In Vivo-like model of glial tumors: Creation of primary cell lines.

e14515 Background: The choice of cell source for 3D bioprinting of in vivo-like models of glial tumors is crucial and must take into account the ability to proliferation and stable metabolism. Oral administration of 5-aminolevulinic acid (5-ALA) in patients prior to surgery increases the fluorescent contrast between tumor and surrounding tissue, but the effect of contrast agents on cells in vitro is unknown. The aim of the study was obtaining viable glial tumor tissues using 5-ALA, as well as the development of a stable primary cell culture for 3D bioprinting. Methods: Tumor tissue was obtained from patients with glioblastoma during surgery under visual control using the Opmi Pentero Blue E400 microscope and 5-ALA. Material was disaggregated on a BD Machine using Medicons 50 μm (BD). Glioblastoma cells were cultured in DMEM/F12 medium with L-glutamine (Gibco) containing 10% fetal bovine serum (Biolot, Russia), 1% non-essential amino acids (NEAA, Sigma-Aldrich) and 0.5% penicillin-streptomycin (Biolot) at 37C. Glial cell lines were characterized immunohistochemically using antibodies to the glial fibrillary acidic protein (GFAP) and proliferation index (Ki-67). Microsatellite analysis was performed using three dinucleotide repeat markers D2S123, D17S250, D5S346 and five mononucleotide loci BAT25, BAT26, NR21, NR24 and NR27. Results: The positive expression of GFAP on the cell processes of the star-like shape was clearly visualized, indicating a morphological feature of glial tumors. The Ki-67 labeling index was 70%. Changes were observed at the D17S250 locus (148-148/148-152) for the glial tumor primary cells after the sixth passage. Microsatellite instability was not observed in the primary cell culture. Conclusions: The accumulation of porphyrins from 5-ALA in glial tumor cells does not prevent the in vitro creation of a cell culture from tumor tissue. Microsatellite analysis showed that the obtained glioblastoma cell lines remain stable for at least 10 passages. Material obtained during resection using 5-ALA is a reliable source of stable glial tumor cell lines.

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Rapid melanization of bomirski amelanotic melanoma cells in cell culture

Bioscience Reports ◽

10.1007/bf01121951 ◽

1983 ◽

Vol 3 (2) ◽

pp. 189-194 ◽

Cited By ~ 16

Author(s):

A. Słominski

Keyword(s):

Cell Culture ◽

Primary Cell Culture ◽

Growth Conditions ◽

Melanoma Cells ◽

Amelanotic Melanoma ◽

Primary Cell ◽

In Vitro Growth

Transfer of Bomirski amelanotic melanoma ceils from in vivo to in vitro growth conditions results in occurrence of rapid melanization in their cytoplasm. The melanized ceils from primary cell culture initiate tumours in hamsters, which do not contain traces of melanin and resemble typical amelanotic melanoma.

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Do alterations in gene expressions influence tumorigenesis in the transmissible venereal tumor in dogs?

Ciência Rural ◽

10.1590/0103-8478cr20200082 ◽

2020 ◽

Vol 50 (11) ◽

Author(s):

Haline Ballestero Fêo ◽

Luis Mauricio Montoya Flórez ◽

Ricardo Seiti Yamatogi ◽

Anderson do Prado Duzanski ◽

João Pessoa Araújo Junior ◽

...

Keyword(s):

Cell Culture ◽

Tumor Suppressor Genes ◽

Primary Cell Culture ◽

Gene Mutations ◽

Tp53 Gene ◽

Primary Cell ◽

Loss Of Function ◽

Gene Expressions

ABSTRACT: Canine transmissible venereal tumor (CTVT) is a transmissible neoplasm, which spreads naturally between dogs through the halogenic transfer of tumor cells, mainly during coitus. It is the oldest known tumoral lineage in nature and reports on gene mutations have been extended. Also, this tumor shares several genetic mutations with some cancers in humans, among them lung carcinomas, melanoma, prostate, breast, among other cancers. Thus, expression of tumor suppressor genes such as TP53, P21, and apoptosis-related genes such as BAX, BCL-2, and BCL-xL, both in vivo and in vitro (primary cell culture) were quantified. In the present study, the comparison of gene expression, the TP53 gene, in most cases, was shown to be high in the majority of tissues (65%) and primary cell culture (100%), while BCL-2, BCL-xL, and BAX presented variation among the animals analyzed. Moreover, in these situations, the results suggested that the apoptotic regulation of these genes did not occur for TP53. The P21 gene was shown to be mostly normal (70%); although, absence (6%) and underexpressions (24%) were also observed. Statistical analysis of the BCL-xL gene demonstrated significant differences between the tissues of the animals when compared to the cell cultures; however, to the other genes, no statistical difference was observed between the groups. Preliminarily, the results suggested the presence of alterations in the gene expressions of the TP53, P21, BAX, BCL-2 and BCL-xL leading to loss of function in these genes, which affect the tumorigenesis of CTVT.

