scholarly journals Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

2021 ◽  
Author(s):  
Aziz UR RAHMAN
Keyword(s):  
Confocal Microscopy ◽  
Drug Uptake ◽  
Live Cells ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Oriented Growth ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Method

Abstract Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

scholarly journals Encapsulated multicellular tumor spheroids as a novel in vitro model to study small size tumors

2003 ◽  
Vol 57 (12) ◽  
pp. 585-588 ◽  
Author(s):  
Elena Markvicheva ◽  
Lina Bezdetnaya ◽  
Artur Bartkowiak ◽  
Annie Marc ◽  
Jean-Louis Gorgen ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Tumor Biology ◽  
Cell Encapsulation ◽  
Large Cell ◽  
In Vitro Models ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Vitro Model

Presently multicellular tumor spheroids (MTS) are being widely used in various aspects of tumor biology, including studies in biology and photodynamic therapy. The cellular organization of spheroids allows the recreation of in vivo small tumors much better than all common two-dimensional in vitro models. The cell encapsulation method could be proposed as a novel technique to quickly and easily prepare a large number of spheroids with narrow size distribution within a desirable diameter range. Moreover, the proposed technique for spheroid generation using encapsulated growing tumor cells could provide entirely new avenues to develop a novel spheroid co-culture model (for instance, the in vitro co-cultvation of tumor cells and monocytes, or epithelial cells, or fibroblasts etc). The current research was aimed at developing a simple and reliable method to encapsulate tumor cells and to cultivate them in vitro. In order to generate spheroids, MCF-7 cells were encapsulated and cultivated in 200 ml T-flasks in a 5% CO2 atmosphere at 37?C for 4-5 weeks. The cell proliferation was easily observed using a light microscope. The cells grew in aggregates increasing in size with time. The cell growth resulted in the formation of large cell clusters (spheroids) which filled the whole microcapsule volume in 4-5 weeks.

scholarly journals Natural and Synthetic Biomaterials for Engineering Multicellular Tumor Spheroids

Polymers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 2506
Author(s):  
Advika Kamatar ◽  
Gokhan Gunay ◽  
Handan Acar
Keyword(s):  
Cancer Research ◽  
Three Dimensional ◽  
3D Culture ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
In Vivo Models ◽  
Advantages And Disadvantages ◽  
Natural And Synthetic

The lack of in vitro models that represent the native tumor microenvironment is a significant challenge for cancer research. Two-dimensional (2D) monolayer culture has long been the standard for in vitro cell-based studies. However, differences between 2D culture and the in vivo environment have led to poor translation of cancer research from in vitro to in vivo models, slowing the progress of the field. Recent advances in three-dimensional (3D) culture have improved the ability of in vitro culture to replicate in vivo conditions. Although 3D cultures still cannot achieve the complexity of the in vivo environment, they can still better replicate the cell–cell and cell–matrix interactions of solid tumors. Multicellular tumor spheroids (MCTS) are three-dimensional (3D) clusters of cells with tumor-like features such as oxygen gradients and drug resistance, and represent an important translational tool for cancer research. Accordingly, natural and synthetic polymers, including collagen, hyaluronic acid, Matrigel®, polyethylene glycol (PEG), alginate and chitosan, have been used to form and study MCTS for improved clinical translatability. This review evaluates the current state of biomaterial-based MCTS formation, including advantages and disadvantages of the different biomaterials and their recent applications to the field of cancer research, with a focus on the past five years.

scholarly journals Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mohammad Azharuddin ◽  
Karin Roberg ◽  
Ashis Kumar Dhara ◽  
Mayur Vilas Jain ◽  
Padraig Darcy ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Head And Neck ◽  
Cancer Cells ◽  
Cell Lines ◽  
Treated Patient ◽  
Drug Uptake ◽  
Multi Drug Resistance ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids

AbstractOne of the hallmarks of cancers is their ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for successful treatment. One of the ways cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3-dimensional (3D) multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistance (MDR) and non-MDR cells does not yet exist. In the present report we describe MCSs obtained from post-diagnosed, pre-treated patient-derived (PTPD) cell lines from head and neck squamous cancer cells (HNSCC) that often develop resistance to therapy. We employed an integrated approach combining response to clinical drugs and screening cytotoxicity, monitoring real-time drug uptake, and assessing transporter activity using flow cytometry in the presence and absence of their respective specific inhibitors. The report shows a comparative response to MDR, drug efflux capability and reactive oxygen species (ROS) activity to assess the resistance profile of PTPD MCSs and two-dimensional (2D) monolayer cultures of the same set of cell lines. We show that MCSs provide a robust and reliable in vitro model to evaluate clinical relevance. Our proposed strategy can also be clinically applicable for profiling drug resistance in cancers with unknown resistance profiles, which consequently can indicate benefit from downstream therapy.

