On the Viability of Tumour Cells in Artificially Produced Suspensions (original) (raw)
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Purity and transplantation properties of tumor cell suspensions
Clinical & Experimental Metastasis, 1985
Cell debris is inevitably produced by the mechanical and enzymatic procedures used to dissociate solid tumors. We investigated the influence of such debris on the i.v. and s.c. transplantability of B 16 melanoma cells. Cell suspensions were centrifuged on a Percoll cushion to remove the debris, and the purified preparations were compared to untreated control suspensions. The i.v. injection of a massive dose (106 cells) of unpurified cells killed the animals within a few minutes, while the same amount of purified cells left the animals unaffected.
Formation of large vacuoles in tumour cells grown in suspension
Acta pathologica et microbiologica Scandinavica. Section A, Pathology, 1980
In suspension cultures of JB-1-E cells approximately 11% of the cells contained a single large vacuole ("balloon cells") or signet-ring cells). Such cells were rarely observed in monolayer cultures, where the cells were attached to a substrate. Only a few "balloon cells" were observed in suspension cultures of HeLa S3 cells. Both cell lines were able to phagocytize latex particles of 5.7 micrometer mean diameter. Nocodazole significantly reduced the formation of "balloon cells" in suspension cultures of JB-1-E cells. Transmission electron microscopy indicated that various cytoplasmic membrane components probably could contribute to the membrane material of the vacuoles. It is believed that the formation of "balloon cells" is due to changes in the regulation of the cellular membrane material.
Clinical & Experimental Metastasis, 1991
Vital microscopic studies on tumour cell lodgement in the rat liver have suggested that tumour cells from culture (CTCs) and from solid tumour (STCs) have different rheological properties. In the present study CTCs and STCs from a rat fibrosarcoma were compared in respect to their morphology, rheology and lodgement properties. The ultrastructural examination showed that the CTCs had a larger mean diameter (14"9 #m) than the STCs (11"2 #m). Both cell types had a very irregular nucleus. Surface irregularities provide the CTCs and STCs with a 'membrane excess', i.e. a larger plasmalemma than required to enclose the cells as smooth spheres, amounting to 46"8% and 60"9~, respectively. These values indicate that both CTCs and STCs can be substantially deformed with preservation of the volume and area. The CTCs were stiffer than the STCs when deformed at constant pressure in 6"5 #m glass pipettes, the nucleus appearing to be a significant hindrance to CTC deformation. Five minutes after intraportal injection of radiolabelled CTCs and STCs a much higher percentage of CTCs (82~) than of STCs (20~) remained lodged in the rat liver. The results indicate that the propagation in culture of fibrosarcoma cells alters the morphology and rheology of the cells such that CTCs are not suited for studies in vivo where the spontaneous intravascular dissemination and organ lodgement of tumour cells is simulated.
Development of suspension cell culture model to mimic circulating tumor cells
Oncotarget, 2018
Circulating tumor cells (CTCs) are essential for the establishment of distant metastasis. Numerous studies have characterized CTCs as metastatic precursors; however, the molecular nature of CTCs has not been completely revealed yet due to the low number of CTCs in the blood stream. As an alternative approach, we developed a long-term suspension cell culture model using human breast cancer cell lines to mimic CTCs. We found that more than 40 passaged suspension cells acquired the ability to enhance metastasis like cancer stem cells. To identify molecular changes acquired during the suspension cell culture, we analyzed metabolic and lipidomic profiles as well as transcriptome in MDA-MB-468 suspension cells. Glutamate and leucine levels increased in suspension cells, and cholesterol synthesis pathway was altered. The inhibition of glutamate metabolic pathway decreased the proliferation of suspension cells compared to that of adherent cells. In the lipidomic profile, PC species containi...
Growth of Cell Colonies in Soft Agar from Biopsies of Different Human Solid Tumors
Cancer Research, 1980
Colony growth in soft agar of cells disaggregated from 87 solid tumor specimens was evaluated. Tumors were disaggre gated by an enzymatic method consisting of microtome slicing of tissues and incubation at 37°for 2 hr in Roswell Park Memorial Institute Tissue Culture Medium 1640 containing 10% fetal bovine serum, 0.8% collagenase II, and 0.002% DNase I. Tumor cells were cultured in a two-layer agar system (0.5% agar feeder, 0.3% agar plating layer) in enriched medium with or without addition of different factors. Fifty-eight of 87 malignant tumors (16 of 22 melanomas, 11 of 16 sarcomas, 8 of 16 pulmonary carcinomas, 8 of 11 colon carcinomas, 5 of 8 breast carcinomas, 2 of 3 carcinomas of unknown site, 2 of 2 ovarian carcinomas, 1 of 1 urinary bladder carcinoma, 0 of 1 kidney carcinoma, 1 of 1 epiglottic carcinoma, 1 of 1 pan creatic carcinoma, 1 of 1 pleural mesothelioma, 1 of 1 lung hemangiopericytoma, 0 of 1 malignant schwannoma, 0 of 1 malignant giant cell tumor, and 1 of 1 neuroblastoma) grew in the soft-agar system yielding an overall cloning success rate of 67%. The number of colonies (30 or more cells) after plating 5 x 105 cells ranged from 50 to 3774 per plate, yielding plating efficiency from 0.01 to 0.8%. Considerable variations in plating efficiency were noticed among tumors of the same type. A linear relationship was obtained between the number of cells plated and the number of colonies formed. Tumor cells washed after enzymatic disaggregation yielded more colonies per plate than did tumor cells plated without washing. Refrigeration of tumor tissue for 24 hr in Roswell Park Memorial Institute Tissue Culture Medium 1640 containing 10% fetal bovine serum did not decrease the number of colonies. The number of colonies formed was not increased with two different cell-conditioned media. Murine and rat red blood cells did not affect tumor colony growth. The cells plucked from individual colonies and stained by the Wright-Giemsa and Papanicolaou methods had the same morphological characteristics as did tumor cells in the original cell suspension. The histological pattern of the original tumors was maintained after passage from agar methyl cellulose into the nude mouse.
