The dissociation of transplantable tumors.

Noel, Jack; Zucker, Robert M.; Wu, Nila C.; Demaray, S. Y.

doi:10.1177/25.7.197162

Cited by 10 publications

(5 citation statements)

References 21 publications

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“…This, we believe, gives a more detailed and accurate representation of the cells in the slices that may be unavailable in the intact tissue. 32 Access to high-quality screening in developed countries has resulted in the detection of breast cancer at an earlier stage, leading to a decrease in the incidence of large tumors and thus less patient tissue available for research purposes. Even though our model uses a large slice of tumor, the bioluminescence detection system we have developed allows the accurate analysis of gene expression over time without sequential sacrifice of tissue, as with previous models, minimizing wastage of valuable patient material.…”

Section: Discussionmentioning

confidence: 99%

Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer

Rajendran

O’Hanlon

Morrissey

et al. 2011

Exp Biol Med (Maywood)

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Preclinical results with various gene therapy strategies indicate significant potential for new cancer treatments. However, many therapeutics fail at clinical trial, often due to differences in tissue physiology between animal models and humans, and tumor phenotype variation. Clinical data relevant to treatment strategies may be generated prior to clinical trial through experimentation using intact patient tissue ex vivo. We developed a novel tumor slice model culture system that is universally applicable to gene delivery methods, using a realtime luminescence detection method to assess gene delivery. Methods investigated include viruses (adenovirus [Ad] and adeno-associated virus), lipofection, ultrasound (US), electroporation and naked DNA. Viability and tumor populations within the slices were well maintained for seven days, and gene delivery was qualitatively and quantitatively examinable for all vectors. Ad was the most efficient gene delivery vector with transduction efficiency >50%. US proved the optimal non-viral gene delivery method in human tumor slices. The nature of the ex vivo culture system permitted examination of specific elements. Parameters shown to diminish Ad gene delivery included blood, regions of low viability and secondary disease. US gene delivery was significantly reduced by blood and skin, while tissue hyperthermia improved gene delivery. US achieved improved efficacy for secondary disease. The ex vivo model was also suitable for examination of tissue-specific effects on vector expression, with Ad expression mediated by the CXCR4 promoter shown to provide a tumor selective advantage over the ubiquitously active cytomegalovirus promoter. In conclusion, this is the first study incorporating patient tissue models in comparing gene delivery from various vectors, providing knowledge on cell-type specificity and examining the crucial biological factors determining successful gene delivery. The results highlight the importance of in-depth preclinical assessment of novel therapeutics and may serve as a platform for further testing of current, novel gene delivery approaches.

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Section: Discussionmentioning

confidence: 99%

Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer

Rajendran

O’Hanlon

Morrissey

et al. 2011

Exp Biol Med (Maywood)

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“…It is hard to believe that the nuclear density in human colorectal tumors, with frequent necrosis and fibrotic areas, is substantially higher than in mouse liver. The average yield of 0.9 x lo8 nuclei per gram of tumor tissue is about twice as high as figures obtained by enzymatic tumor digestion of transplantable mouse tumors (14,15), and orders of magnitude higher than the nuclear yield reported for a technique using tissue homogenization (12). A trypsin technique on human mammary tumors yielded about 30 times fewer cells and a simple mechanical disaggregation about six times fewer (7).…”

Section: Discussionmentioning

confidence: 93%

“…Or, in other words: How representative is the sample introduced into the flow cytometer? Few papers deal with these problems, and a simple figure giving the number of cells obtained per gram of tissue is only rarely reported for the various isolation procedures (4,7,12,14,15,19). It is of interest to know whether one technique yields significantly more cells than another.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometry of human colorectal tumors: Nuclear isolation by detergent technique

Petersen¹

1985

Cytometry

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A simple one-step technique for detergent isolation and DNA staining of nuclei from mouse colon and from human colorectal tumors was investigated. Nuclear yield increased with treatment time, up to 24 h. There were only minor differences when detergent concentrations from 0 to 0.6% were used. The lowest (0.03%) concentration was most effective. No loss of nuclei was effected by cell lysis and no selectivity was observed for isolation of certain cell-cycle phases or ploidy classes from heterogeneous tumors. The nuclei were stable in the stain-detergent solution for 24 h, but lymphocytes were sensitive to the possible proteolytic activity of one of the two commercial RNase preparations. Of the total number of parenchymal nuclei in mouse liver, as estimated by a stereological method, approximately 60% were isolated by the procedure (approximately 0.9 x 10* nuclei/g tissue). From mouse colon the average nuclear yield was 1.8 x 10s/g, and from human colorectal tumors 0.9 x l@/g (ranges 0.3-1.9 x 10' ). Microscopic examination of undissolved tissue fragments from the preparation of tumors and mouse colon showed a high selectivity for isolation of epithelial and neoplastic nuclei, leaving the stroma with its nuclei almost intact.

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“…The large number of enzymes available for tissue dissociation is beyond the scope of this review; however, most enzymatic digestions can be enhanced by utilising rotation, providing continuous agitation of the suspension. 81 This movement can improve both the speed and quality of the digestion reducing the incidence of cell clustering and cell death in a given cell suspension. Other simple methods to ensure achievement of a single-cell suspension include passing the cells through a filter with a pore size of between 30 and 70 µm, thus removing the vast majority of cell clusters, and suspending cells at densities below 1 × 10 6 cells/mL to reduce the potential for aggregation.…”

Section: Tissue Dissociation and Cell Clustersmentioning

confidence: 99%

Cell separation: Terminology and practical considerations

et al. 2012

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Cell separation is a powerful tool in biological research. Increasing usage, particularly within the tissue engineering and regenerative medicine communities, means that researchers from a diverse range of backgrounds are utilising cell separation technologies. This review aims to offer potential solutions to cell sorting problems and to clarify common ambiguities in terminology and experimental design. The frequently used cell separation terms of ‘purity’, ‘recovery’ and ‘viability’ are discussed, and attempts are made to reach a consensus view of their sometimes ambiguous meanings. The importance of appropriate experimental design is considered, with aspects such as marker expression, tissue isolation and original cell population analysis discussed. Finally, specific technical issues such as cell clustering, dead cell removal and non-specific antibody binding are considered and potential solutions offered. The solutions offered may provide a starting point to improve the quality of cell separations achieved by both the novice and experienced researcher alike.

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The dissociation of transplantable tumors.

Cited by 10 publications

References 21 publications

Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer

Preclinical evaluation of gene delivery methods for the treatment of loco-regional disease in breast cancer

Flow cytometry of human colorectal tumors: Nuclear isolation by detergent technique

Cell separation: Terminology and practical considerations

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