Establishment and Analysis of a 3D Co-Culture Spheroid Model of Pancreatic Adenocarcinoma for Application in Drug Discovery
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Spheroid
3D cell culture
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Abstract Objective: The spheroid model provides a physiological platform to study cancer cell biology and drug sensitivity. Usage of bovine collagen I for spheroid assays is costly especially when experiments are conducted in 24-well plates, as high volume of bovine collagen I is needed. The aim of the study was to downsize spheroid assays to a microfluidic 3D cell culture chip and compare the growth, invasion and response to drug/compound of spheroids embedded in the 3D chip to spheroids embedded in 24-well plates. Results: Spheroids generated from nasopharyngeal carcinoma cell line HK-1 continuously grew and invaded into collagen matrix in a 24-well plate. Similar observations were noticed with spheroids embedded in the 3D chip. Large spheroids in both 24-well plate and the 3D chip disintegrated and invaded into the collagen matrix. Preliminary drug sensitivity assays showed that the growth and invasion of spheroids were inhibited when spheroids were treated with combination of cisplatin and paynantheine at high concentrations, in a 24-well plate. Comparable findings were obtained when spheroids were treated with the same drug combination in the 3D chip. Moving forward, spheroid assays could be performed in the 3D chip in a more high-throughput manner with minimal time and cost.
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Abstract Objective The spheroid model provides a physiological platform to study cancer cell biology and drug sensitivity. Usage of bovine collagen I for spheroid assays is costly especially when experiments are conducted in 24-well plates, as high volume of bovine collagen I is needed. The aim of the study was to downsize spheroid assays to a microfluidic 3D cell culture chip and compare the growth, invasion and response to drug/compound of spheroids embedded in the 3D chip to spheroids embedded in 24-well plates. Results Spheroids generated from nasopharyngeal carcinoma cell line HK-1 continuously grew and invaded into collagen matrix in a 24-well plate. Similar observations were noticed with spheroids embedded in the 3D chip. Large spheroids in both 24-well plate and the 3D chip disintegrated and invaded into the collagen matrix. Preliminary drug sensitivity assays showed that the growth and invasion of spheroids were inhibited when spheroids were treated with combination of cisplatin and paynantheine at high concentrations, in a 24-well plate. Comparable findings were obtained when spheroids were treated with the same drug combination in the 3D chip. Moving forward, spheroid assays could be performed in the 3D chip in a more high-throughput manner with minimal time and cost.
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Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments.
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Gastric cancer (GC) is highly deadly. Three-dimensional (3D) cancer cell cultures, known as spheroids, better mimic tumor microenvironment (TME) than standard 2D cultures. Cancer-associated fibroblasts (CAF), a major cellular component of TME, promote or restrain cancer cell proliferation, invasion and resistance to drugs. We established spheroids from two human GC cell lines mixed with human primary CAF. Spheroid organization, analyzed by two-photon microscopy, showed CAF in AGS/CAF spheroids clustered in the center, but dispersed throughout in HGT-1/CAF spheroids. Such differences may reflect clonal specificities of GC cell lines and point to the fact that GC should be considered as a highly personalized disease.
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Three-dimensional (3D) cell culture has become increasingly adopted as a more accurate model of the complex in vivo microenvironment compared to conventional two-dimensional (2D) cell culture. Multicellular spheroids are important 3D cell culture models widely used in biological studies and drug screening. To facilitate simple spheroid manipulation, magnetic spheroids were generated from magnetically labeled cells using a scaffold-free approach. This method is applicable to a variety of cell types. The spheroids generated can be targeted and immobilized using magnetic field gradients, allowing media change or dilution to be performed with minimal disruption to the spheroids. Cells in magnetic spheroids showed good viability and displayed typical 3D morphology. Using this platform, a 28 day study was carried out using doxorubicin on magnetic MCF-7 spheroids. The results provided a proof-of-principle for using magnetic tumor spheroids in therapeutic studies. They can offer beneficial insights that help to bridge the gap between in vitro and in vivo models. Furthermore, this platform can be adapted for high-throughput screening in drug discovery.
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Abstract Three-dimensional (3D) cancer cell models, such as 3D multicellular spheroids, are superior models of in vivo systems than the more popular two-dimensional (2D) cell culture. 3D cancer spheroids provide a closer reflection of tumor gene expression and biology than 2D cultures. This is particularly relevant for drug development and precision medicine programs were cell culture conditions that better reflect the 3D tumor environments, including matrix stiffness, would be highly advantageous. A key challenge for the routine application of embedded 3D cancer spheroids in precision and drug programs is the lack of high throughput and uniform production of spheroids in a biocompatible matrix. To address this, we have developed a high-throughput method of producing 3D multicellular cancer spheroids embedded inside a tissue-like matrix using a custom-built drop-on-demand 3D bioprinter. The 3D bioprinter is capable of printing a cell-suspension ink with up to 300 million cells/mL and a cell viability greater than 95%. We have validated the 3D bioprinting using glioblastoma, neuroblastoma and lung cancer cells. The 3D bioprinted spheroids were confirmed to maintain all the biological characteristics typically found in a cancer spheroid. In particular, the 3D bioprinted cancer spheroids were shown to be viable and proliferating, preserved the apoptotic and cancer-stemness characteristics and using super-resolution lattice-light sheet microscopy, we showed the spheroids maintained the same structural integrity as manually formed spheroids. The potential application of the 3D bioprinted spheroids for high-throughput drug screening in 3D environments was demonstrated using doxorubicin as the model drug. 3D-bioprinting of patient-derived cancer cells for precision medicine applications is now underway. High-throughput embedded 3D bioprinting has enormous potential to accelerate precision medicine, drug discovery and cancer biology. Citation Format: Lakmali Atapattu, Robert Utama, Aiden O'Mahony, Christopher Fife, Jongho Baek, Théophile Allard, Kieran O'Mahony, Julio Ribeiro, Katharina Gaus, Justin Gooding, Maria Kavallaris. Precision medicine: High-throughput 3D bioprinting of embedded multicellular cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5022.
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Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model.
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Abstract A new channel‐free water‐in‐oil (WO) droplet 3D cell culture method is proposed to address the challenges while maintaining the advantages of the conventional 3D cell culture methods. The proposed WO method can fundamentally solve the constraint of spheroids size, a common challenge in conventional 3D culture, by using droplet size controllability. The 3D cell culture performance of the WO method is verified by comparing it with the conventional 3D cell culture methods. A systematic investigation of the culture conditions of the WO method confirms the working range of cell concentration and droplet size, as well as the scalability of spheroid size. Adjusting droplet size and cell concentration enables rapid spheroid formation with large and high cell concentration droplets or fast spheroid growth with small and low cell concentration droplets, providing control over the spheroid size and growth rate according to the purpose. Furthermore, long‐term culture is demonstrated for 1 month with the proposed method, showing the largest spheroid culture and demonstrating the possibility that this method can be used not only for spheroid formation but also for organoid studies. Finally, if a WO‐based automated 3D cell culture system is developed, it will be a useful tool for organoid research.
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