scholarly journals Stroma-Rich Co-Culture Multicellular Tumor Spheroids as a Tool for Photoactive Drugs Screening

2019 ◽  
Vol 8 (10) ◽  
pp. 1686 ◽  
Author(s):  
Ilya Yakavets ◽  
Samuel Jenard ◽  
Aurelie Francois ◽  
Yulia Maklygina ◽  
Victor Loschenov ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Indocyanine Green ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
High Throughput Screening ◽  
Chlorin E6 ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids

Conventional 3D multicellular tumor spheroids of head and neck squamous cell carcinoma (HNSCC) consisting exclusively of cancer cells have some limitations. They are compact cell aggregates that do not interact with their extracellular milieu, thus suffering from both insufficient extracellular matrix (ECM) deposition and absence of different types of stromal cells. In order to better mimic in vivo HNSCC tumor microenvironment, we have constructed a 3D stroma-rich in vitro model of HNSCC, using cancer-associated MeWo skin fibroblasts and FaDu pharynx squamous cell carcinoma. The expression of stromal components in heterospheroids was confirmed by immunochemical staining. The generated co-culture FaDu/MeWo spheroids were applied to study penetration, distribution and antitumor efficacy of photoactive drugs such as Temoporfin and Chlorin e6 used in the photodynamic therapy flow cytometry and fluorescence microscopy techniques. We also investigated the distribution of photodiagnostic agent Indocyanine Green. We demonstrated that the presence of stroma influences the behavior of photoactive drugs in different ways: (i) No effect on Indocyanine Green distribution; (ii) lower accumulation of Chlorin e6; (iii) better penetration and PDT efficiency of Temoporfin. Overall, the developed stroma-rich spheroids enlarge the arsenal of in vitro pre-clinical models for high-throughput screening of anti-cancer drugs.

scholarly journals Multicellular tumor spheroids- an effective in vitro model for understanding drug resistance in head and neck cancer

2019 ◽  
Author(s):  
Mohammad Azharuddin ◽  
Karin Roberg ◽  
Ashis Kumar Dhara ◽  
Mayur Vilas Jain ◽  
Padraig D’arcy ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Head And Neck ◽  
Cancer Cells ◽  
Present Report ◽  
Treatment Success ◽  
Integrated Approach ◽  
Drug Uptake ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids

AbstractA hallmark of cancer is the ability to develop resistance against therapeutic agents. Therefore, developing effective in vitro strategies to identify drug resistance remains of paramount importance for treatment success. A way cancer cells achieve drug resistance is through the expression of efflux pumps that actively pump drugs out of the cells. To date, several studies have investigated the potential of using 3D multicellular tumor spheroids (MCSs) to assess drug resistance; however, a unified system that uses MCSs to differentiate between multi drug resistant (MDR) and non-MDR cells does not exist. In the present report, we have used MCSs obtained from post-diagnosed, pre-treated (PDPT) patient derived head and neck squamous cancer cells that often become treatment resistant, to develop an integrated approach combining clinical drug response and cytotoxicity screening, real-time drug uptake monitoring, and drug transporter activity assessment using flow cytometry in the presence and absence of their respective specific inhibitors. The present report shows a comparative response to MDR, drug efflux capability, and reactive oxygen species (ROS) activity to assess the resistance profile of PDPT patient-derived MCSs and two-dimensional cultures of the same set of cells. We show that MCSs serve as robust and reliable models for the clinical evaluation of drug resistance. Our proposed strategy can thus have potential clinical applicability for profiling drug resistance in cancers with unknown resistance profiles, which consequentially can indicate benefit from downstream therapy.

Neuroprotective effect of dauricine in cortical neuron culture exposed to hypoxia and hypoglycemia: involvement of correcting perturbed calcium homeostasis

2007 ◽  
Vol 85 (6) ◽  
pp. 621-627 ◽  
Author(s):  
Yan-Hong Li ◽  
Pei-Li Gong
Keyword(s):  
Cerebral Ischemia ◽  
Cortical Neurons ◽  
Focal Cerebral Ischemia ◽  
Neuroprotective Effect ◽  
Primary Cultures ◽  
Trypan Blue Exclusion ◽  
Trypan Blue Exclusion Method ◽  
Fluorescence Measurements

