primary cell culture

Glioblastoma multiforme (GBM) is incurable with routine treatments. Ascorbic acid (Asc) has antioxidant and anti-cancer properties. However, its specific anti-cancer mechanisms are only partially understood. In this study, the effect of Asc on the c-Myc, HIF-1α, and lnc-SNHG16 genes in GBM cells and their exosomes was investigated. Cells isolated from the tissue were characterized by the immunocytochemistry method (GFAP+). The cell-doubling time was determined, and FBS-free medium supplemented with Asc (5 mM) was added to the cells. The extracted exosomes in the cell culture medium were scanned by electron microscopy, Zetasizer, and BCA assay. The expression of lnc-SNHG16 in the exosomes and c-Myc and HIF-1α in the treated and control cells was evaluated by real-time PCR. The interactions between Asc and the c-Myc and HIF-1α proteins were studied using the molecular docking method. The cells showed 90–100% GFAP+ in passage 4, with a cell-doubling time of 4.8 days. Exosomal vesicles measuring 98.25–105.9 were observed. Zetasizer results showed a sharp pick at 90 nm. Protein quantitation showed 3.812 µg/ml protein in the exosomes. Lnc-SNHG16 expression was reduced (P = 0.041), and c-Myc was upregulated (P = 0.002). The expression of HIF-1α was not significant in the treated cells. Also, Asc was able to interact and affect c-Myc and HIF-1α. Asc exerts its effect by reducing lnc-SNHG16 expression in exosomes, upregulating c-Myc in GBM cells, and interacting with HIF-1α and c-Myc. Further research is necessary to achieve a full understanding of these findings.

Mesenchymal stem cells (MSCs) with their spindle like shapes are a lineage of stem cells with the capacity to self-renew and differentiate into osteoblasts, adipocytes, and chondrocytes and with CD105, CD73, and CD90 expression and the lack of CD34, CD14, CD45, and HLA-DR expression. The immunomodulatory, angiogenic, antiapoptotic, antimicrobial, and antioxidative characteristics of these cells made them more attractive in the field of cell-therapy for several autoimmune and inflammatory diseases, including diabetes, neurological disorders, sepsis, cardiac ischemia, and GvHD. For this reason, various protocols have been proposed to isolate mesenchymal stem cells from different tissue sources, such as adipose tissue (AT), umbilical cord (UC), Wharton’s jelly (WJ), bone marrow (BM), dental pulp, and even menstrual fluid. Considering the ease of access to the umbilical cord tissue and the fact that this tissue is rich in MSCs with embryonic origin and higher proliferation rate and lower senescence of the cells, the umbilical cord became a suitable source for explant MSC culture. In this study, we decided to introduce an explant culture protocol of MSCs that is less expensive and cost-effective achieving a high yield of MSCs.

Keratocytes are the major components of the human corneal stromal cell. Cell therapy by keratocytes can be used in some corneal diseases. Because keratocytes are mitotically quiescent; therefore, the cultivation of these cells is associated with challenges. The present study aimed to isolate, culture, and validate keratocyte cells from discarded corneal tissue based on optimizing some cultivation conditions. In this experimental study, keratocytes were isolated from discarded corneal tissue. Different culture medium composition such as amniotic membrane extract, time, and the role of coating scaffolds was evaluated. Real-time PCR of specific genes were used to confirm the primary keratocyte cells compared to corneal epithelial cells. The specific genes were keratocan, lumicane, aldehyde dehydrogenase three members of family A1 (ALDH3A1), and CD34. In addition, immunocytochemistry (ICC) was used to confirm the expression of specific keratocan and lumican markers. Keratocytes was isolated and cultured in the culture medium containing amniotic membrane extract. Based on analyses, keratocan, lumicane, ALDH3A1, and CD34 gene expression in keratocytes was significantly higher than in the epithelial cells. Moreover, keratocan and lumican expression was detected in 92.5% and 91.1% of the cells, respectively. According to the results, the addition of amniotic membrane extract significantly increased the growth of keratocytes. Our findings in this study showed that discarded corneal tissue can be used as a suitable source for obtaining keratocyte cells needed in corneal tissue engineering.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

Cell culture is an important technique in cellular and molecular biology. There are two basic systems for culturing cells, adherent (monolayer) culture and suspension culture. In adherent cell culture, cellular adhesion molecules play a vital role in many physiological processes. Many cells, especially primary-obtained cells, hardly attach to plates, so coating agents are used for cell attachment and growth. There is no striking report on whether all cells need coated plates.

We investigated whether a coating plate is essential for primary spinal cord-obtained neuron culture.

Cells were separated and seeded in tissue culture plates coated with either Poly(L-lysine) or Poly(D-lysine), as well as without coating containing DMEM-F12 media with 10% FBS.

Interestingly, we found that neuron cells more potently attached to coating free plates.

These results provide strong evidence helping the researchers to optimize primary spinal cord cultures.

