Background: Culture of cells and tissues is a standard research method practiced in many laboratories. In most of the cases, these cultures are being used as substrates for cell products or as investigative tools for delving the mechanism of gene expression, cell proliferation and transformation. Primary monolayer cell culture has been beneficial to study the general biology of both oral and skin keratinocytes. There are two different techniques of primary cell cultures followed, which include direct explant and enzymatic techniques. Aims: The aim of the study was to optimize the culture of keratinocytes obtained from patients with normal oral mucosa by direct explant technique. Materials and Methods: Keratinocytes were isolated from 15 patients and were cultured in vitro and observed under an inverted microscope. The cultured cells were characterized by immunocytochemistry method using pan-cytokeratin. Results: The total success rate of primary culture of the oral epithelial cells by direct explant technique was 88.6%. No contamination of microorganisms in the primary cell cultures was obtained. Conclusion: Within the limited numbers of samples used in the current pilot study, we conclude that the direct explant technique appears to be a simple and successful technique for the isolation of oral mucosal keratinocytes if we follow the appropriate protocol.

Exosomes are nano-sized particles which belong to the family of extracellular vesicles (EVs) that are produced in the endosomal compartment of those cells which mediate intercellular communication. These particles can be found abundantly in the biological body fluids such as urine, blood, saliva, cerebrospinal fluid and breast milk. These vesicles can transfer genetic materials such as the microRNAs, noncoding RNAs, DNA and lipids by means of direct or indirect cell-to-cell interaction. Consequently, there has been lot of growing interest related to cancer exosomes as biomarkers and as potential therapeutics. There are studies done which demonstrate the exosomes in relation to cancer, by targeting specific cells and also promote the tumor progression. The other part of the spectrum has stressed the importance of exosomes stability and its potential role in targeting cancer cells through drug delivery system of anticancer molecules. The dichotomy allied with exosomes and their role in oral squamous cell carcinoma biomarkers or as therapy enhancement will be highlighted.

Objective: Cancer stem cells (CSCs) belong to a subpopulation of undifferentiated cells present within tumors that have the potential to regenerate, differentiate, maintenance of pluripotency, drug resistance, and tumorigenicity when transplanted into an innate host. These can influence the growth and behavior of these tumors and are used to investigate the initiation, progression, and treatment strategies of laryngeal cancer. Research on CSC science and targeted therapies were hinge on their isolation and/or enrichment procedures. The object of the study is to isolate cancer stem cells from primary laryngeal carcinoma (CSCPLC) by tumor spheres enrichment. We checked the properties of self-renewal, stemness, clonogenicity, and chemotherapeutic resistance. Materials and Methods: We performed tumor sphere formation assay (primary, secondary, and tertiary) chemotherapy resistance by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were performed to evaluate the CSC cells. Immunofluorescence for stem cell markers (CD133+, CD44+) and gene expression of stem cell markers for CD133+, CD44+, OCT4, SOX2, and NANOG was done using the real-time polymerase chain reaction technique. Results: We were able to isolated CSC subpopulations from PLC cell lines by the tumor sphere method. These cells exhibited good primary, secondary, and tertiary tumor sphere formation efficiency and also disclosed a resistant index of more than 2. Immunofluorescence for stem cell markers (CD133+ and CD44+) confirms the presence of CSC. There was significantly higher mRNA expression of stem cell markers in CSC enriched subpopulations compared to the parental cell lines. Conclusion: We conclude that tumor spheres enrichment is an efficient, economical, and reliable approach for the isolation and characterization of CSC from PLC cell lines. These cells demonstrated the properties of self-renewal, stemness, clonogenicity, and chemotherapeutic resistance.

Aim: The cancer stem cells (CSCs) are known to be responsible for drug resistance and cancer relapse in the treatment of cancer. Identification and isolation of CSCs and study of their properties will play a crucial role in developing an effective drug against these targets. The aim of the study was to isolate CSCs from primary cancer by the tumorspheres enrichment method, to confirm by indirect immunofluorescence and gene expression of stem cell markers by using real-time polymerase chain reaction (RT-PCR) technique. Materials and methods: In this in vitro study, we enriched oral CSCs through tumorsphere formation assay from seven primary cultures of OSCC patients with defined serum media. The expression and localization of the cell surface markers of CD133 and CD44 were tested by indirect immunofluorescence. Gene expression of stem cell markers such as CD44, CD133, Oct4, Sox2, and Nanog were quantified by RT-PCR technique. One-way analysis of variance was applied to analyze gene expression. Results: Tumorsphere formation has been used to isolate the CSCs from the OSCC tissue culture. Both CD133 and CD44 antibody confirmed the presence of CSCs through indirect immunofluorescence. In comparison to parental cell lines, the expression levels of CD133, CD44, Oct4, Sox2, and Nanog stem cell were significantly higher in CSC-enriched subpopulations. Conclusions: The cost-effective spheroid enrichment and the indirect immunofluorescence methods are useful for the isolation of CSCs from the primary tumor.

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