Establishment Of Primary Cell Cultures Research Articles

Establishment of Primary Cell Cultures from Canine Oral Melanomas via Fine-Needle Aspiration: A Novel Tool for Tumorigenesis and Cancer Progression Studies.

Oral melanomas are the most common oral malignancies in dogs and are characterized by an aggressive nature, invasiveness, and poor prognosis. With biological and genetic similarities to human oral melanomas, they serve as a valuable spontaneous comparative model. Primary cell cultures are widely used in human medicine and, more recently, in veterinary medicine to study tumorigenesis, cancer progression, and innovative therapeutic approaches. This study aims to establish two- and three-dimensional primary cell lines from oral canine melanomas using fine-needle aspiration as a minimally invasive sampling method. For this study, samples were collected from six dogs, represented by four primary oral melanomas and five lymph nodal metastases. The cells were digested to obtain single-cell suspensions, seeded in flasks, or processed with Matrigel® to form organoids. The cell cultures were characterized through flow cytometry using antibodies against Melan-A, PNL2, and Sox-10. This technique offers a minimally invasive means to obtain cell samples, particularly beneficial for patients that are ineligible for surgical procedures, and enables the establishment of in vitro models crucial for comparative studies in mucosal melanoma oncology. To the best of our knowledge, this is the first work establishing neoplastic primary cell cultures via fine-needle aspiration in dogs.

A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature

Studying disease-related changes in the brain vasculature is warranted due to its crucial role in supplying oxygen and nutrients and removing waste and due to the anticipated vascular dysfunction in brain diseases. To this end, we have developed a protocol for fast and simple isolation of brain vascular fragments without the use of transgenic reporters. We used it to isolate and analyze 22,515 cells by single-cell RNA sequencing. The cells distributed into 23 distinct clusters corresponding to all known vascular and perivascular cell types in the brain. Western blot analysis also suggested that the protocol is suitable for proteomic analysis. We further adapted it for the establishment of primary cell cultures. The protocol generated highly reproducible results. In conclusion, we have developed a simple and robust brain vascular isolation protocol suitable for different experimental modalities, such as single-cell analyses, western blotting, and primary cell culture.

The establishment of primary cell culture from canine mammary gland tumor

BACKGROUND: In dogs, an insufficient variety of cell lines commercially available or difficulties in obtaining the existing cell lines developed from various studies results in a limited number of cytotoxicity and related molecular studies integrated with clinical practice. Hence, the doses of many drugs or supportive treatments used in canine tumor cases are adjusted based on studies in humans. OBJECTIVE: A cell line was established from a benign mixed tumor of the canine mammary gland. METHODS: Following surgical removal of the tumor, mechanical dissociation, and PBS washing, a culture process of the tumor cells was performed, including the passaging, freezing, and thawing stages. After several passages, the morphological characteristics of the cells at the logarithmic growth phase were observed under a phase-contrast microscope. RESULTS: The microscopy of the cells cultured on plastic dishes revealed monolayer colonies. The average passage time, which was 5–6 days in the first three passages, decreased to 2–3 days after the third passage. Microscopic examination of tumor cells revealed an adherent, stellated, and spindle-shaped structure. CONCLUSIONS: No difference was observed in the viability and morphology of the cells thawed even after the long period of freezing (∼18 months). The different canine cell lines can provide promising molecular applications that can be adapted into practical clinics in veterinary science.

Establishment of primary cell culture of Ruditapes decussatus haemocytes for metal toxicity assessment.

In ecotoxicology, in vitro testing on cell cultures represents an ideal alternative to in vivo strategies for emerging contaminants. These tests have limited use particularly with marine invertebrates like the clams Ruditapes decussatus. In the present study, a primary culture of R. decussatus haemocytes was realized for the first time in order to determine the effect of metals (copper, zinc, and cobalt) on haemocyte parameters like viability and phagocytosis. Results showed that (i) among the studied medium, the modified Leibovitz (L-15) is the best for R. decussatus haemocytes primary culture. (ii) The primary culture system used here represents a suitable in vitro model for assessing cytotoxic responses, (iii) a decrease of cell viability and phagocytosis after 24 h exposure to 100 μg mL-1 CoSO4 and an increase of phagocytosis after 24 h exposure to 50 μg mL-1CuSO4.

Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology.

BackgroundEpendymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs.ResultsWe elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth.ConclusionsWe demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs.MethodsIn this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

Establishment of Primary Cell Culture From Ascitic Fluid and Solid Tumor Obtained From Epithelial Ovarian Carcinoma Patients.

Ovarian cancer is the seventh leading cause of cancer death worldwide. This is mainly due to late diagnosis and high rate of relapse and resistance following chemotherapy. In the present study, we describe simple and cost-effective method to establish primary culture from ascitic fluid and solid tumor obtained from epithelial ovarian carcinoma patient, which may provide a better tool for in vitro testing of drug sensitivity and designing individualized treatment protocol. Complete Dulbecco modified Eagle medium (DMEM) was prepared by supplementing DMEM with 10% fetal bovine serum and antibiotics (ciprofloxacin and amphotericin B). Establishment of primary culture of ovarian cancer cells from ascites fluid and solid tumor was done by using complete DMEM media. Primary cultures of ovarian cancer cells were established from ascitic fluid and solid tumor tissue. Of the 7 ascitic fluid samples, we were able to establish 5 primary cultures of ovarian cancer cells. All the 7 samples were diagnosed as serous papillary adenocarcinoma. Some fibroblasts were also attached to culture flask on day 4; they were removed by exposing them to trypsin for a brief period. On day 7, grape-like clusters were visualized under inverted microscope. The cells became confluent on the 10th and 11th day and showed cobblestone appearance, which is a hallmark of ovarian cancer cells. Senescent irregularly shaped cells that have ceased dividing were seen after 8 to 10 passages. This study highlights the fact that establishing primary cultures from ascitic fluid or solid tumor tissue may help us to understand the molecular profile of the cancer cells, which allow us to select the best chemotherapeutic agent for ovarian cancer patients and thus take a step toward patient-tailored therapy so that patients are not exposed to drugs to which they are not likely to respond.

Integration of genomic, transcriptomic and functional profiles of aggressive osteosarcomas across multiple species

In complex, highly unstable genomes such as in osteosarcoma, targeting aberrant checkpoint processes (metabolic, cell cycle or immune) may prove more successful than targeting specific kinase or growth factor signaling pathways. Here, we establish a comparative oncology approach characterizing the most lethal osteosarcomas identified in a biorepository of tumors from three different species: human, mouse and canine. We describe the development of a genetically-engineered mouse model of osteosarcoma, establishment of primary cell cultures from fatal human tumors, and a biorepository of osteosarcoma surgical specimens from pet dogs. We analyzed the DNA mutations, differential RNA expression and in vitro drug sensitivity from two phenotypically-distinct cohorts: tumors with a highly aggressive biology resulting in death from rapidly progressive, refractory metastatic disease, and tumors with a non-aggressive, curable phenotype. We identified ARK5 (AMPK-Related Protein Kinase 5, also referred to as NUAK Family Kinase 1) as a novel metabolic target present in all species, and independent analyses confirmed glucose metabolism as the most significantly aberrant cellular signaling pathway in a model system for highly metastatic tumors. Pathway integration analysis identified Polo Like Kinase 1 (PLK1)-mediated checkpoint adaptation as critical to the survival of a distinctly aggressive osteosarcoma. The tumor-associated macrophage cytokine CCL18 (C-C Motif Chemokine Ligand 18) was significantly over-expressed in aggressive human osteosarcomas, and a clustering of mutations in the BAGE (B Melanoma Antigen) tumor antigen gene family was found. The theme of these features of high risk osteosarcoma is checkpoint adaptations, which may prove both prognostic and targetable.