We have previously reported that dauricine protects brain tissues from focal cerebral ischemia. To corroborate this effect, neurotoxicity due to hypoxia and hypoglycemia was assessed in primary cultures of rat cortical neurons by using a trypan blue exclusion method. To further clarify the mechanism, the intracellular Ca2+ concentration ([Ca2+]i) and mitochondrial membrane potential (ΔΨm) of dissociated rat cortical cells were monitored by fura-2 fluorescence measurements and flow cytometry, respectively. The results showed that 1 and 10 μmol/L dauricine significantly enhanced neuronal survival during 4 h of hypoxia and hypoglycemia. Dauricine inhibited the increase in [Ca2+]i and decrease in ΔΨm induced by 30 min of hypoxia and hypoglycemia. When exploring the pathway, we found that 1 μmol/L dauricine inhibited the [Ca2+]i increase induced by 7.5 nmol/L thapsigargin in either the presence or absence of extracellular Ca2+ and by 1 mmol/L l-glutamate in the presence of extracellular Ca2+. These results suggest that dauricine prevents neuronal loss from ischemia in vitro, which is in accordance with our previous research in vivo. In addition, by inhibiting Ca2+ release from the endoplasmic reticulum and Ca2+ influx from the extracellular space, dauricine suppressed the increase in [Ca2+]i and, subsequently, the decrease in ΔΨm induced by hypoxia and hypoglycemia. This effect may underlie the mechanism of action of dauricine on cerebral ischemia.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Pia Montanucci ◽  
Silvia Terenzi ◽  
Claudio Santi ◽  
Ilaria Pennoni ◽  
Vittorio Bini ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Chemical Properties ◽  
Live Cells ◽  
High Performing ◽  
Physical Chemical ◽  
Degenerative Disorders ◽  
Selection Of ◽  
Better Than

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation,in vitroandin vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules’ basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

scholarly journals Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karl Olofsson ◽  
Valentina Carannante ◽  
Madoka Takai ◽  
Björn Önfelt ◽  
Martin Wiklund
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Drug Distribution ◽  
In Vitro Models ◽  
Biological Information ◽  
Dimensional Structure ◽  
Multicellular Tumor Spheroids ◽  
Tumor Spheroids ◽  
Nuclear Segmentation

AbstractMulticellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

Very high dilutions of dexamethasone inhibit its pharmacological effects in vivo

2001 ◽  
Vol 90 (04) ◽  
pp. 198-203 ◽  
Author(s):  
LV Bonamin ◽  
KS Martinho ◽  
AL Nina ◽  
F Caviglia ◽  
RGW Do Rio
Keyword(s):  
Inflammatory Cells ◽  
Experimental Models ◽  
Tumour Cells ◽  
Pharmacological Effects ◽  
Trypan Blue Exclusion ◽  
Male Adult ◽  
Trypan Blue Exclusion Method ◽  
High Dilutions ◽  
Ascitic Tumour

AbstractWe evaluated the interaction of dexamethasone 10−17 and 10−33 M (equivalent to 7cH and 15cH) with dexamethasone in pharmacological concentrations, using as experimental models: acute inflammation induced by carrageenan, Ehrlich ascitic tumour, and migration of tumour infiltrating leukocytes (TIL). Male adult BALB/c mice (n=7 per group) were used in all experiments. Carrageenan (1%) was injected into the footpad for oedema evaluation and into the peritoneal cavity (i.p.), for differential counting of inflammatory cells. Ehrlich ascitic tumour cells (107 viable cells/ml) were injected i.p. and tumour cells were counted after 6 days, by the Trypan blue exclusion method. The differential TIL was counted using smears stained by hematoxylin–eosin. Treatments were made immediately after carrageenan inoculation or once a day, during Ehrlich tumour development, until the animals were killed. Animals were treated with the following preparations: (1) phosphate buffer saline (PBS) solution; (2) dexamethasone (0.5 mg/kg for inflammation model or 4 mg/kg for tumour model) mixed with dexamethasone 7cH or 15cH; (3) dexamethasone (same doses) mixed in PBS. Homeopathic dexamethasone partially blocked the anti-inflammatory effect of pharmacological dexamethasone with regard to paw oedema (two-way ANOVA, P≤0.0008) and polymorphonuclear cell migration (χ2, P=0.0001). No important differences were observed between experimental and control groups, in relation to Ehrlich tumour cells viability or count, or bodyweight, but potentised dexamethasone restored control levels of TIL viability, compared to mice treated with pharmacological doses of dexamethasone (χ2, P≤0.001). The results demonstrate that a potentised substance may change its own pharmacological effects and suggest that ultradilutions effects act mostly on host response.

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