Abstract:The investigation is focused on the development of a compartmentalized microfluidic device for coculturing the cells of crucial retinal cellular layers and assessing cellto-cell interactions. A perfusion-based microfluidic co-culture device was employed and computationally validated for determining the pressure drop and fluid flow rate within the device microchannels. Fabrication was performed using PDMS polymer and coating of fibronectin and collagen facilitated adherence of the cells over the glass surface. … Show more
“…Initially, the membrane of the cell insert was coated with a coating solution comprising of collagen peptide and fibronectin to establish the triple co‐culture model. [ 15 ] The inverted insert, coated with the protein cocktail, was incubated for 45 min and further ARPE19 cells were plated on the bottom surface of the insert. The inverted insert was further incubated for 4 h for the cells to attach to the membrane, in humidified environment containing 5% CO 2 .…”
Section: Resultsmentioning
confidence: 99%
“…The composition was optimized based on previous experiments conducted for establishing a microfluidic model. [ 15 ] A uniform layer, represented by green dye, was visible even under the influence of gravitational force and the cells continued to grow on the inverted membrane till confluency was achieved. The Rb cells were separately isolated and assessed for viability and protein expression, via cell counting and flow cytometer, respectively.…”
Section: Resultsmentioning
confidence: 99%
“…Further such an in vitro Rb model can be established using a microfluidic platform to thoroughly mimic the physiological events and derive a more reliable method for orthogonal bio‐chemical and cellular evaluations. [ 15 ] The triple co‐culture model is a reproducible and robust platform for testing of newer therapeutic molecules.…”
Section: Discussionmentioning
confidence: 99%
“…The cells were seeded in the respective media and were allowed to grow for 72 h. The redox dye EZBlue was metabolically reduced by only the live cells, giving a pink resorufin product. The cells were then incubated in the presence of EZBlue reagent for 4 h. [ 15 ] The absorbance of reduced dye was analyzed at 570 and 600 nm, using plate reader, to calculate the proliferation of respective cell types (VICTOR Nivo, Perkin Elmer, USA).…”
Background: Retinoblastoma (Rb) is a rare cancer of the retina that occurs during early childhood. The disease is relatively rare but aggressive, accounting for ∼3% of childhood cancers. Treatment modalities encompass the administration of large doses of chemotherapeutic drugs, which result in multiple side-effects. Therefore, it is essential to have safe and effective newer therapies and suitable physiologically relevant, alternative-to-animal, in vitro cell culture-based models to enable rapid and efficient evaluation of potential therapies.
Methodology:This investigation was focused on the development of a triple co-culture model comprising Rb, retinal epithelium, and choroid endothelial cells, using a protein coating cocktail, to recapitulate this ocular cancer under in vitro conditions. This resulting model was used for screening drug toxicity, based on the growth profile of Rb cells, using carboplatin as the model drug. Further, a combination of bevacizumab and carboplatin was evaluated using the developed model, to lower the concentration of carboplatin and thereby reduce its physiological side-effects.
Major Results:The effect of drug treatment on the triple co-culture was assessed by increase in the apoptotic profile of Rb cells. Further, the barrier properties were found to be lower with a decrease in the angiogenetic signals that included expression of vimentin. Measurement of cytokine levels signified reduced inflammatory signals due to the combinatorial drug treatment.
Conclusions:These findings validated that the triple co-culture Rb model was suitable for evaluating anti-Rb therapeutics and could thereby decrease the immense load on animal trials, which are the primary screens employed for evaluating retinal therapies.