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Expression of Selected Connexin and Aquaporin Genes and Real-Time Proliferation of Porcine Endometrial Luminal Epithelial Cells in Primary Culture Model

BioMed Research International ◽

10.1155/2020/7120375 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Katarzyna Wojtanowicz-Markiewicz ◽

Magdalena Kulus ◽

Sandra Knap ◽

Ievgenia Kocherova ◽

Maurycy Jankowski ◽

...

Keyword(s):

Cell Culture ◽

Epithelial Cells ◽

Primary Cell Culture ◽

Water Movement ◽

Inorganic Ions ◽

Primary Cell ◽

Lag Phase ◽

Luminal Epithelial Cells

Luminal epithelial cells are the first embryonic-maternal contact site undergoing very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume, with the help of aquaporins that provide a transcellular path of rapid water movement during the secretion and absorption of fluids, as well as connexins enabling the flow of inorganic ions and small molecules. In this work, we have examined how AQPs and Cx’s behave in luminal epithelium primary cell culture. Cells obtained from porcine specimen during slaughter were primarily in vitro cultured for 7 days. Their proliferation patterns were then analyzed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes of gene of interest expression were analyzed on each of the seven days of the porcine luminal primary cell culture. Our study showed that the significant changes were noted in the case of Cx43, whose level of protein expression and distribution increases after 120 hours of culture, when the cells enter the lag phase, and maintains an upward trend until the end of the culture. We noted an increase in AQP4, AQP7, AQP8, and AQP11 levels throughout the entire culture period, while the largest differences in expression were found in AQP3, AQP4, and AQP10. The obtained results could become a point of reference for further in vivo and clinical research. Experiments conducted with these proteins showed that they influence the endometrial fluid content during the oestrous cycle and participate in the process of angiogenesis, which intensifies during endometrial development.

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PRIMARY CELL CULTURE OF AEDES ALBOPICTUS MIDGUT CELLS: A PROSPECTIVE MODEL FOR IN VITRO STUDY OF ARBOVIRUSES

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2017.v10i10.19136 ◽

2017 ◽

Vol 10 (10) ◽

pp. 223

Author(s):

Enakshi Roy ◽

Moonmoon Sinha ◽

Shailja Katoch ◽

Urmita Chakraborty ◽

Satadal Das ◽

...

Keyword(s):

Cell Culture ◽

Cell Line ◽

Cell Lines ◽

Primary Cell Culture ◽

In Vitro Study ◽

Primary Cell ◽

Human Beings ◽

Mosquito Midgut ◽

Columnar Cells

Objective: Midgut cells play a key role in the propagation of mosquito borne Arboviruses. The existing mosquito cell lines for studying viral pathogenesis are derived either from larvae or from eggs since there is no cell line available from the mosquito midgut. Therefore, to delineate the in situ viral interaction which naturally occurs within the mosquito midgut and represent cellular pathogenesis in human beings, the present work was aimed to develop a primary cell line from the midgut cells of Aedes albopictus.Methods: The midgut cells of A. albopictus were collected, cultured and incubated at 28°C to study the growth after every 24 hrs for 7 days.Result: The primary cell culture showed an increasing growth pattern of columnar cells up to 48 hrs followed by decrease in cell population afterward. However, the number of stem cells increased significantly throughout the study period, and their population outnumbered the columnar cells after 72 hrs. There was no significant change of goblet cells and regenerating cells which were scanty in number throughout the experiment.Conclusion: The present method will help to develop the individual cell lines from mosquito midgut and study the host pathogen interaction in arboviral diseases in future.

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Preclinical evidence of ET-743 as a potential chemotherapy option for the treatment of biliary carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.4_suppl.193 ◽

2013 ◽

Vol 31 (4_suppl) ◽

pp. 193-193

Author(s):

Francesco Leone ◽

Caterina Peraldo-Neia ◽

Giuliana Cavalloni ◽

Marco Soster ◽

Loretta Gammaitoni ◽

...