Oral Squamous Cell Carcinoma (OSCC) is the most frequent oral cancer worldwide. It is known as the eighth most common cancer in men and as the fifth most common cancer in women. Cytogenetic and biochemical studies in recent decades have emphasized the necessity of providing an appropriate tool for such researches. Cancer cell culture is a useful tool for investigations on biochemical, genetic, molecular and immunological characteristics of different cancers, including oral cancer. Here, we explain the establishment process of five primary oral cancer cells derived from an Iranian population.
The specimens were obtained from five oral cancer patients. Enzymatic, explant culture and magnetic-activated cell sorting (MACS) methods were used for cell isolation. After quality control tests, characterization and authentication of primary oral cancer cells were performed by short tandem repeats (STR) profiling, chromosome analysis, species identification, and monitoring the growth, morphology and the expression of CD326 and CD133 markers.
Five primary oral cancer cells were established from an Iranian population. The flow cytometry results showed that the isolated cells were positive for CD326 and CD133 markers. Furthermore, the cells were free from mycoplasma, bacterial and fungal contamination. No misidentified or cross-contaminated cells were detected by STR analysis.
Human primary oral cancer cells provide an extremely useful platform for studying carcinogenesis pathways of oral cancer in Iranian population. They may be helpful in explaining the ethnic differences in cancer biology and the individuality in anticancer drug response in future studies.

Glutamate is a major excitatory neurotransmitter in mammalian central nervous system. Excessive glutamate releasing overactivates its receptors and changes calcium homeostasis that in turn leads to a cascade of intracellular events causing neuronal degeneration. In current study, we used neural stem cells conditioned medium (NSCs-CM) to investigate its neuroprotective effects on glutamate-treated primary cortical neurons. Embryonic rat primary cortical cultures were exposed to different concentrations of glutamate for 1 hour and then they incubated with NSCs-CM. Subsequently, the amount of cell survival in different glutamate excitotoxic groups were measured after 24 h of incubation by trypan blue exclusion assay and MTT assay. Hoechst and propidium iodide were used for determining apoptotic and necrotic cell death pathways proportion and then the effect of NSCs-CM was investigated on this proportion. NSCs conditioned medium increased viability rate of the primary cortical neurons after glutamate-induced excitotoxicity. Also we found that NSCs-CM provides its neuroprotective effects mainly by decreasing apoptotic cell death rate rather than necrotic cell death rate. The current study shows that adult neural stem cells could exert paracrine neuroprotective effects on cortical neurons following a glutamate neurotoxic insult.

S100ß a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100ß. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100β in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day''s embryos. Cell cultures were exposed to lithium (1mM) or vehicle for 1day (acute) or 7 days (chronic). RT-PCR and ELISA determined S100β mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100β in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05). Acute and chronic lithium treatments exerted no significant effects on intracellular S100β protein levels in astrocytes, and extracellular S100β protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100β mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100ß protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100ß level, with no effect on astrocytes or interaction between neurons and astrocytes.

Current anti-cancer drug therapy results in systemic side effects due to non-specific uptake by normal healthy noncancerous tissues. To alleviate this difficulty, many attempts have been devoted to the development of new delivery systems such as polymeric Nanoparticles (NPs). In this study, we prepared ICD-85 NPs based on sodium alginate and analyzed the cytotoxic activity of ICD-85 NPs relative to free ICD-85 on primary lamb kidney cells. ICD-85 loaded sodium alginate nanoparticles were prepared by ionic gelation method and were characterized by the particle size, size distribution and Fourier Transform Infrared (FT-IR) spectroscopy. The in vitro cytotoxicity was evaluated by MTT assay and membrane integrity was evaluated by measuring Lactate Dehydrogenase (LDH) activity. The morphological alterations of untreated and treated cells were assessed by light inverted microscope. MTT assay showed that ICD-85 NPs could significantly decrease the in vitro cytotoxicity on primary lamb kidney cells compared to the free ICD-85. The IC10 value at 72 hours was increased from 9±2.7 μg/ml for free ICD-85 to 52±4.3 μg/ml for ICD-85 NPs. LDH assay demonstrated that free ICD-85 had dose-dependent cytotoxicity on primary lamb kidney cells while ICD-85 NPs exhibited significantly decreased cytotoxicity at equivalent concentrations. Moreover, morphological analysis showed no significant difference between control and treated cells with ICD-85 NPs. Based on the results obtained in the present study it can be concluded that encapsulation of ICD-85 with sodium alginate nanoparticles can reduce its necrotic effect on primary lamb kidney cells.

Antibiotic supplements are regularly used in neuronal culture media to control contamination; however، they can interfere with the neuronal excitability and affect electrophysiological properties. Therefore، in this study، the effect of penicillin/streptomycin supplements on the spontaneous electrophysiological activity of hippocampal pyramidal neurons was examined. Electrophysiological whole-cell patch-clamp recordings from rat hippocampal pyramidal cells in primary culture were performed to investigate the effects of antibiotic supplements on the intrinsic excitability of cultured cells. The present findings indicated that presence of antibiotic supplements (penicillin/streptomycin) in the culture medium altered the intrinsic electrical activity of hippocampal pyramidal neurons in primary culture. These alterations included: 1) depolarized resting membrane potential; 2) a significant enhancement in the after-hyperpolarization amplitude; 3) a significant increase in the area under the action potential and in the decay and rise time of the action potential; 4) a significant broadening of action potential and 5) a significant reduction in the firing frequency. These findings suggest that addition of antibiotic supplements to culture media influences the neuronal excitability and alters the electrophysiological properties of cultured neurons، possibly through changing the ionic conductance underlying neuronal excitability.