteogenic superiority of intra-operatively available SVF as compared with donor-matched ATMSCs. Consequently, further studies should focus on optimizing the efficacy of these cell populations for implementation in orthotopic bone fracture or defect treatment.

Strategies for optimizing CITE-seq for human islets and other tissues.

Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor-matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

Enzymatic isolation of articular chondrons: is it much different than that of chondrocytes?

In native articular cartilage, chondrocytes are completely capsulated by a pericellular matrix (PCM), together called the chondron. Due to its unique properties (w.r.t. territorial matrix) and importance in mechanotransduction, the PCM and chondron may be important in regenerative strategies. The current gold standard for the isolation of chondrons from cartilage dates from 1997. Although previous research already showed the low cell yield and the heterogeneity of the isolated populations, their compositions and properties have never been thoroughly characterized. This study aimed to compare enzymatic isolation methods for chondrocytes and chondrons and characterize the isolation efficiency and quality of the PCM. Bovine articular cartilage was digested according to the 5-hour gold standard chondron isolation method (0.3% dispase + 0.2% collagenase II), an overnight chondron isolation (0.15% dispase + 0.1% collagenase II), and an overnight chondrocyte isolation (0.15% collagenase II + 0.01% hyaluronidase). Type VI collagen staining, fluorescence-activated cell sorting (FACS) analysis, specific cell sorting and immunohistochemistry were performed using a type VI collagen staining, to study their isolation efficiency and quality of the PCM. These analyses showed a heterogeneous mixture of chondrocytes and chondrons for all three methods. Although the 5-hour chondron isolation resulted in the highest percentage of chondrons, the cell yield was significantly lower compared to the other isolation methods. FACS, based on the type VI collagen staining, successfully sorted the three identified cell populations. To maximize chondron yield and homogeneity, the overnight chondron enzymatic digestion method should be combined with type VI collagen staining and specific cell sorting.

Chondrocyte Isolation and Expansion.

Chondrocyte isolation requires a combination of enzymatic and mechanical digestion of cartilaginous tissues in order to release the chondrocytes. Extracted primary chondrocytes will then adhere to standard tissue culture plastics, typically in small clusters, over a period of a few days in monolayer culture. Chondrocyte populations are expanded in a basal medium containing serum, supplemented with ascorbic acid, antibiotics, and sometimes antifungal agents and growth factors. Here we describe the standard research grade and good manufacturing practice (GMP) protocols used for the isolation and expansion of chondrocytes by the Oswestry/Keele University Orthopaedic Research (OsKOR) group and John Charnley GMP and MHRA licensed laboratory, both based at the RJAH Orthopaedic Hospital, Oswestry, UK.

A Platform of Patient-Derived Microtumors Identifies Individual Treatment Responses and Therapeutic Vulnerabilities in Ovarian Cancer.

Simple SummaryFor personalized oncology, it is crucial to develop appropriate patient-derived tumor models that allow individualized validation of the most effective cancer therapy. The objective of this study was to develop and characterize a new patient-derived ovarian cancer tumor model composed of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TIL). In contrast to other preclinical tumor models, such as patient-derived organoids, PDM are generated within 24 h from fresh ovarian tumor samples. From immunohistochemical comparison with the original primary tumor, we conclude that the histopathological features of the original tumor are essentially preserved. Importantly, we successfully identified treatment-sensitive and treatment-resistant tumor models for standard platinum-based therapy by reverse-phase protein array (RPPA) analysis of PDM. Furthermore, we were able to evaluate the efficacy of cancer immunotherapy by co-culturing PDM and autologous TILs. PDM and TILs may therefore serve as a preclinical platform to identify individualized, tailored cancer treatments in the future.In light of the frequent development of therapeutic resistance in cancer treatment, there is a strong need for personalized model systems representing patient tumor heterogeneity, while enabling parallel drug testing and identification of appropriate treatment responses in individual patients. Using ovarian cancer as a prime example of a heterogeneous tumor disease, we developed a 3D preclinical tumor model comprised of patient-derived microtumors (PDM) and autologous tumor-infiltrating lymphocytes (TILs) to identify individual treatment vulnerabilities and validate chemo-, immuno- and targeted therapy efficacies. Enzymatic digestion of primary ovarian cancer tissue and cultivation in defined serum-free media allowed rapid and efficient recovery of PDM, while preserving histopathological features of corresponding patient tumor tissue. Reverse-phase protein array (RPPA)-analyses of >110 total and phospho-proteins enabled the identification of patient-specific sensitivities to standard, platinum-based therapy and thereby the prediction of potential treatment-responders. Co-cultures of PDM and autologous TILs for individual efficacy testing of immune checkpoint inhibitor treatment demonstrated patient-specific enhancement of cytotoxic TIL activity by this therapeutic approach. Combining protein pathway analysis and drug efficacy testing of PDM enables drug mode-of-action analyses and therapeutic sensitivity prediction within a clinically relevant time frame after surgery. Follow-up studies in larger cohorts are currently under way to further evaluate the applicability of this platform to support clinical decision making.

