Alginate-based 3D cell culture technique to evaluate the half-maximal inhibitory concentration: an in vitro model of anticancer drug study for anaplastic thyroid carcinoma

Abstract Background Three-dimensional (3D) cell culture methods are identified for simulating the biological microenvironment and demonstrating more similarity to in vivo circumstances. Anaplastic thyroid carcinoma (ATC) is a lethal endocrine malignancy. Despite different treatment approaches, no improvement in the survival rate of the patients has been shown. In this study, we used the 3D in vitro ATC model to investigate the cytotoxic effect of BI-847325 anticancer drug in two-dimensional (2D)- and 3D- cultured cells. Methods Human ATC cell lines, C643 and SW1736, were cultured in one percentage (w/v) sodium alginate. Spheroids were incubated in medium for one week. The reproducibility of the fabrication of alginate beads was evaluated. Encapsulation of the cells in alginate was examined by DAPI (4′,6-diamidino-2-phenylindole) staining. Survival of alginate-encapsulated cells was evaluated by CFSE (5,6-Carboxyfluorescein N-hydroxysuccinimidyl ester) staining. The population doubling times of C643 and SW1736 cell lines cultured in 2D monolayer as well as in 3D system were calculated. The cytotoxic effect of BI-847325 on 2D- and 3D- cultured cell lines was assessed for 24–72 h by MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay. Finally, the 3D culture results were compared with the 2D culture method. Results The half-maximal inhibitory concentration (IC50) values of BI-847325 were higher in 3D culture compared to 2D culture. The cytotoxicity data indicated that 3D in vitro models were more resistant to chemotherapy agents. Conclusions The findings of this study are beneficial for developing in vitro ATC 3D models to analyze the efficacy of different chemotherapy drugs and formulations.

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Practical Applications of 3D Cell Culture Biotechnologies for Translational Oncology and Personalized Therapy

Biotekhnologiya ◽

10.21519/0234-2758-2020-36-3-3-15 ◽

2020 ◽

Vol 36 (3) ◽

pp. 3-15

Author(s):

D.A. Chudakova ◽

E.Yu. Skorova ◽

I.V. Reshetov

Keyword(s):

Cell Culture ◽

Extracellular Matrix ◽

3D Culture ◽

3D Cell Culture ◽

Personalized Therapy ◽

3D Bioprinting ◽

Culture Systems ◽

Culture Models ◽

2D And 3D

The creation of in vitro three-dimensional cellular model systems (in vitro 3D cultures) is a fast-growing leading-edge segment of the biotechnological industry. We have examined in this work the key 80 articles published after 2008, and focused on applications of in vitro 3D culture in translational oncology. We described a broad range of 3D culture systems, including models with and without extracellular matrix (ECM). 3D culture models based on decellularized ECM were discussed in more detail. The role of ECM in pathogeneis of malignant neoplasms, in particular, in the phenomenon of the tumor resistance to chemotherapy, was evaluated. 2D and 3D culture systems were compared, and natural and synthetic ECM were described, as well as the model creation based on 3D bioprinting. Particular attention was paid to in vitro models of various cancers, including those at the metastatic stage, based on 3D cell cultures, which maximally mimic the in vivo tumor behavior. The prospects of the practical application of 3D cell culture models in preclinical drag screening and in personalized therapy were discussed. We also presented our data on in vitro 2D and 3D culturing of human cells on various substrates. 3D cellular models, 3D bioprinting, biotechnology, extracellular matrix, cancer, translational medicine, personalized medicine, drag development, in vitro, ex vivo, oncology The authors are grateful to Dr. E. Shabalina for providing part of the experimental data and to OKA-Biotech Company for the samples of recombinant Funding-The work was supported by a Grant from the Russian Science Foundation (no. 18-15-00391). doi: 10.21519/0234-2758-2020-36-3-3-15

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A rhabdomyosarcoma hydrogel model to unveil cell-extracellular matrix interactions

Biomaterials Science ◽

10.1039/d1bm00929j ◽

2021 ◽

Author(s):

Mattia Saggioro ◽

Stefania D'Agostino ◽

Anna Gallo ◽

Sara Crotti ◽

Sara D'Aronco ◽

...

