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A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

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Related in: MedlinePlus

This figure shows the phase contrast and fluorescent microscope images of Y79 cells grown on Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticles. A: Phase contrast microscope picture of Gelatin-coated PLGA microparticles under 10× magnification. B: Phase-contrast images showing cells attached to the microparticles (white arrows pointing Y79 cells attached to microparticles) forming a three-dimensional growth over the microparticles under 10× magnification. C: Phase contrast microscopic image of 3-D growth of Y79 cells over scaffold microparticles (black arrow pointing to Y79 cells attaché to microparticles) under 40× magnification. D: Fluorescent microscopic image showing the 3-D growth of Y79 cells (labeled with Celltracker dye) over scaffold microparticle (white arrows pointing Y79 cells attached to microparticle) under 40× magnification.
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f5: This figure shows the phase contrast and fluorescent microscope images of Y79 cells grown on Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticles. A: Phase contrast microscope picture of Gelatin-coated PLGA microparticles under 10× magnification. B: Phase-contrast images showing cells attached to the microparticles (white arrows pointing Y79 cells attached to microparticles) forming a three-dimensional growth over the microparticles under 10× magnification. C: Phase contrast microscopic image of 3-D growth of Y79 cells over scaffold microparticles (black arrow pointing to Y79 cells attaché to microparticles) under 40× magnification. D: Fluorescent microscopic image showing the 3-D growth of Y79 cells (labeled with Celltracker dye) over scaffold microparticle (white arrows pointing Y79 cells attached to microparticle) under 40× magnification.

Mentions: The optimized composition of microparticles, which consisted of 5% gelatin and 1.25% chitosan, demonstrated cell growth from an initial seeding cell density of 0.05×106 to a cell density of 0.4×106 cells/mg of microparticles, which is an eightfold increase in cell density after 6 days of culture. Cells attached more to the microparticles rather than to the surface of the nontissue culture Petri dish. Initially, cells were seen attached to the microparticle surface, and with time cells engulfed the microparticles completely, forming a 3-D tumor-like structure (Figure 5A-C phase contrast and Figure 5D fluorescence-labeled Y79 cells). Either a single microparticle or a cluster of two to three microparticles were witnessed to form the 3-D tumor-like structure.


A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

This figure shows the phase contrast and fluorescent microscope images of Y79 cells grown on Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticles. A: Phase contrast microscope picture of Gelatin-coated PLGA microparticles under 10× magnification. B: Phase-contrast images showing cells attached to the microparticles (white arrows pointing Y79 cells attached to microparticles) forming a three-dimensional growth over the microparticles under 10× magnification. C: Phase contrast microscopic image of 3-D growth of Y79 cells over scaffold microparticles (black arrow pointing to Y79 cells attaché to microparticles) under 40× magnification. D: Fluorescent microscopic image showing the 3-D growth of Y79 cells (labeled with Celltracker dye) over scaffold microparticle (white arrows pointing Y79 cells attached to microparticle) under 40× magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369889&req=5

f5: This figure shows the phase contrast and fluorescent microscope images of Y79 cells grown on Poly(D,L-lactide-co-glycolide) (PLGA) scaffold microparticles. A: Phase contrast microscope picture of Gelatin-coated PLGA microparticles under 10× magnification. B: Phase-contrast images showing cells attached to the microparticles (white arrows pointing Y79 cells attached to microparticles) forming a three-dimensional growth over the microparticles under 10× magnification. C: Phase contrast microscopic image of 3-D growth of Y79 cells over scaffold microparticles (black arrow pointing to Y79 cells attaché to microparticles) under 40× magnification. D: Fluorescent microscopic image showing the 3-D growth of Y79 cells (labeled with Celltracker dye) over scaffold microparticle (white arrows pointing Y79 cells attached to microparticle) under 40× magnification.
Mentions: The optimized composition of microparticles, which consisted of 5% gelatin and 1.25% chitosan, demonstrated cell growth from an initial seeding cell density of 0.05×106 to a cell density of 0.4×106 cells/mg of microparticles, which is an eightfold increase in cell density after 6 days of culture. Cells attached more to the microparticles rather than to the surface of the nontissue culture Petri dish. Initially, cells were seen attached to the microparticle surface, and with time cells engulfed the microparticles completely, forming a 3-D tumor-like structure (Figure 5A-C phase contrast and Figure 5D fluorescence-labeled Y79 cells). Either a single microparticle or a cluster of two to three microparticles were witnessed to form the 3-D tumor-like structure.

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

Show MeSH
Related in: MedlinePlus