Establishment of a primary culture of polymorphous low grade adenocarcinoma cells

ObjectiveThe aim of the present study was to establish a primary cell culture derived from polymorphous low grade adenocarcinoma (PLGA). DesignThe neoplastic cells were derived from a 57-year-old female patient diagnosed with PLGA. A fragment of the tumor was collected and submitted to enzymatic digestion followed by centrifugation on a Percoll gradient. The cell population was characterized by means of immunofluorescence and detection of PRKD1 gene mutations. ResultsEpifluorescence analysis of the primary culture revealed that the malignant epithelial cells were predominantly polygonal in shape and positive for cytokeratin 7, vimentin, and S100. The doubling time of the cell culture was 86.73h. The restriction digestion assay showed that the neoplastic cells possess PRKD1 gene mutations. ConclusionThe establishment of primary cell culture derived from PLGA should be considered a useful tool for molecular analysis of this salivary gland tumor.

Assessment of Radiobiological α/β Ratio in Lung Cancer and Fibroblast Cell Lines Using Viability Assays.

Altered fractionation is an area of intense clinical research in radiation oncology. Estimation of the α/β ratio of individual carcinomas after establishment of primary cell cultures from tumor biopsies may prove of importance in the individualization of radiotherapy schemes. Here we proposed a simple method to estimate the α/β ratio in cultured cell lines (two lung carcinomas: A549 and H1299; one lung fibroblast cell line: MRC5), using viability assays. For the A549 cell line, the α/β ratio ranged from 14-25 Gy, for H1299 from 11-43 Gy and for the MRC5 fibroblast cell line this was far lower, ranging from 0.69 to 6 Gy. The α/β ratio decreased when extracted from comparisons of lower dose per fraction schemes. The α/β ratio of a cell line can be easily defined after simple viability/dose fractionation experiments.

Evaluation of Various Tissues of the Caerulean Damsel, Pomacentrus caeruleus for Initiating In Vitro Cell Culture Systems

Explantation and trypsinisation methods for tissue dissociation were attempted for the establishment of primary cell cultures from the caerulean damsel, Pomacentrus caeruleus. Among the tissues taken, fin, liver and caudal peduncle showed good attachment with emergence of cells. The cells were best suited to grow in Leibovitz’s L-15 basal medium supplemented with foetal bovine serum (initially 20 % which was later reduced to 5–10 % during subsequent passages) at an ambient temperature of 28 ± 2 °C and pH 7.2 ± 0.2. These cultures persisted at temperatures from 17 to 32 °C, and proliferated at temperatures from 24 to 30 °C. The cells have been cryopreserved successfully with a survival rate of 80 %. Results suggest that fin, caudal peduncle and liver cell cultures have potential for development into cell lines.

Method of isolation and characterization of Girardia tigrina stem cells.

Tissue regeneration is widely studied due to its importance for understanding the biology of stem cells, aiming at their application in medicine for therapeutic and various other purposes. The establishment of experimental models is necessary, as certain invertebrates and vertebrates have different regeneration abilities depending on their taxon position on the evolutionary scale. Planarians are an efficacious in vivo model for stem cell biology, but the correlation between planarian cellular and molecular neoblast pluripotency mechanisms and those of mammalian stem cells is unknown. The present study had the following objectives: i) Establish Girardia tigrina cell culture, ii) determine the time required for complete cell disintegration and iii) obtain neoblasts by cell subdivision. Twenty-four specimens were deprived of food for seven days. After this time, disintegration was performed by incubation protected at three temperatures for 48 h in an antibiotic, antimycotic and trypsin solution, after which the suspension was homogenized and centrifuged. Histopaque® 1077 was used for cell separation and interphases were collected and monitored by optical and fluorescence microscopy. Optical microscopy analysis informed the nucleus-to-cytoplasm ratio, cell morphology and cell size. Under fluorescence microscopy, interphase 1 (I1) was subdivided into two groups and neoblasts were marked for characterization; one group was stained with 4',6-diamidino-2-phenylindole and the other was immunolabeled with octamer-binding transcription factor 4 (OCT4) and isolated and observed after 10 days of cultivation. Neoblasts predominated in I1 with a small amount of other cell types. In conclusion, sample disintegration with a trypsin and antibiotic solution was effective at 18˚C and Iscove's modified Dulbecco's medium supplemented with fetal bovine serum was adequate for the establishment of primary cell cultures after 48-h incubation and centrifugation. Antibody anti-OCT4 was used for the characterization of stem cells and was successfully labeled with concentrated neoblasts on interphase 1.

Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5μm diameter). Molecular analyses conducted on primary cultures, maintained for 2weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

Isolation of a novel herpesvirus from a Pacific white-sided dolphin

During establishment of primary cell culture from the kidney of a dead Pacific white-sided dolphin (Lagenorhynchus obliquidens), a cytopathic effect was observed. Polymerase chain reaction with a set of herpesvirus consensus primers yielded a fragment of the expected size. Nucleotide sequencing of the product indicated that the isolated virus was closely related to an alphaherpesvirus detected in a bottlenose dolphin in the United States, but the sequence identity at the protein level was low (86.6 %). Phylogenetic analysis of the encoded sequence confirmed that the new isolate belonged to the subfamily Alphaherpesvirinae and clustered together with other cetacean alphaherpesviruses. The complete gene encoding glycoprotein B (2,757 bp) was amplified from the novel isolate; the encoded protein was compared with the corresponding protein of other herpesviruses, revealing that this virus belongs to the genus Varicellovirus. Taken together, these results suggest that this virus corresponds to a novel herpesvirus capable of infecting Pacific white-sided dolphins.

Cryopreservation of cartilage cell and tissue for biobanking

Preservation of cell and tissue samples from endangered species is a part of biodiversity conservation strategy. Therefore, setting up proper cell and tissue cryopreservation methods is very important as these tissue samples and cells could be used to reintroduce the lost genes into the breeding pool by nuclear transfer. In this study, we investigated the effect of vitrification and slow freezing on cartilage cell and tissue viability for biobanking. Firstly, primary adult cartilage cells (ACCs) and fetal cartilage cells (FCC) were cryopreserved by vitrification and slow freezing. Cells were vitrified after a two-step equilibration in a solution composed of ethylene glycol (EG), Ficoll and sucrose. For slow freezing three different cooling rates (0.5, 1 and 2°C/min) were tested in straws. Secondly, the tissues taken from articular cartilage were cryopreserved by vitrification and slow freezing (1°C/min). The results revealed no significant difference between the viability ratios, proliferative activity and GAG synthesis of cartilage cells which were cryopreserved by using vitrification or slow freezing methods. Despite the significant decrease in the viability ratio of freeze–thawed cartilage tissues, cryopreservation did not prevent the establishment of primary cell cultures from cartilage tissues. The results revealed that the vitrification method could be recommended to cryopreserve cartilage tissue and cells from bovine to be used as alternative cell donor sources in nuclear transfer studies for biobanking as a part of biodiversity conservation strategy. Moreover, cartilage cell suspensions were successfully cryopreserved in straws by using a controlled-rate freezing machine in the present study.

Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues

The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.

Cardosins improve neuronal regeneration after cell disruption: a comparative expression study

The establishment of primary cell cultures is invaluable for studying cell and molecular biological questions. Although primary cell cultures more closely resemble and function like in the native environment, during the culture establishment the cells undergo several changes including the damage sustained during their removal from original tissue. The resultant cells have to rebalance the expression of their processing molecules to ascertain matrix signalling that ensure cell adaptation and consequent proliferation. Hence, we used cardosin, a novel plant enzyme for tissue disaggregation, for isolating and culturing neuronal cells from embryonic rats. The present investigation reports the molecular events, mainly related with matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) expression, which could substantiate the superior neurite outgrowth and dendritic extension previously described. It was observed that 24 h after primary culture establishment, MMP-2 and MMP-9 messenger RNA (mRNA) are significantly upregulated, while the expression of TIMP-1 and TIMP-2 is unaltered. Regarding the role of laminin in neuronal pathfinding, it was found that the use of anti-laminin antibody and arginine-glycine-aspartate (RGD) peptide exerted inhibitory effects on neurite outgrowth after mechanical lesion where the expression of MMP-9 and TIMP-1 is upregulated under non-permissive conditions in response to mechanical injury.