Keyword(s):

Cell Culture ◽

Extracellular Matrix ◽

Three Dimensional ◽

3D Culture ◽

3D Cell Culture ◽

Culture Systems ◽

Matrix Interactions ◽

In Vitro Systems ◽

Extracellular Matrix Interactions

Three-dimensional (3D) culture systems are progressively getting attention given their potential in overcoming limitations of the classical 2D in vitro systems. Among different supports for 3D cell culture, hydrogels (HGs)...

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Conjunctival melanoma and electrochemotherapy: preliminary results using 2D and 3D cell culture models in vitro

Acta Ophthalmologica ◽

10.1111/aos.13993 ◽

2018 ◽

Vol 97 (4) ◽

pp. e632-e640 ◽

Cited By ~ 4

Author(s):

Miltiadis Fiorentzis ◽

Periklis Katopodis ◽

Helen Kalirai ◽

Berthold Seitz ◽

Arne Viestenz ◽

...

Keyword(s):

Cell Culture ◽

3D Cell Culture ◽

Conjunctival Melanoma ◽

Preliminary Results ◽

Culture Models ◽

2D And 3D ◽

Cell Culture Models

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In Vitro Identification and Characterization of CD133pos Cancer Stem-Like Cells in Anaplastic Thyroid Carcinoma Cell Lines

PLoS ONE ◽

10.1371/journal.pone.0003544 ◽

2008 ◽

Vol 3 (10) ◽

pp. e3544 ◽

Cited By ~ 69

Author(s):

Giovanni Zito ◽

Pierina Richiusa ◽

Alessandra Bommarito ◽

Elvira Carissimi ◽

Leonardo Russo ◽

...

Keyword(s):

Thyroid Carcinoma ◽

Cell Lines ◽

Carcinoma Cell ◽

Anaplastic Thyroid Carcinoma ◽

Carcinoma Cell Lines ◽

Identification And Characterization ◽

Thyroid Carcinoma Cell

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Application of Three-dimensional (3D) Tumor Cell Culture Systems and Mechanism of Drug Resistance

Current Pharmaceutical Design ◽

10.2174/1381612825666191014163923 ◽

2019 ◽

Vol 25 (34) ◽

pp. 3599-3607 ◽

Cited By ~ 3

Author(s):

Adeeb Shehzad ◽

Vijaya Ravinayagam ◽

Hamad AlRumaih ◽

Meneerah Aljafary ◽

Dana Almohazey ◽

...

Keyword(s):

Cell Culture ◽

Cancer Cells ◽

Anticancer Drugs ◽

Three Dimensional ◽

3D Culture ◽

3D Cell Culture ◽

Cancer Therapeutics ◽

In Vivo Model

: The in-vitro experimental model for the development of cancer therapeutics has always been challenging. Recently, the scientific revolution has improved cell culturing techniques by applying three dimensional (3D) culture system, which provides a similar physiologically relevant in-vivo model for studying various diseases including cancer. In particular, cancer cells exhibiting in-vivo behavior in a model of 3D cell culture is a more accurate cell culture model to test the effectiveness of anticancer drugs or characterization of cancer cells in comparison with two dimensional (2D) monolayer. This study underpins various factors that cause resistance to anticancer drugs in forms of spheroids in 3D in-vitro cell culture and also outlines key challenges and possible solutions for the future development of these systems.

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Improved In Vitro Anti-Tumoral Activity, Intracellular Uptake and Apoptotic Induction of Gemcitabine-Loaded Pegylated Unilamellar Liposomes

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2008.065 ◽

2008 ◽

Vol 8 (4) ◽

pp. 2102-2113 ◽

Cited By ~ 40

Author(s):

Christian Celia ◽

Maria Grazia Calvagno ◽

Donatella Paolino ◽

Stefania Bulotta ◽

Cinzia Anna Ventura ◽

...

