Rapid Staining of Circulating Tumor Cells in Three-Dimensional Microwell Dialysis (3D-μDialysis) Chip.
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ABSTRACT:
SUBMITTER: Cho W
PROVIDER: S-EPMC5595982 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Study questionCan we reconstitute physiologically relevant 3-dimensional (3D) microengineered endometrium in-vitro model?Summary answerOur representative microengineered vascularised endometrium on-a-chip closely recapitulates the endometrial microenvironment that consists of three distinct layers including epithelial cells, stromal fibroblasts and endothelial cells in a 3D extracellular matrix in a spatiotemporal manner.What is known alreadyOrgan-on-a-chip, a multi-channel 3D microfluidic cell culture system, is widely used to investigate physiologically relevant responses of organ systems.Study design, size, durationThe device consists of five microchannels that are arrayed in parallel and partitioned by array of micropost. Two central channels are for 3D culture and morphogenesis of stromal fibroblast and endothelial cells. In addition, the outermost channel is for the culture of additional endometrial stromal fibroblasts that secrete biochemical cues to induce directional pro-angiogenic responses of endothelial cells. To seed endometrial epithelial cells, on Day 8, Ishikawa cells were introduced to one of the two medium channels to adhere on the gel surface. After that, the microengineered endometrium was cultured for an additional 5-6 days (total ∼ 14 days) for the purpose of each experiment.Participants/materials, setting, methodsMicrofluidic 3D cultures were maintained in endothelial growth Medium 2 with or without oestradiol and progesterone. Some cultures additionally received exogenous pro-angiogenic factors. For the three distinct layers of microengineered endometrium-on-a-chip, the epithelium, stroma and blood vessel characteristics and drug response of each distinct layer in the microfluidic model were assessed morphologically and biochemically. The quantitative measurement of endometrial drug delivery was evaluated by the permeability coefficients.Main results and the role of chanceWe established microengineered vascularised endometrium-on-chip, which consists of three distinct layers: epithelium, stroma and blood vessels. Our endometrium model faithfully recapitulates in-vivo endometrial vasculo-angiogenesis and hormonal responses displaying key features of the proliferative and secretory phases of the menstrual cycle. Furthermore, the effect of the emergency contraception drug levonorgestrel was evaluated in our model demonstrating increased endometrial permeability and blood vessel regression in a dose-dependent manner. We finally provided a proof of concept of the multi-layered endometrium model for embryo implantation, which aids a better understanding of the molecular and cellular mechanisms underlying this process.Large scale dataN/A.Limitations, reasons for cautionThis report is largely an in-vitro study and it would be beneficial to validate our findings using human primary endometrial cells.Wider implications of the findingsOur 3D microengineered vascularised endometrium-on-a-chip provides a new in-vitro approach to drug screening and drug discovery by mimicking the complicated behaviours of human endometrium. Thus, we suggest our model as a tool for addressing critical challenges and unsolved problems in female diseases, such as endometriosis, uterine cancer and female infertility, in a personalised manner.Study funding/competing interest(s)This work is supported by funding from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) to Y.J.K. (No. 2018R1C1B6003), to J.A. (No. 2020R1I1A1A01074136) and to H.S.K. (No. 2020R1C1C100787212). The authors report no conflicts of interest.
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Project description:The performance of current microfluidic methods for exosome detection is constrained by boundary conditions, as well as fundamental limits to microscale mass transfer and interfacial exosome binding. Here, we show that a microfluidic chip designed with self-assembled three-dimensional herringbone nanopatterns can detect low levels of tumour-associated exosomes in plasma (10?exosomes??l-1, or approximately 200 vesicles per 20??l of spiked sample) that would otherwise be undetectable by standard microfluidic systems for biosensing. The nanopatterns promote microscale mass transfer, increase surface area and probe density to enhance the efficiency and speed of exosome binding, and permit drainage of the boundary fluid to reduce near-surface hydrodynamic resistance, thus promoting particle-surface interactions for exosome binding. We used the device for the detection-in 2??l plasma samples from 20 ovarian cancer patients and 10 age-matched controls-of exosome subpopulations expressing CD24, epithelial cell adhesion molecule and folate receptor alpha proteins, and suggest exosomal folate receptor alpha as a potential biomarker for early detection and progression monitoring of ovarian cancer. The nanolithography-free nanopatterned device should facilitate the use of liquid biopsies for cancer diagnosis.
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Project description:Glioblastoma multiforme (GBM) is a rapidly progressive and deadly form of brain tumor with a median survival rate of ~15 months. GBMs are hard to treat and significantly affect the patient's physical and cognitive abilities and quality of life. Temozolomide (TMZ)-an alkylating agent that causes DNA damage-is the only chemotherapy choice for the treatment of GBM. However, TMZ also induces autophagy and causes tumor cell resistance and thus fails to improve the survival rate among patients. Here, we studied the drug-induced programmed cell death and invasion inhibition capacity of TMZ and a mevalonate cascade inhibitor, simvastatin (Simva), in a three-dimensional (3D) microfluidic model of GBM. We elucidate the role of autophagy in apoptotic cell death by comparing apoptosis in autophagy knockdown cells (Atg7 KD) against their scrambled counterparts. Our results show that the cells were significantly less sensitive to drugs in the 3D model as compared to monolayer culture systems. An immunofluorescence analysis confirmed that apoptosis is the mechanism of cell death in TMZ- and Simva-treated glioma cells. However, the induction of apoptosis in the 3D model is significantly lower than in monolayer cultures. We have also shown that autophagy inhibition (Atg7 KD) did not change TMZ and Simva-induced apoptosis in the 3D microfluidic model. Overall, for the first time in this study we have established the simultaneous detection of drug induced apoptosis and autophagy in a 3D microfluidic model of GBM. Our study presents a potential ex vivo platform for developing novel therapeutic strategies tailored toward disrupting key molecular pathways involved in programmed cell death and tumor invasion in glioblastoma.
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