Primary Trophoblast Cultures: Characterization of HLA Profiles and Immune Cell Interactions

IntroductionTrophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells.MethodsAfter enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT.ResultsThe trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-α and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR.ConclusionWe verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions.

So, you want to create a frog cell line? A guide to establishing frog skin cell lines from tissue explants

Skin is an important interface with the external environment and investigating amphibian skin cell biology will improve our understanding of how environmental factors such as pathogens and pollutants are contributing to global amphibian declines. There is a critical need for in vitro systems to facilitate conservation research in model and non-model amphibians and the creation of new amphibian cell lines will play a significant role in reducing or even replacing the use of live animals for in vivo studies by providing an in vitro alternative. Here, we detail an adapted protocol for the generation of spontaneously arising cell lines from frog skin tissues, without the need for immortalization steps. Expanding the amphibian invitrome will foster and expedite new research in amphibian gene function, cellular responses, host-pathogen interactions, and toxicology. The following customizations to traditional tissue explant generation procedures have facilitated the successful generation of adherent skin epithelial-like cell lines from Xenopus laevis and can be further adapted for use with different frog species, such as Rana sylvatica, and different tissues:•Osmotic adjustment of culture medium and solutions for different amphibian species.•Use of small tissue explants, instead of enzymatic digestion of tissues, and gentle spotting of these tissue explants onto the growth surface of tissue culture flasks to promote better tissue adherence.•Partial replacement of medium to allow accumulation of potential endogenous growth factors in cultures.

Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering.

The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity. Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

Human fibroblast-like synoviocyte isolation matter: a comparison between cell isolation from synovial tissue and synovial fluid from patients with rheumatoid arthritis.

Cell culture technology has become a popular method in the field of cell biology, pharmacology, and medical researches. Primary cells represent the normal physiological condition of human cells. Fibroblasts are the most common native cells of connective tissue that play a crucial role in the entire pathogenesis of various disorders, such as rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), which overlie the loose connective tissue of the synovial sublining, are known to be the central mediators of joint damage. The most routine approach for the isolation of FLS is an enzymatic digestion of synovial tissue. This experimental study is designed to introduce an easy, fast, and high-throughput method compared with enzymatic digestion for isolation of FLS. The synovial tissue and synovial fluid (SF) samples were collected from eight patients with RA who underwent routine knee replacement surgery. Synovial tissue was incubated with collagenase VIII enzyme, while SF was washed with a similar volume of phosphate-buffered saline. The cells were further subcultured and stored based on the standard protocols. The purity of isolated synoviocytes was confirmed using flow cytometry analysis. Isolation of FLS from SF was more successful with a faster rate, 3-5 days after culture. The morphological assessment and flow cytometry analysis confirmed the purity of SF-derived cells in passage 4. SF could be a more accessible source of FLS than synovial tissue. Obtaining primary FLS from SF is a simple, fast, and cost-effective way to have a large-scale cell during a short time.

The impact of stress on the transcriptomic signature of iNKT1 cells

Invariant natural killer T (iNKT) cells develop in thymus before emigrating and settling peripheral tissues and organs. In contrast to regular naïve T cells, most iNKT cells do not continuously recirculate but are rather sessile and can adopt phenotypically as well as functionally to their tissue environment. To explore this in more detail, we focused on the most widely distributed CD4+iNKT1 cells and compared the transcriptome of cells isolated from liver and spleen. Whereas there are only very few genuine differences in the transcriptomes of CD4+iNKT1 cells of these two organs, the mode of cell isolation left clear marks in the transcriptomic signature. In contrast to liver cell isolated in the cold, cells prepared by enzymatic tissue digestion upregulated quickly a series of genes known to respond to stress. Therefore, to avoid erroneous conclusions, a comparison of expression profiles must take into consideration the history of cell preparation.