Keyword(s):

Thyroid Carcinoma ◽

Cytotoxic Effect ◽

Anaplastic Thyroid Carcinoma ◽

Chemotherapeutic Agents ◽

Free Drug ◽

Parp Cleavage ◽

Significant Activity ◽

Intracellular Uptake ◽

Cell Mortality

Anaplastic thyroid carcinoma is one of the most aggressive and lethal solid carcinomas affecting humans. A major limit of the chemotherapeutic agents is represented by their low therapeutic index. In this work, we investigated the possibility of improving the anti-tumoral activity of gemcitabine by using pegylated unilamellar liposomes. Liposomes were made up of 1,2-dipalmitoyl-sn-glycero-3-phospocholine monohydrate/cholesterol/N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6:3:1 molar ratio) and they were prepared with a pH gradient to improve the gemcitabine loading capacity. The anti-tumoral efficacy of the liposomal formulation was tested in vitro on human anaplastic thyroid carcinoma cells (ARO) in culture, comparing the effects with those of the free drug. Gemcitabine-loaded unilamellar liposomes had a mean size ∼200 nm with a zeta potential ∼–2mV. The liposomal carrier noticeably improved the anti-tumoral activity of gemcitabine against ARO cells in terms of both dose-dependent cytotoxic effect and of drug exposition effect. Namely, gemcitabine-loaded liposomes showed a cytotoxic effect (58.2% increase of cell mortality at 1 μM with respect to free drug) after 12 h incubation, while the free drug showed a significant activity only after 72 h incubation. Moreover, a significant effect on the cell mortality appeared at 0.1 μM and 100% mortality was detected at a concentration of 1 μM of gemcitabine-loaded liposomes, while the free drug elicited the same effect at a concentration of 100 μM. The improved anti-tumoral activity of gemcitabine determined by the liposomal carrier was due to a greater intracellular uptake. The intracellular gemcitabine levels as a function of time showed a sinusoidal profile with peaks after 2 h, 6 h and 11 h, related to the cellular cycle of ARO. PARP cleavage and DNA fragmentation analysis provided clear evidence of the apoptosis induction in ARO cells by treatment with liposomally entrapped gemcitabine after 72 h incubation. Thus, gemcitabine-loaded liposomes may have a potential therapeutic relevance for the treatment of anaplastic thyroid carcinoma.

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Comparison of antioxidant activity between cyanidin-3-O-glucoside (C3G) liposome and cyanidin-3-O-glucoside (C3G) in 2D and 3D cell cultures

10.1101/314369 ◽

2018 ◽

Author(s):

Tisong Liang ◽

Rongfa Guan ◽

Guozhou Cao ◽

Haitao Shen ◽

Zhenfeng Liu ◽

...

Keyword(s):

Cell Culture ◽

Antioxidant Activity ◽

Cell Cultures ◽

Cell Model ◽

3D Cell Culture ◽

Culture Model ◽

Mda Content ◽

2D And 3D ◽

Cells Cultured

ABSTRACTThe 2D cell culture is the predominant in vitro model for numerous studies. However, 2D cell cultures may not accurately reflect the functions of three-dimensional (3D) tissues, which have extensive cell–cell and cell–matrix interactions; thus, using 2D cell cultures may lead to inaccurate experimental results. Therefore, to obtain adequate and detailed information about the antioxidant activity of cyanidin-3-O-glucoside (C3G) and C3G liposomes in the 2D and 3D cell culture models, we used in this study H2O2to construct the cell damage model and assess the antioxidant activity of C3G and C3G liposomes on Caco-2 cells cultured in the 3D model. We also measured the cell viability, cell morphology, and activity of glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content of Caco-2 cells treated with H2O2, C3G, and C3G liposomes. Results showed that cells cultured in the 3D culture model formed a 3D structure and tight spheroids and showed increased cell activity and IC50. The C3G and C3G liposomes can enhance the activity of GSH, SOD, and T-AOC but decrease the MDA content. At the same time, the effect was more obvious in the 3D cell culture model than in the cells cultured in the 2D model. This study revealed that the results obtained from the 2D cell model may be inaccurate compared with the results obtained from the 3D cell model. A realistic mechanism study of antioxidant activity of C3G and C3G liposomes in the 3D cell model, which acts as an intermediate stage bridging the in vitro 2D and in vivo models, was observed.

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RGDS-Modified Superporous Poly(2-Hydroxyethyl Methacrylate)-Based Scaffolds as 3D In Vitro Leukemia Model

International Journal of Molecular Sciences ◽

10.3390/ijms22052376 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2376

Author(s):

Hana Svozilová ◽

Zdeněk Plichta ◽

Vladimír Proks ◽

Radana Studená ◽

Jiří Baloun ◽

...

