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ABSTRACT
In this study, we have developed a modified microfluidic T-junction tubing apparatus to fabricate high-throughput hydrogel-encapsulated personalized breast cancer organoids. We have used mineral oil as a continuous phase and decellularized adipose tissue hydrogel (DAT) encapsulated patient-derived cancer cells as a dispersed phase to generate homogeneous organoids. After assembly, organoids were cultured for 21 days in a 96-well plate format, followed by extensive phenotypic and genotypic characterization, and dose-responsive chemotherapeutic drug cytotoxic evaluation for six different drugs. We demonstrate the successful development of a high-throughput microfluidic platform for generating functional personalized breast cancer organoid models using DAT hydrogel. This innovative approach holds significant potential for future applications in developing personalized cancer organoids and targeted therapeutics.
KEYWORDS: Decellularized adipose tissue (DAT), Microfluidic T-junction, Tumor microenvironment (TME), Personalized cancer organoids.
INTRODUCTION
Recent research has profoundly influenced the development of personalized cancer organoids with complex TME in in-vitro tumor models towards personalized medicine for different cancers [1]. In this prospect, droplet microfluidics has played a significant role in developing high-throughput personalized microtumor models with greater accuracy, homogeneity, and reproducibility [2]. These developed personalized cancer models help us to understand the intricate biological complexity of the patient's tumor growth, differentiation, and metastasis. Thus, developing microfluidic 3D breast cancer organoid models with patient-derived cancer cells with reinforced ECM hydrogel as a component of the TME is essential to improve our comprehensive understanding of cancer biology and screen the most effective and safe chemotherapeutic drugs for individual cancer patients.
EXPERIMENTAL
We used mineral oil as a continuous phase and decellularized adipose tissue hydrogel (DAT) encapsulated cells as a dispersed phase to generate homogeneous cell-encapsulated droplets. After fabrication, we cultured them with essential growth factors in media for 21 days to develop personalized breast cancer organoids. We have thoroughly characterized the fabricated organoids by performing cell viability, metabolic activity, morphological analysis, histological staining, immunostaining for specific marker proteins, and gene expression analysis for specific tumor microenvironment genes by qRT-PCR experiments. We then evaluated the dose-dependent cytotoxicity and calculated the respective IC50 values for six different chemotherapeutic drugs on the developed organoids by Live/dead imaging and MTT assay.
RESULTS AND DISCUSSION
The developed microfluid system is a non-lithography-based device that is easy to assemble and use with a syringe pump. We can use the T-junction multiple times after cleaning and reassembling with the same fittings and PTFE tubes. These advantages make it a cost-effective device that any research laboratory can use for multiple applications to develop personalized organoids. Biological characterization of the developed organoid model shows excellent biocompatibility, metabolic activity, breast cancer-specific marker protein expression, key TME Gene expression, and different dose-response cytotoxicity of anticancer drugs for individual patients. In the future, we aim to validate our DAT hydrogel-based personalized breast cancer organoid models with multiple omics data, followed by biobanking for different breast cancer subtypes. In the future, hospitals and pharmaceutical laboratories could adopt this microfluidic method as a tool for developing personalized cancer organoids as 3D in vitro cancer models.
REFERENCES
[1] doi.org/10.1016/j.cell.2017.11.010.
[2] doi.org/10.1039/D2LC00493C.
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