Singapore-MIT Alliance for Research & Technology

Biosystems and Micromechanics

#04-13/14 Enterprise Wing and #B-101
Singapore 138602

BioSystems and Micromechanics (BioSyM) - IRG

Research Projects Under Thrust 3: In-Vitro Cellular Systems Engineering

Thrust 3  focuses on understanding of emergent behaviours of cell clusters in-vitro through analysis based on integrated nano / microscale optical, microfluidics and mechanical experiments and modelling. Such understanding can possibly be exploited for drug screening / development, regenerative medicine and cancer treatment.

1. Microfluidic Co-culture Platform for Studying Epithelial Mesecnchymal Transistions (EMT) in 3D Scaffolds

(Roger Kamm (MIT/SMART-BioSyM), Jean Paul Thiery (A*STAR IMCB), Sun Wei (SMART-BioSyM), Ruby Yun Ju Huang (CSI/A*STAR IMCB), Weimiao Yu (A*STAR IMCB), Ting Yuan Tu (NUS/SMART-BioSyM), Sim Weng Jing (A*STAR IMCB) and Meng Kang (CSI)

Epithelial–mesenchymal transition (EMT) is an indispensable mechanism during morphogenesis, as without mesenchymal cells, tissues and organs will never be formed. Transitions between epithelial and mesenchymal states have crucial roles in embryonic development and without EMT, in which polarized epithelial cells are converted into motile cells, multicellular organisms would be incapable of progressing past the blastula stage of embryonic development. EMT provides a new basis for understanding the progression of carcinoma towards dedifferentiated and more malignant states. Some of these subpopulations may exhibit more differentiated features, whereas others have characteristics of stem cells. In recognition of the importance of these tumor-associated phenotypes in metastasis and cancer-related mortality, targeting the products of such cellular plasticity is an attractive but challenging approach that is likely to lead to improved clinical management of cancer patients.

The main challenge is to unravel how growth factors, scatter factors and ECM components cooperate to induce EMT. By understanding the processes that trigger EMT, we might be able to prevent it. This therapeutic strategy has the potential to block metastasis and, perhaps, also prevents cancer recurrence, because micro-metastases often remain after conventional surgery, radiotherapy and/or chemotherapy. 

The research goal is to develop an intermediate drug assay model capable of monitoring the inhibition of cancer cells break loose from the primary tumor (EMT) in 3D culture, aiming to prevent cancer metastasis. We optimized the microfluidic device co-culturing cancer and endothelial cells for observing EMT that leads to the 3D dissemination of cancer cells. With quantitative data analysis methods developed, our model is able to assess the efficacy of anti-invasion cancer drugs at various doses, thus validating the system for drug screening.

Selected publications

  • Amir Aref, Ruby Huang, Jean Paul Thiery and Roger Kamm, "Transitions between epithelial and mesenchymal states in Microfluidic platform: Aquisition of malignant and stem cell traits", 6th World Congress of Biomechanics, 1-6 August 2010, Singapore, Proceedings pp.553-554
  • A.R.Aref, R.Y.J. Huang, W. Kang, S.W. Jing, J.P.Thiery, R.D.Kamm, "Microfluidic Co-culture Platform for Studying EMT in 3D Scaffolds", 14th International Biotechnology Symposium and Exhibition, 14-18 September 2010, Rimini, Italy.

2. Control of sprout development in angiogenesis

(Harry Asada (SMART-BioSyM), Roger Kamm (MIT/SMART-BioSyM), Ong L.Sharon (SMART-BioSyM), Min Cheol Kim (SMART-BioSyM), Waleed Farhat (MIT), Levi Wood (MIT), Alisha Schor (MIT) and Devin Niel (MIT)

Angiogenesis is the process of generating a vascular network from an existing blood vessel. A population of Endothelial Cells (ECs) residing in a blood vessel can sprout out and create a new vascular network when exposed to growth factors. Cells communicate with each other and interact with the matrix field in a stochastic manner, leading to pattern formation as a result of collective cell behaviors.

Project 2
Project 2
Angiogenic sprouting process. (a) ECs residing in a blood vessel sprout out in response to growth factor molecules. (b) A leading cell, called a tip cell, detects gradients in certain growth factors and migrates toward the source while stalk cells follow behind the tip. (c) New sprouts branch at multiple stages to form a new vascular network.
In vitro micro-fluidic device for the angiogenic EC sprouting

Our goal is to establish feedback control to reduce variance in angiogenic development and form desired vascular geometries and patterns. We have developed an open-loop map between system cues (exogenous inputs) and angiogenic response over 2-4 days and automated confocal image processing methods to evaluate the data. We have proposed models and control algorithms to control sprout elongation rate by modulating the chemotactic gradient. Combining these relationships will be essential to providing a closed loop control scheme that regulates sprout geometry.