Keyword(s):

Cell Lines ◽

Cultured Cells ◽

Ammonium Oxalate ◽

3D Culture ◽

3D Cell Culture ◽

Lymphocytic Leukemia ◽

Interleukin 4 ◽

Hydroxyethyl Methacrylate ◽

Cell Functions

Superporous poly(2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N-(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.

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A Microfluidic Chip Embracing a Nanofiber Scaffold for 3D Cell Culture and Real-Time Monitoring

Nanomaterials ◽

10.3390/nano9040588 ◽

2019 ◽

Vol 9 (4) ◽

pp. 588 ◽

Cited By ~ 2

Author(s):

Jeong Hwa Kim ◽

Ju Young Park ◽

Songwan Jin ◽

Sik Yoon ◽

Jong-Young Kwak ◽

...

Keyword(s):

Cell Culture ◽

Real Time ◽

Microfluidic Chip ◽

Three Dimensional ◽

3D Culture ◽

3D Cell Culture ◽

Culture Conditions ◽

Real Time Monitoring ◽

Nanofiber Scaffold

Recently, three-dimensional (3D) cell culture and tissue-on-a-chip application have attracted attention because of increasing demand from the industries and their potential to replace conventional two-dimensional culture and animal tests. As a result, numerous studies on 3D in-vitro cell culture and microfluidic chip have been conducted. In this study, a microfluidic chip embracing a nanofiber scaffold is presented. A electrospun nanofiber scaffold can provide 3D cell culture conditions to a microfluidic chip environment, and its perfusion method in the chip can allow real-time monitoring of cell status based on the conditioned culture medium. To justify the applicability of the developed chip to 3D cell culture and real-time monitoring, HepG2 cells were cultured in the chip for 14 days. Results demonstrated that the cells were successfully cultured with 3D culture-specific-morphology in the chip, and their albumin and alpha-fetoprotein production was monitored in real-time for 14 days.

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In Vitro Characterization of Cisplatin and Pemetrexed Effects in Malignant Pleural Mesothelioma 3D Culture Phenotypes

Cancers ◽

10.3390/cancers11101446 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1446 ◽

Cited By ~ 2

Author(s):

Eleftherios D. Papazoglou ◽

Rajesh M. Jagirdar ◽

Olympia A. Kouliou ◽

Eleanna Pitaraki ◽

Chrissi Hatzoglou ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Malignant Pleural Mesothelioma ◽

3D Culture ◽

Collagen Gel ◽

3D Cell Culture ◽

Pleural Mesothelioma ◽

Tumor Spheroid ◽

Collagen Gel Contraction

Malignant pleural mesothelioma (MPM) is an aggressive cancer with poor prognosis. The main treatment for MPM is doublet chemotherapy with Cisplatin and Pemetrexed, while ongoing trials test the efficacy of pemetrexed monotherapy. However, there is lack of evidence regarding the effects of Cisplatin and Pemetrexed on MPM cell phenotypes, especially in three-dimensional (3D) cell cultures. In this study, we evaluated the effects Cisplatin and Pemetrexed on cell viability using homologous cell derived extracellular matrix (hECM) as substratum and subsequently in the following 3D cell culture phenotypes: tumor spheroid formation, tumor spheroid invasion, and collagen gel contraction. We used benign mesothelial MeT-5A cells as controls and the MPM cell lines M14K (epithelioid), MSTO (biphasic), and ZL34 (sarcomatoid). Cell viability of all cell lines was significantly decreased with all treatments. Mean tumor spheroid perimeter was reduced after treatment with Pemetrexed or the doublet therapy in all cell lines, while Cisplatin reduced the mean spheroid perimeter of MeT-5A and MSTO cells. Doublet treatment reduced the invasive capacity of spheroids of cell lines into collagenous matrices, while Cisplatin lowered the invasion of the MSTO and ZL34 cell lines, and Pemetrexed lowered the invasion of MeT-5A and ZL34 cell lines. Treatment with Pemetrexed or the combination significantly reduced the collagen gel contraction of all cell lines, while Cisplatin treatment affected only the MeT-5A and M14K cells. The results of the current study can be used as an in vitro 3D platform for testing novel drugs against MPM for ameliorating the effects of first line chemotherapeutics.

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