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The Nobel Prize awarded gene editing system, CRISPR-Cas9, is probably one of the greatest achievements of the last decades. CRISPR-Cas9 can introduce irreversible genomic changes in its target DNA by simple specifying a 20-nucleotide sequence within its RNA guide. Due to its simplicity, efficacy, and relative low cost in comparison with other genome editing systems, it has become the most common gene editing system used in research laboratories. Here we describe a step-by-step protocol to produce genetically edited primary oral keratinocytes using the CRISPR-Cas9 system.

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Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal-glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10-P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-ßIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and ßIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and ßIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal-glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.

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BACKGROUND: GnRH analogs are widely used as neoadjuvant agents for radiotherapy in prostate cancer (PCa) patients, with well-documented effects in reducing tumor bulk and increasing progression-free survival. GnRH analogs act locally in the prostate by triggering apoptosis of PCa cells via activation of the GnRH receptor (GnRHR). During PCa progression, the distribution of GnRHR within the cell is altered, with reduced expression in the cell membrane and remaining sequestered in the endoplasmic reticulum. Pharmacoperone IN3 is able to relocalize GnRHR to the cell membrane. The aim of this study was to evaluate the effect of radiation on PCa cells pretreated with leuprolide, alone or in combination with IN3, as radiosensitizers. MATERIAL AND METHODS: PC3 and human PCa primary cell cultures were treated with IN3 for 24 h, followed by different doses of leuprolide for 48 h and, finally, single doses of radiation (3, 6, and 9 Gy). After radiation, cell survival, apoptosis, cell cycle distribution, and colony growth were evaluated. RESULTS: Radiation reduced cell survival and increased apoptosis in a dose-dependent manner. This effect was also directly related to leuprolide concentration. Pretreatment with IN3 enhanced apoptosis and decreased cell survival, also observing a higher proportion of cells arrested in G2. CONCLUSION: Neoadjuvant leuprolide increases radiation-mediated apoptosis of PCa cells. This effect was enhanced by pretreatment with pharmacoperone IN3. Clinical use of IN3 as a radiosensitizer combined with androgen deprivation therapy to improve survival of patients with PCa remains to be evaluated.

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This study aimed to establish and characterize primary cell cultures and xenografts derived from penile carcinoma (PeCa) in order to provide experimental models for cellular processes and efficacy of new treatments. A verrucous squamous cell carcinoma (VSCC) was macrodissected, dissociated, and cultivated in KSFM/DF12 medium. Cell cultures were evaluated at passage 5 (P5) using migration and invasion assays and were serially propagated, in vivo, in BALB/c nude mice until passage 3 (X1-X3). Immunophenotypic characterization of cultures and xenografts was performed. Genomic (CytoScan HD, Affymetrix) and transcriptomic profiles (HTA 2.0 platform, Affymetrix) for VSCC, cell cultures, and xenografts were assessed. P5 cells were able to migrate, invade the Matrigel, and produce tumors in immunodeficient mice, demonstrating their malignant potential. The xenografts unexpectedly presented a sarcomatoid-like carcinoma phenotype. Genomic analysis revealed a high similarity between the VSCC and tumor-derived xenograft, confirming its xenograft origin. Interestingly, a subpopulation of P5 cells presented stem cell-related markers (CD44(+)CD24(-) and ALDH1(high)) and sphere-forming capacity, suggesting their potential xenograft origin. Cell cultures and xenografts retained the genomic alterations present in the parental tumor. Compared to VSCC, differentially expressed transcripts detected in all experimental conditions were associated with cellular morphology, movement, and metabolism and organization pathways. Malignant cell cultures and xenografts derived from a verrucous penile carcinoma were established and fully characterized. Nevertheless, xenograft PeCa models must be used with caution, taking into consideration the selection of specific cell populations and anatomical sites for cell/tumor implantation.

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Introducción: el tejido adiposo humano está compuesto por diferentes tipos celulares, y ha sido objeto de múltiples estudios en los últimos años debido a su acción en diversas funciones. Métodos: se realizó una búsqueda bilbiográfica en PubMed, Scielo, Science Direct and Google Scholars y se reporta la experiencia de los autores. Resultados: los primeros modelos de estudio fueron con tejido de roedores, y permitieron la comprensión del metabolismo de carbohidratos y ácidos grasos y facilitaron el estudio de la biología del adipocito, junto a estudios histológicos y el aislamiento de adipocitos maduros. Posteriormente se realizaron estudios con células precursoras (preadipocitos) que propiciaron el aislamiento de la línea celular 3T3-1L murina. En Latinoamérica, se han realizado diversos estudios con líneas celulares y con células madre mesenquimales precursoras de adipocitos para estudiar el efecto de hormonas y otras sustancias y para genotipificación. En Colombia se han realizado estudios con adipocitos 3T3-L1 para determinar los efectos de medicamentos y sustancias en estas células. En el laboratorio de Fisiología Celular de la Universidad Tecnológica de Pereira el proceso de obtención de muestras ha evidenciado dificultades por tratarse de tejido humano, pero el protocolo de aislamiento y cultivo pudo ser estandarizado a lo largo de seis años de experimentación; se aislaron preadipocitos y adipocitos maduros que permitieron estudiar los efectos de hormonas, realizar caracterización electrofisiológica y estudiar la fisiología del calcio. Conclusiones: este es un campo de investigación muy relevante debido a la implicación de este tipo celular en funciones metabólicas sistémicas y su relación con patologías de alta prevalencia como la obesidad y el síndrome metabólico.

Introduction: The human adipose tissue is composed of different cell types. It has been subject of several studies in the past years due to its role on diverse functions. Methods: A search in the PubMed, Scielo, Science direct and Google Scholars databases was performed and the authors experience was reported. Results: The first model used was rodent fat, which allowed a better comprehension on carbohydrate and fatty acid metabolism and enhanced research in adipocyte cell biology in addition with histological studies and mature adipocyte isolation. Afterwards, learning about precursor cell (pre-adipocytes) promoted the isolation of murine 3T3-L1 cell line. In Latin America research has been conducted using cell lines and adipocyte precursor mesenchymal stem cells to describe effects of hormones and perform DNA sequencing. In Colombia, studies in 3T3-L1 cell line aimed to stablish the effects of different compounds on these cells. In the Cell Physiology Laboratory of the Universidad Tecnológica de Pereira, sample collection process has shown difficulties because the source was human tissue; nevertheless isolation and cell culture protocols were standardized throughout the last six years of experimentation. Pre-adipocytes and mature adipocytes were isolated to study the effects of hormones, perform electrophysiological characterization and study calcium physiology. Conclusions: This is a relevant research field since these cells have important systemic metabolic functions and they have a clear relationship with high-prevalence pathologies such as obesity and the metabolic syndrome.

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Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

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