Isolation Of Primary Cells Research Articles

Simultaneous isolation of primary human hepatocytes and non-parenchymal liver cells for the establishment of functional co-culture and tissue engineering liver models

Simultaneous isolation of primary human hepatocytes and non-parenchymal liver cells for the establishment of functional co-culture and tissue engineering liver models

Isolation, culture and identification of primary human pericardial interstitial cells

Objective To establish a method for isolation,culture and identification of primary human pericardial interstitial cells( PICs). Methods Human pericardial tissues were cut into 1 mm × 1 mm × 1 mm sized pieces,digested with type Ⅱ collagenase,hyaluronic acid enzyme and trypsin to isolate the interstitial cells. The cells were cultured and cell morphology was observed. The cell doubling time, flow cytometry and immunohistochemical staining were applied for cell identification. Results Adherent cells were observed 24 h after culturing. The cells reached 80% confluences and could be subcultured after about 10 days. The cells isolated and cultured were fibroblast-like or spindle-like,with obvious nuclei,clear nucleolus,and a greater proportion of nucleus and cytoplasm. Flow cytometry results showed no cell surface markers CD34 and CD45. Immunocytochemistry showed negative CK staining,and positive vimentin and α-SMA staining. Cells isolated from 8 of the 10 pericardial tissues were successfully cultured. Conclusion The present method can effectively isolate and culture primary human PICs,which lays a foundation for further in vitro study of constrictive pericarditis.

Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells

[Abstract] The intestine, together with skin and blood, belongs to the organs with the highest cell turnover, which makes it a perfect model to study cellular processes, such as proliferation and differentiation. Epithelial cell turnover in intestine is possible due to the presence of intestinal stem cells, which are located at the bottom of the crypt. Here, we recapitulate a detailed protocol for the isolation and culture procedures of primary epithelial intestinal cells in a three dimensional (3D) in vitro system, described for the first time by Hans Clevers group (Sato et al., 2009). This specific 3D culture preserves intestinal stem cells, which give rise to differentiated progeny for example goblet cells. The culture has many applications and represents a useful model to study stem cell biology, epithelial cell regeneration, and transplantation studies. Moreover, the presented 3D culture can be used to investigate the barrier function of intestinal epithelial cells, as well as heterotypic cell interactions between epithelial cells and stromal cells.

Direct reprogramming of porcine fibroblasts to neural progenitor cells

Direct reprogramming of porcine fibroblasts to neural progenitor cells

Isolation and culture of primary human nasal epithelial cells from anesthetized nasal epithelia

Conclusion: Using a local anesthetic agent before obtaining nasal biopsies by nasal brushing makes the sampling procedure smooth, avoids lacrimation, nasal itching/irritation, and/or sneezing and provides enough viable cells to establish primary cultures. Objectives: To examine the use of local anesthesia to avoid the irritation experienced by the subject when nasal biopsies are obtained by nasal brushing in order to culture viable nasal epithelial cells. Methods: Nasal epithelial cells were collected from the mid-part of the inferior turbinate of healthy volunteers by brushing with interdental brushes, after spraying a topical anesthetic on the nasal mucosa. Immunocytochemistry was performed to assess the purity of epithelial cells. Results: Cell samples ranging from 1.16 × 105 to 3.06 × 105 cells/per sample were obtained. Of 11 samples, 7 formed confluent cultures, while the remaining 4 samples showed only patches of epithelial cells. Neither fungal nor bacterial contamination posed a problem. Immunocytochemistry of the cytospin slides confirmed the presence of epithelial cells in the cultures. No adverse effects were experienced by the volunteers.

Characteristics of human infant primary fibroblast cultures from Achilles tendons removed post-mortem

Primary cell cultures were investigated as a tool for molecular diagnostics in a forensic setting. Fibroblast cultures had been established from human Achilles tendon resected at autopsies, from cases of sudden infant death syndrome and control infants who died in traumatic events (n=41). After isolation of primary cultures cells were stored at −135°C, and re-established up to 15 years later for experimental intervention. Growth characteristics in cultures were evaluated in relation to the age of the donor, the post mortem interval before sampling, and the storage interval of cells before entry into the study. High interpersonal variation in growth rates and cell doubling time was seen, but no statistically significant differences were found with increasing age of the donor (mean 19 weeks), length of post-mortem interval prior to sampling (6–100h), or increase in years of storage. Fibroblast cultures established from post-mortem tissue are renewable sources of biological material; they can be the foundation for genetic, metabolic and other functional studies and thus constitute a valuable tool for molecular and pathophysiological investigations in biomedical and forensic sciences.

Harvesting, Isolation, and Functional Assessment of Primary Vagal Ganglia Cells

Airway sensory nerves play an important defensive role in the lungs, being central in mediating protective responses like cough and bronchoconstriction. In some cases, these responses become excessive, hypersensitive, and deleterious. Understanding the normal function of airway nerves and phenotype changes associated with disease will help in developing new therapeutics for treating chronic obstructive pulmonary disease and chronic cough. Guinea pigs, and to a lesser extent ferrets, are commonly employed for studying the cough reflex because they have a cough response similar to humans. While rats and mice do not exhibit a cough response, they do possess sensory nerves that respond to the same range of tussive stimuli as guinea pigs and humans. Described in this unit are protocols for harvesting guinea pig, mouse, and rat sensory nerve cell bodies to assess molecular and functional changes associated with pulmonary disease, and to identify new targets for therapeutic intervention.