European Journal of Cancer, 1999
cytoskeletal alterations. The influence of broad-spectrum protein tyrosine kinase (PTK) inhibitor genistein on integrin-mediated dynamic adhesion to ECM components was investigated. Methods: HT-29 colon carcinoma cells were used to study dynamic cell adhesion to collagen in a parallel plate laminar flow chamber. Wall shear adhesion threshold (WSAT), dynamic adhesion rate (DAR) and adhesion stabilization rate (ASR) were determined to differentiate initial adhesion events and adhesion stabilization. These data were compared to static adhesion rates and cell spreading. Results: Genistein interfered with early events of a2bl-integrin-mediated adhesion under flow conditions, but not with secondary adhesion stabilization and cell spreading. This drug lead to an increased rate of adhesion under static conditions, but the same treatment inhibited dynamic adhesion of HT-29 cells, DAR was significantly reduced using genistein-pretreated cells, whereas WSAT and ASR did not show differences between treated and untreated cells. Conclusions: Genistein-sensitive PTK appear to be involved in initial events of stabilization of integrin-mediated cell adhesion to ECM. Dynamic conditions of fluid flow may have substantial influence on integrin-mediated signal transduction involved in adhesion stabilization of HT-29 cells.
Journal of Immunotherapy, 2012
Adoptive cell therapy (ACT) of metastatic melanoma with autologous tumor infiltrating lymphocytes (TIL) is clinically effective, but TIL production can be challenging. Here we describe a simplified method for initial TIL culture and rapid expansion in gas-permeable flasks. TIL were initially cultured from tumor digests and fragments in 40 mL capacity flasks with a 10 cm 2 gaspermeable silicone bottom, G-Rex10. A TIL rapid expansion protocol (REP) was developed using 500 mL capacity flasks with a 100 cm 2 gas-permeable silicone bottom, G-Rex100. TIL growth was successfully initiated in G-Rex10 flasks from tumor digests from 13 of 14 patients and from tumor fragments in all 11 tumor samples tested. TIL could then be expanded to 8-10×10 9 cells in a two-step REP which began by seeding 5 × 10 6 TIL into a G-Rex100 flask, followed by expansion at day 7 into 3 G-Rex100 flasks. To obtain the 30 to 60 × 10 9 cells used for patient treatment we seeded 6 G-Rex100 flasks with 5×10 6 cells and expanded into 18 G-Rex100 flasks. Large scale TIL REP in gas-permeable flasks requires approximately 9 to 10 liters of media, about 3 to 4 times less than other methods. In conclusion, TIL initiation and REP in gas-permeable G-Rex flasks require fewer total vessels, less media, less incubator space and less labor than initiation and REP in 24-well plates, tissue culture flasks and bags. TIL culture in G-Rex flasks will facilitate the production of TIL at the numbers required for patient treatment at most cell processing laboratories.
Cell subpopulations dispersed from solid tumours and separated by centrifugal elutriation
British journal of cancer, 1981
The degree of non-neoplastic host-cell infiltration was assessed in 3 in vivo-in vitro tumour models commonly used in radiobiological studies: EMT6/Ro mammary carcinoma, 9L/Ro tumour and KHT sarcoma. While the 2 former tumour models have been shown to be moderately to highly immunogenic when grown s.c., the KHT sarcoma is apparently non-immunogenic. Using differential staining on single-cell suspensions from enzymatically dissociated solid tumours, all 3 tumour types were found to contain large proportions (30-60%) of non-neoplastic host cells. The actual host-cell component found in the cell suspensions differed both in type and percentage for the 3 tumours studied. These host and neoplastic cells in the cell suspensions prepared from the solid tumours could be readily separated by centrifugal elutriation. After separation the clonogenic potential of the neoplastic cells was assessed, and was found to be higher than the clonogenic capacity of the unseparated cell suspension by a fa...