K E Y W O R D Sdisease model, pre-clinical drug testing, retinal cells, retinoblastoma, triple culture
INTRODUCTIONRetinoblastoma (Rb) is a type of ocular cancer that is particularly prevalent in children up to 5 years of age. This form of cancer accounts for ∼3% of the pediatric cancer population. [1] Its progression is activated due to the mutation in the Rb gene, responsible for the transcription tumor suppressor Rb protein. [2] Rb tumors occur either as endophytic or exophytic tumors. Endophytic tumors arise in the inner retinal layer, whereas exophytic tumors develop in the outer retinal layer further extending the sub-retinal space beneath the retina. [3] The currently available treatment options for Rb can be classified as (1) focal therapy including cryotherapy, conventional laser photocoagulation, transpupillary thermotherapy, photodynamic therapy (in vitro study), and plaque radiotherapy; (2) external beam radiotherapy;(3) systemic chemotherapy; and (4) enucleation. Proper management of Rb, by tumor reduction, excision, and curbing the side effects of
“…Initially, the membrane of the cell insert was coated with a coating solution comprising of collagen peptide and fibronectin to establish the triple co‐culture model. [ 15 ] The inverted insert, coated with the protein cocktail, was incubated for 45 min and further ARPE19 cells were plated on the bottom surface of the insert. The inverted insert was further incubated for 4 h for the cells to attach to the membrane, in humidified environment containing 5% CO 2 .…”
Section: Resultsmentioning
confidence: 99%
“…The composition was optimized based on previous experiments conducted for establishing a microfluidic model. [ 15 ] A uniform layer, represented by green dye, was visible even under the influence of gravitational force and the cells continued to grow on the inverted membrane till confluency was achieved. The Rb cells were separately isolated and assessed for viability and protein expression, via cell counting and flow cytometer, respectively.…”
Section: Resultsmentioning
confidence: 99%
“…Further such an in vitro Rb model can be established using a microfluidic platform to thoroughly mimic the physiological events and derive a more reliable method for orthogonal bio‐chemical and cellular evaluations. [ 15 ] The triple co‐culture model is a reproducible and robust platform for testing of newer therapeutic molecules.…”
Section: Discussionmentioning
confidence: 99%
“…The cells were seeded in the respective media and were allowed to grow for 72 h. The redox dye EZBlue was metabolically reduced by only the live cells, giving a pink resorufin product. The cells were then incubated in the presence of EZBlue reagent for 4 h. [ 15 ] The absorbance of reduced dye was analyzed at 570 and 600 nm, using plate reader, to calculate the proliferation of respective cell types (VICTOR Nivo, Perkin Elmer, USA).…”
Background: Retinoblastoma (Rb) is a rare cancer of the retina that occurs during early childhood. The disease is relatively rare but aggressive, accounting for ∼3% of childhood cancers. Treatment modalities encompass the administration of large doses of chemotherapeutic drugs, which result in multiple side-effects. Therefore, it is essential to have safe and effective newer therapies and suitable physiologically relevant, alternative-to-animal, in vitro cell culture-based models to enable rapid and efficient evaluation of potential therapies.
Methodology:This investigation was focused on the development of a triple co-culture model comprising Rb, retinal epithelium, and choroid endothelial cells, using a protein coating cocktail, to recapitulate this ocular cancer under in vitro conditions. This resulting model was used for screening drug toxicity, based on the growth profile of Rb cells, using carboplatin as the model drug. Further, a combination of bevacizumab and carboplatin was evaluated using the developed model, to lower the concentration of carboplatin and thereby reduce its physiological side-effects.
Major Results:The effect of drug treatment on the triple co-culture was assessed by increase in the apoptotic profile of Rb cells. Further, the barrier properties were found to be lower with a decrease in the angiogenetic signals that included expression of vimentin. Measurement of cytokine levels signified reduced inflammatory signals due to the combinatorial drug treatment.
Conclusions:These findings validated that the triple co-culture Rb model was suitable for evaluating anti-Rb therapeutics and could thereby decrease the immense load on animal trials, which are the primary screens employed for evaluating retinal therapies.
K E Y W O R D Sdisease model, pre-clinical drug testing, retinal cells, retinoblastoma, triple culture
INTRODUCTIONRetinoblastoma (Rb) is a type of ocular cancer that is particularly prevalent in children up to 5 years of age. This form of cancer accounts for ∼3% of the pediatric cancer population. [1] Its progression is activated due to the mutation in the Rb gene, responsible for the transcription tumor suppressor Rb protein. [2] Rb tumors occur either as endophytic or exophytic tumors. Endophytic tumors arise in the inner retinal layer, whereas exophytic tumors develop in the outer retinal layer further extending the sub-retinal space beneath the retina. [3] The currently available treatment options for Rb can be classified as (1) focal therapy including cryotherapy, conventional laser photocoagulation, transpupillary thermotherapy, photodynamic therapy (in vitro study), and plaque radiotherapy; (2) external beam radiotherapy;(3) systemic chemotherapy; and (4) enucleation. Proper management of Rb, by tumor reduction, excision, and curbing the side effects of
“…Studies confirming the functionality of this retinal coculture model have highlighted the potential use of microfluidic-based coculture models for replicating retinal diseases (Figure 4), as well as for culturing spheroids under perfusion conditions. 66,67 Figure 4.Proof-of-concept of the retina-on-a-chip model, for use as a preclinical tool.…”
Section: Session 3: Alternative Models For Drug Treatmentmentioning
Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.
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