73 : Regulation and function of IL-1F11-producing lung resident cells

The lung, essential for providing sufficient oxygen to the host harbors a large mucosal surface that is constantly exposed to respiratory viruses and bacteria. Recognition and rapid clearance of pathogens by the innate immune system provide the first line of defense, however, excessive activation can lead to pathological inflammatory consequences. Although our knowledge of immune maintenance and response in the lung is limited, it is becoming increasingly clear that non-hematopoietic cells including endothelial and epithelial cells exert key functions in regulating innate and adaptive immunity. Interestingly, these cells have been shown to constitute a major source of IL-1F11, a key immune mediator in initiating early lung inflammation and regulating late immune responses to viral challenge. Here, we describe advanced isolation and culture methods of primary murine lung resident cells and detail their qualitative traits upon infection with the respiratory pathogen Influenza A virus (IAV). We further examine IL-1F11 expression and release and its contribution to inflammatory lung pathology and immunity to IAV. Our observations highlight the critical involvement of IL-1F11 in lung immunity to viral infection and identify lung resident cells as central regulators of excessive immunopathology.

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10(6) acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca(2+)-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca(2+)-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca(2+)-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca(2+)-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca(2+)-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Gene Delivery to the Airway

This unit describes generation of and gene transfer to several commonly used airway models. Isolation and transduction of primary airway epithelial cells are first described. Next, the preparation of polarized airway epithelial monolayers is outlined. Transduction of these polarized cells is also described. Methods are presented for generation of tracheal xenografts, as well as both ex vivo and in vivo gene transfer to these xenografts. Finally, a method for in vivo gene delivery to the lungs of rodents is included. Methods for evaluating transgene expression are given in the support protocols.

Effective method for the isolation and proliferation of primary lung cancer cells from patient lung tissues

We have developed a technique for isolating and culturing primary lung cancer cells extracted from patient tissue to facilitate anti-cancer drug development. Patient-derived lung cancer tissues were mechanically dissociated to 40-100 μm. Dispase was then used to isolate cultured lung cancer cell populations, which were re-plated on Matrigel-coated dishes containing N2-supplemented medium and growth factors. This method allows pure populations of primary non-small cell lung cancer cells to be grown in vitro. The isolated cells exhibited hallmark cancerous properties such as abnormal chromosomes and in vivo tumor formation. The cell lines generated through this procedure may help to advance our knowledge of certain forms of lung cancer and may also be useful for developing patient-specific anti-cancer drug screening procedures.

Response to microtubule-interacting agents in primary epithelial ovarian cancer cells

BackgroundOvarian cancer constitutes nearly 4% of all cancers among women and is the leading cause of death from gynecologic malignancies in the Western world. Standard first line adjuvant chemotherapy treatments include Paclitaxel (Taxol) and platinum-based agents. Taxol, epothilone B (EpoB) and discodermolide belong to a family of anti-neoplastic agents that specifically interferes with microtubules and arrests cells in the G2/M phase of the cell cycle. Despite initial success with chemotherapy treatment, many patients relapse due to chemotherapy resistance. In vitro establishment of primary ovarian cancer cells provides a powerful tool for better understanding the mechanisms of ovarian cancer resistance. We describe the generation and characterization of primary ovarian cancer cells derived from ascites fluids of patients with epithelial ovarian cancer.MethodsChemosensitivity of these cell lines to Taxol, EpoB and discodermolide was tested, and cell cycle analysis was compared to that of immortalized ovarian cancer cell lines SKOV3 and Hey. The relationship between drug resistance and αβ-tubulin and p53 status was also investigated.ResultsAll newly generated primary cancer cells were highly sensitive to the drugs. αβ-tubulin mutation was not found in any primary cell lines tested. However, one cell line that harbors p53 mutation at residue 72 (Arg to Pro) exhibits altered cell cycle profile in response to all drug treatments. Immortalized ovarian cancer cells respond differently to EpoB treatment when compared to primary ovarian cancer cells, and p53 polymorphism suggests clinical significance in the anti-tumor response in patients.ConclusionsThe isolation and characterization of primary ovarian cancer cells from ovarian cancer patients’ specimens contribute to further understanding the nature of drug resistance to microtubule interacting agents (MIAs) currently used in clinical settings.

PL.84 Short-Term Culture of Human Ecto-Cervical Epithelial Cells for Genomic, Proteomic and Functional Studies

Background Understanding cell physiology is limited by reliance on tumour-derived immortalised cell lines. Primary cell culture models may offer more relevant mechanistic insight into cell physiology but are often difficult...

