Jianhua Qin Dalian Institute of Chemical Physics Chinese Academy of Sciences
Outline What and why microfluidics? Cancer microenvironment on chip Multicellular organism on chip Summary and futures
Microfluidics (or Lab on a Chip) Fluidic flow Low Reynold number Laminar flow (viscous force) Mass transport (local diffusion) Manipulating small volume of liquid in microchannel (1-1000um)
Why microfluidics? miniaturization integration Low consumption High throughput To integrate chemical and biological processes into a single device.
Interdisciplinary science Biology Chemistry Bioengineering Physics Medicine Pharmacy
Enable technology in life sciences High throughput droplet PCR Nature biotechnol 2009 Recreating the perivascular niche ex vivo Biotechnology and Bioengineering 2010 Integrated sandwich immunoassay Analytical Chemistry 2005 Modeling organ-level lung functions on a chip Science 2010
Enable technology in life sciences HIV tests (blood, plasma or oral fluid) Cholesterol test Intestine (blood) Urine infection Desired point of care (POC) analyser Area of application: ambulance sport center police health center at home
Unique platform for cells-based studies Comparable dimension to cell size Well-defined controllable conditions Amenable for high throughput assay mimicking physiological in vivo environment Low reagents and cost Cell - tissue - organ
What we have done on chip? Microfluidic platform (a) Biomimetic microsystem Microfluidic chips Drug screening Functional elements Droplet control (b) Microvalve Enzymatic reaction Droplet trapping 1 mm (c) Biomaterial synthesis Medical diagnosis Cell pattern Cell culture Cell trapping Bio-inspired applications A B Worm control 2D Separation C
Cell-based studies on chip Cell metabolism Cell-based drug screening organism-based drug screening GPCR receptor screening Modeling wound healing Stem cell differentiation
Complex cancer microenvironment Major factor that affect the efficacy of anticancer drug action Cell-cell, cell-matrix, cell-molecules
Why do people die from cancer? Hallmark of cancer Metastasis! Extracellular matrix (ECM) Stromal cells How neighboring cells involve in cancer invasion is not clear.
Mimicking cancer microenvironment Oral cancer normal salivary gland tissue Cancer tissue ECM fibroblast BM CAFs adenoid cystic cancer(acc) normal glandular epithelium cancer nest Construct cancer microenvironment adenoid cystic cancer cells (ACCs) cancer associated fibroblasts (CAFs) extracellular matrix Current methods to model cancer microenvironment are technically challenging!
Mimicking cancer microenvironment on chip Co-culturing cancer cells and neighboring cells in 3D matrix Schematics of 3D co-culture chip Outlet B Outlet A Inlet A Inlet B (a) Chip fabrication Coating negative photoresist UV Photolithography (first) UV Photolithography (second) (b) Removing photoresist Casting PDMS Peeling PDMS from master PDMS Glass Sealing PDMS layer with glass
Identification of neighboring stromal cells adenoid cystic carcinoma (ACC) tissue Identification of cancer-associated fibroblasts (CAFs) identification Vimentin α-sma Cytokeratin Digestion Centrifuge and collect pellet purification Isolation 24 h Hoechst -PI Fibroblasts purification 6 days
CAFs promoted cancer invasion ACC-M / CAFs ACC-M / HFL1 ACC-M / BME day 0 day 2 day 4 day 6 cancer cells escaped from primary sites in a spheroid fashion spheroid invaded into CAFs-containing matrix instead of normal fibroblast
CAFs promoted cancer invasion Invasive distance and area of ACC-M increased significantly after co-culture with CAFs
MMPs involve in cancer invasion Soluble Membrane Matrix Metalloproteinase MMPs Destroy matrix barriers Degradation to reveal cryptic sites E-cadherin Disruption of Cell-matrix adhesion CD44 Disruption of cell-cell adhesion Release of growth factor
GM6001 (6 days) Invasion area (μm 2 ) Invasion distance (μm) MMPs inhibitors repress CAFs induced invasion ACC-M / CAFs (6 days ) 250 0 μg/ml 200 150 10 μg/ml 50 μg/ml 100 μg/ml 100 50 0 50000 40000 30000 20000 10000 0 10 50 100 1 2 3 4 GM6001 [μg/ml] GM6001 inhibited CAF-promoted invasion in 3D matrix 0 0 10 50 100 1 2 3 4 GM6001 [μg/ml] Neighboring cells CAFs might be a target for cancer treatment. Suitable platform for screening anticancer drugs in physiological conditions. Quantitive data could be obtained in real time.
Multicellular organism on chip Genetics Neurology Aging Small size (1mm) Hermaphroditic Tiny worm-c.elegans Transparent Short life cycle (3.5 d) Complete genome sequence, 70% of human genes have homolog in C. elegans Simple nervous system Drug screening Neurodegenerative disease
Model organism in genetics and neurology 2002 Nobel Prize in Physiology or Medicine Apoptosis Sydney Brenner John Sulston Bob Horvitz 2006 Nobel Prize in Physiology or Medicine RNA interference (RNAi) Andrew Fire Craig C. Mello
Current techniques Manual handling Low throughput No integration Time consuming Petri dish Microfluidic chip Matched size Flexible integration High throughput Individual control
Model for Parkinson s disease (PD) C. elegans PD model normal PD shaking, rigidity slow movement dopaminergic neuronal loss
Recreating Parkinson s disease model on chip Neurotoxin (6-OHDA) induced responses in worm oil inlet neurotoxin worm inlet waste 2 waste 1 Droplet generator Floatage trapper array immobilizing array droplet as bioreactor single worm analysis multiparameter evaluation high throughput screening
Trapping droplets encapsulated with worms Droplets encapsulated with worms Cross section of the chip worm was trapped into the trapper due to the floatage (density: aqueous < oil phase)
Individual worm immobilizing and imaging (i) (ii) (iii) (iv) 1 mm Process of worm immobilization Bright field fluorescence 500 µm 500 μm photograph of eight individual worms immobilized in the immobilization channels
Induced mobility defect in mutant strains CL2166 UA57 Normal movement expressing specific GFP in all eight DA neurons expressing oxidative stress inducible GFP normal movement Abnormal movement worms stroke frequency decreased after treated with 6-OHDA
Induced neuron degeneration in UA57 strain 0mM 6-OHDA 10mM 6-OHDA Loss of GFP expression in the specific DAergic neurons was observed. The degree of neuron degeneration was closely related to mobility disorder.
6-OHDA induced oxidative stress in CL2166 strain The increase of GFP expression was associated with neurotoxin concentrations. Oxidative stress was closely related to mobility disorder. Possible for single animal assay with high throughput Potential platform for multiple conditions screening of anti-pd drugs
Summary and future Microfluidics represents an attractive platform for bioengineering and cancer biology. It is able to mimic more closely the physiological environment that is normally encountered by cells/tissues in the body or even construct organ on a chip. It is of great hope that such technologies will support the use for high throughput drug screening and disease diagnostics. BM CAFs cancer nest
Acknowledgements 1 Ministry of Science and Technology, China 2 Natural Science Foundation of China (NSFC) 3 Chinese Academy of Sciences (CAS) 4 Dalian Institute of Chemical Physics, CAS
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