Keyword(s):

Cell Cycle ◽

Cell Culture ◽

Dna Damage ◽

Clinical Trials ◽

Cell Lines ◽

Primary Cell Culture ◽

Primary Cell ◽

Term Survival ◽

Anti Tumor Activity

193 Background: The standard chemotherapy for unresectablebiliary tract carcinoma (BTC) is based on gemcitabine and platinum compounds. However, these combinations have not been shown to be effective in improving long-term survival. Thus, there is a real need to find new strategies that would impact in a significant way on clinical outcome. Ecteinascidin-743 (ET-743), a compound isolated from the marine tunicate Ecteinascidia turbinata. ET-743, is approved for the treatment of ovarian cancer and soft tissue sarcoma. Phase II and III clinical trials are ongoing for the treatment of different solid tumors. No preclinical data are available about the efficacy of ET-743 in BTC. In a phase I study, one patient received ET-743 plus capecitabine and experienced a long lasting complete metabolic response. Here, we investigated the antitumor activity of ET-743 in preclinical BTC models. Methods: Four BTC cell lines TFK1, EGI-1, HuH28 and TGBC1 were used to evaluate the effect of ET-743 on proliferation, cell cycle, apoptosis and on the activation of DNA damage proteins. The effect on proliferation was also investigated on a primary cell culture of a gallbladder carcinoma (GBC) resistant to gemcitabine and oxaliplatin. On the same cells, the inhibition of VEGF secretion mediated by ET-743 was analyzed by ELISA. The anti-tumor activity of ET-743 was tested on EGI-1 xenografts in NOD/SCID mice. Results: In vitro, ET-743 is able to markedly reduce cell proliferation of BTC cell lines through cell cycle blockage on G0/G1 phase and to inhibit the growth of primary cell culture derived from GBC patient. Moreover, ET-743 promotes apoptosis by caspase 3 activation, activates proteins involved in DNA damage and reduces VEGF secretion. In the in vivo model, ET-743 is able to slow tumor growth in BTC xenograft. The mechanism of anti-tumor activity involves DNA damage, the induction of hypoxia transcription factor-1, and angiogenesis inhibition. ET-743 has no significant effect on apoptosis in vivo. Conclusions: These data suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials of ET-743 in BTC patients.

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Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma

10.21203/rs.3.rs-36929/v2 ◽

2020 ◽

Author(s):

zhichao xue ◽

Vivian Wai Yan Lui ◽

Yongshu Li ◽

Jia Lin ◽

Chanping You ◽

...

Keyword(s):

Cell Death ◽

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Chemotherapeutic Drugs ◽

Induce Cell Cycle Arrest ◽

Ki 67 ◽

Concurrent Treatment ◽

Xenograft Models

Abstract Background: Recent genomic analyses revealed that druggable molecule targets were detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6–cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. Methods: We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response–related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. Results: In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro . Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo , as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro . Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo . This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. Conclusions: Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

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Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture

BMC Cancer ◽

10.1186/s12885-020-06929-8 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

L. Ehlen ◽

J. Arndt ◽

D. Treue ◽

P. Bischoff ◽

F. N. Loch ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cell Culture ◽

Primary Cell Culture ◽

Primary Cell ◽

In Vitro Modeling ◽

Novel Methods

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TMOD-01. CHARACTERIZATION OF PATIENT-DERIVED PRIMARY CELL LINES AND XENOGRAFTS FOR GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noz175.1100 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi262-vi262 ◽

Cited By ~ 1

Author(s):

Noriyuki Kijima ◽

Daisuke Kanematsu ◽

Tomoko Shofuda ◽

Masahiro Nonaka ◽

Ryoichi Iwata ◽

...

Keyword(s):

Cell Culture ◽

Cell Surface ◽

Cell Lines ◽

Primary Cell Culture ◽

Surface Marker ◽

Primary Cell ◽

Cell Surface Marker ◽

High Expression ◽

Glioblastoma Patient ◽

Primary Cell Lines

Abstract Patient-derived primary cell culture and xenograft are essential tools for translational research for glioblastoma. However, characteristics of each patient derived cell line and xenograft is not extensively studied. In this study, we aim to analyze the characteristics of our glioblastoma patient-derived cell lines and xenografts based on cell surface markers and their differentiation patterns. We have established 20 glioblastoma primary cell culture lines by serum free medium containing EGF and bFGF and found that primary cell culture lines could be classified based on the expression of CD133 and CD44. Four cell lines had high expression of both CD133 and CD44. Eleven cell lines had high expression of only CD44, three cell lines had high expression of only CD133, two cell lines had low expression of both CD133 and CD44. In addition when we induce differentiation, these cell lines showed differentiation to both glial and neuronal differentiation, but differentiation patterns were different depending on each cell line. Four cell lines showed predominant neuronal differentiation and others showed predominant glial differentiation. We next investigated in vivo characteristics of glioblastoma patient derived xenografts from these established cell lines. We have injected these cell lines into NOD/Shi-scid IL2Rγ KO mouse and histopathologically analyzed characteristics of xenografts. Each xenograft well recapitulated histological features of original patients’ tumors and tumor cells remarkably invade through subventricular zone. These results suggest that glioblastoma patient derived primary cell lines and xenografts have different characteristics of cell surface marker expressions and differentiation patterns, thus can classify these cell lines depending on cell surface marker expressions and differentiation patterns. Further analysis is needed to examine the biological importance of the differences in cell surface marker expressions and differentiation patterns.

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