Establishment of primary cell cultures from fish calcified tissues

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case.

Establishment of Primary Cell Culture from Hepatopancreas of Penaeus monodon for the Study of White Spot Syndrome Virus (WSSV)

A primary cell culture system was developed from the cells of hepatopancrea s of Penaeus monodon. Minced tissues of hepatopancrea s were seeded (10 6 cells·cm 3 ) and incubated at 28 o C in single strength Leibovitz’s L-15 medium (pH 7.2) supplemented with 10% fetal bovine serum and antibiotics (ciprofloxicin 100 mg·ml; penicillin 100 IU·ml; streptomycin 100 mg·ml and nystatin 25 mg·ml). The cells showed good attachment to the culture vessels by 24h and formed a confluent monolayer within 72 h. The primary cell culture could be maintained up to a maximum period of 12 weeks by subculturing seven times. When whitespot syndrome virus (WSSV) was inoculated in the hepatopancreas cell culture, characteristic cytopathic effects which include rounding of cells, detachment, lysis of cell sheet with circular clear areas of depletion of cells were observed within 120 h.

Bioengineering organs using small intestinal submucosa scaffolds: in vivo tissue-engineering technology.

Tissue engineering is a promising field of research that has the potential to revolutionize urinary bladder reconstruction. Currently, two techniques for the induction of bladder regeneration are being researched. The first, the in vivo technique, involves the use of a biodegradable scaffold that the host bladder can use to remodel and regenerate. This technique takes advantage of the cell's natural ability to heal or regenerate itself back to a normal state. The second technology, the in vitro technique, involves establishment of primary cell cultures from the host's native bladder. These cells are seeded on a biodegradable scaffold to create a composite graft that is then transplanted back into the host for continuation of the regeneration process. Clearly, both techniques have advantages and disadvantages, and both will have some role in future urinary reconstruction. To date, the most successful results utilizing in vivo techniques have been with small intestinal submucosa (SIS). In this article, we discuss in vivo tissue engineering technology and the preclinical studies that have been performed utilizing SIS for urinary tract regeneration.

Establishment of primary cell culture from stria vascularis explants. Morphological and functional characterization.

To provide the prerequisite for long-term study of the inner ear related to structural and functional integrity, tissue of stria vascularis with spiral ligament was isolated from Wistar rat cochleas and cultured using the explant-culture technique. The following culture media were used: EMEM with Hepes buffer, hydrocortisone (400 ng/ml), transferrin (5 micrograms/ml). triiodothyronine (10(-9) M), cholera toxin (10(-10) M), insulin (5 micrograms/ml), and epidermal growth factor (10 ng/ml). To characterize the cells growing out from the explant, immunofluorescence with cytokeratin (cytokeratin 18) and ultrastructural examination with SEM and TEM were performed. The marginal cell function was investigated by expression of Na+, K(+)-ATPase antisera against beta 2 subunit of rat Na+, K(+)-ATPase and P-NPPase. We were able to maintain the cultured cells for 3 weeks or more. Monolayered marginal cells were observed beyond 14 days in vitro and the expression of cytokeratin 18 was especially enhanced. The cultured marginal cells were almost identical to in vivo cells both as regards ultrastructural features and Na+, K(+)-ATPase activity. The present results suggest that the primary explant culture technique is a reliable in vitro model of strial marginal cells. However, establishment of the cell line is needed for long-term study.