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Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies

Yap NY, Ong TA, Morais C, Pailoor J, Gobe GC, Rajandram R

Cell Biol Int, 2019 Jun;43(6):715-725.
PMID: 31062478 DOI: 10.1002/cbin.11150

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan-cytokeratin, cytokeratin 8 and E-cadherin whereas fibroblast cells expressed high α-smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.

Matched MeSH terms: Primary Cell Culture/methods*
Fulltext Amenable epigenetic traits of dental pulp stem cells underlie high capability of xeno-free episomal reprogramming

Thekkeparambil Chandrabose S, Sriram S, Subramanian S, Cheng S, Ong WK, Rozen S, et al.

Stem Cell Res Ther, 2018 03 20;9(1):68.
PMID: 29559008 DOI: 10.1186/s13287-018-0796-2

BACKGROUND: While a shift towards non-viral and animal component-free methods of generating induced pluripotent stem (iPS) cells is preferred for safer clinical applications, there is still a shortage of reliable cell sources and protocols for efficient reprogramming.
METHODS: Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13-18 days).
RESULTS: The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation.
CONCLUSION: In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs.

Matched MeSH terms: Primary Cell Culture/methods
Fulltext Evaluating Feasibility of Human Tissue Engineered Respiratory Epithelium Construct as a Potential Model for Tracheal Mucosal Reconstruction

Mohd Yunus MH, Rashidbenam Z, Fauzi MB, Bt Hj Idrus R, Bin Saim A

Molecules, 2021 Nov 06;26(21).
PMID: 34771136 DOI: 10.3390/molecules26216724

The normal function of the airway epithelium is vital for the host's well-being. Conditions that might compromise the structure and functionality of the airway epithelium include congenital tracheal anomalies, infection, trauma and post-intubation injuries. Recently, the onset of COVID-19 and its complications in managing respiratory failure further intensified the need for tracheal tissue replacement. Thus far, plenty of naturally derived, synthetic or allogeneic materials have been studied for their applicability in tracheal tissue replacement. However, a reliable tracheal replacement material is missing. Therefore, this study used a tissue engineering approach for constructing tracheal tissue. Human respiratory epithelial cells (RECs) were isolated from nasal turbinate, and the cells were incorporated into a calcium chloride-polymerized human blood plasma to form a human tissue respiratory epithelial construct (HTREC). The quality of HTREC in vitro, focusing on the cellular proliferation, differentiation and distribution of the RECs, was examined using histological, gene expression and immunocytochemical analysis. Histological analysis showed a homogenous distribution of RECs within the HTREC, with increased proliferation of the residing RECs within 4 days of investigation. Gene expression analysis revealed a significant increase (p < 0.05) in gene expression level of proliferative and respiratory epithelial-specific markers Ki67 and MUC5B, respectively, within 4 days of investigation. Immunohistochemical analysis also confirmed the expression of Ki67 and MUC5AC markers in residing RECs within the HTREC. The findings show that calcium chloride-polymerized human blood plasma is a suitable material, which supports viability, proliferation and mucin secreting phenotype of RECs, and this suggests that HTREC can be a potential candidate for respiratory epithelial tissue reconstruction.

Matched MeSH terms: Primary Cell Culture/methods

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