Isolation, Purification, and Culture of Primary Murine Microglia Cells

Isolation, Purification, and Culture of Primary Murine Microglia Cells

The Aryl Hydrocarbon Receptor (AhR) Regulates the Production of Bipotential Hematopoietic Progenitor Cells

Abstract 766The evolutionarily conserved aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix Per-ARNT-Sim family, has been studied for its role in environmental chemical-induced toxicity. Recent studies now demonstrate that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. To better understand the possible role of the AhR in hematopoiesis, we developed a novel, human, induced pluripotent stem cell (iPSC) platform based on the step-wise directed differentiation of hematopoietic progenitor cells. Our in silico analysis of transcriptional profiles of 71 primary human hematopoietic cell isolates indicated AhR-upregulation at both the hematopoietic stem cell and bi-potential megakaryocyte-erythroid progenitor (MEP) stages. This result, together with the absence of an in vitro model system enabling production of large numbers of primary human MEPs capable of differentiating into megakaryocytes (Mks) and erythroid lineage cells, motivated us to determine if AhR modulation could facilitate both MEP expansion and Mk and erythroid cell differentiation.Our results indicate that AhR has a physiological and functional role in normal hematopoietic development, and that modulation of the receptor in bi-potential hematopoietic progenitors can direct cell fate. We demonstrate a novel methodology for the directed differentiation of pluripotent stem cells in chemically defined, serum and feeder-free culture conditions into MEPs capable of final specification into Mks and/or erythroid-lineage cells. The use of a non-toxic aryl hydrocarbon receptor agonist in our directed differentiation scheme significantly increases the number of MEPs and resultant cells. Following the addition of potent AhR ligands to our cultures, we observed exponential expansion of MEPs from fifteen thousand to one billion cells in two weeks, with the role of AhR in the MEP population confirmed using a specific AhR inhibitor. This logarithmic expansion of cells appears to be a function of decreased cell death and is consistent with previous studies which suggest that the AhR can control apoptosis. In addition to allowing for the exponential expansion of MEPs, our results demonstrate that modulation of AhR can direct cell fate, with AhR agonism permissive to erythroid differentiation and antagonism favoring Mk specification. Although erythropoietin (EPO) and thrombopoietin (TPO) stimulate RBC and Mk production respectively, AhR may play a cytokine-independent role in the specification of these lineages and warrants further study.These results demonstrate a new platform for studying human red blood cell and Mk development that allows for exponentially greater production of RBCs and Mks in comparison to existing methodologies. This strategy relies on the first of its kind definition of the role of the AhR receptor in normal hematopoietic development using specialized ligands in hematopoietic progenitor cells. A useful outcome for this work will be the utilization of this in vitro platform for the clinically relevant production of blood products. An iPSC-based system, such as the one described here in which sufficient numbers of cells can be produced, should facilitate future clinical adaptation involving the transfusion of iPSC-derived red blood cells and platelets without problems related to immunogenicity, contamination, or supply. Disclosures:No relevant conflicts of interest to declare.

Nature versus nurture: does human skin maintain its stratum corneum lipid properties in vitro?

Human skin equivalents (HSEs) mimic human skin closely, but show differences in their stratum corneum (SC) lipid properties. The aim of this study was to determine whether isolation of primary cells, which is needed to generate HSEs, influence the SC lipid properties of HSEs. For this purpose, we expanded explants of intact full thickness human skin and isolated epidermal sheets in vitro. We investigated whether their outgrowths maintain barrier properties of human skin. The results reveal that the outgrowths and human skin have a similar morphology and expression of several differentiation markers, except for an increased expression of keratin 16 and involucrin. The outgrowths show a decreased SC fatty acid content compared with human skin. Additionally, SC lipids of the outgrowths have a predominantly hexagonal packing, whereas human skin has the dense orthorhombic packing. Furthermore, the outgrowths have lipid lamellae with a slightly reduced periodicity compared with human skin. These results demonstrate that the outgrowths do not maintain all properties observed in human skin, indicating that changes in properties of HSEs are not caused by isolation of primary cells, but by culture conditions.

Development and Characterization of a Clinically Compliant Xeno-Free Culture Medium in Good Manufacturing Practice for Human Multipotent Mesenchymal Stem Cells

Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.

Isolation and characterization of primary skeletal muscle satellite cells from rats

The purpose of this study was to isolate and characterize skeletal muscle satellite cells from rats using tissue block culture method. Specific Pathogen Free (SPF) level Sprague-Dawley (SD) rats were used to isolate skeletal muscle satellite cells. Morphology, expression and distribution of α-actin and Desmin within the cytoplasm of skeletal muscle satellite cells were compared with those of C2C12 myoblasts. The results showed that tissue block culturing method achieved robust proliferation and excellent differentiation of skeletal muscle satellite cells. Immunofluorescence and immunohistochemistry results showed that α-actin and Desmin proteins were expressed in the cytoplasm of both skeletal muscle satellite cells and myoblasts. We concluded that tissue block culturing method can obtain highly purified skeletal muscle satellite cells with robust proliferation and excellent differentiation capabilities.