Selected publications

  • Wood, L., Kamm, R., Asada, H., "Stochastic Modeling and Identification of Emergent Behaviours of an Endothelial Cell Population in Angiogenic Pattern Formation", International Journal of Robotics Research (Published Online, March 2011).

3. Visualization of Angiogenic Pattern Formation Applying Hybrid Stochastic Dynamic Modeling to the Endothelial Cells Migration in the Microfluidic in vitro Environment

(Harry Asada (SMART-BioSyM), Roger Kamm (MIT/SMART-BioSyM), Peter Chen (NUS), Kim Min Cheol (SMART-BioSyM), Choong Kim (SMART-BioSyM), Levi Wood (MIT) and Devin Niel (MIT)

The objective of this project is to develop a computational methods that can visualize tempo-spatial distribution of the multiple cells in the event of the angiogenic sprouting formation governed by novel dynamic modelling of integrated cell migration based experimental observations of focal adhesion dynamics, cytoskeleton remodelling and actin motor activity.

The dynamics of the tip cell and stalk cells migration in the gel matrix field are computationally modeled as a 3-dimensional stochastic agent that detects the gradient of growth factors (VEGF) by extending its filopodia in diverse direction and 2-dimensional stochastic agents that craw on the surface of conduit wall, respectively. The objective of this project is to develop a computational tool that can visualize tempo-spatial distribution of the multiple cells in the event of the angiogenic sprouting formation that governed by novel hybrid stochastic dynamics based experimental observations.

4. Spatio-Temporal Image Analysis of Cell Sprouting with Bayesian Estimation

(Harry Asada (SMART-BioSyM), Ang Marcelo (NUS), Ong L.Sharon (SMART-BioSyM)

The project goal is to develop an automated analysis of the growth of endothelial cell sprouts in in-vitro experimentation using time-lapse and fixed images from confocal microscopy to aid the understanding of cell behaviors and interactions. We approach these multi-cell tracking challenges via probabilistic methodologies. A Kalman filtering combined with Multiple Hypothesis Testing (MHT) and smoothing/retrodiction is proposed to allow tracking of varying cell dynamics and account for clutter due to close contact cells. In addition to that, probabilistic techniques are used to incorporate fixed end-point imaging data with time-lapse information in a mathematically consistent manner.

Selected publications

  • Lee-Ling S. Ong, Marcelo H. Ang Jr., and H. Harry Asada, "Tracking of Cell Population from Time Lapse and End Point Confocal Microscopy Images with Multiple Hypothesis Kalman Smoothing Filters", In: Proceedings of the IEEE Computer Society Conference on Computer Vision and Pattern Recognition Workshops, (CVPRW), 2010, Pages 71—78, San Francisco, CA, 13-18 June 2010.

5. Manipulation of Stiffness Gradients in Extracellular Microenvironment through Stochastic Control of Magnetic-Particle Ensemble

(Peter Chen (NUS), Harry Asada (SMART-BioSyM), Sahan Christie Bandara Herath (NUS)

The mechanical properties of microstructures that surround cells play an important role in determining the behavior of a cell population, including differentiation, proliferation, and apoptosis. The objective of this project is to develop engineering approaches to directly manipulate the extracellular microenvironment in order to produce desired changes in its stiffness.

6. A new in-vivo-mimic 3D monitoring microfluidic platform: migratory behavior via release of angiogenic growth factors from cell encapsulated beads

(Roger Kamm (MIT/SMART-BioSyM), Harry Asada (MIT/SMART-BioSyM), Seok Chung (Korea Univ.), Choong Kim (SMART-BioSyM), Kim Min Cheol (SMART-BioSyM)

Cell encapsulations are promising for the purpose of cellular therapy and tissue engineering. This technology is based on the immobilization of different types of cells within a polymeric matrix surrounded by a semipermeable membrane for the long-term release of therapeutics. The research goal is to develop an in vivo-mimic model capable of 3D monitoring of cellular behaviours using microencapsulation & microfluidic technologies. We target to gain more information on cellular behaviors, such as cell growth and cell migration, depending on the release of angiogenic growth factors from cell encapsulated beads with our microfluidic platform integrated cell encapsulation technology.

7. Prolyl Hydroxylases-inhibitors Induced Angiogenesis in a Microfluidic Device

(Roger Kamm (MIT/SMART-BioSyM), Michael Raghunath (NUS) and Lim Sei Hien (NUS/SMART-BioSyM)

Angiogenesis has been implicated in more than 70 disorders so far such as the ischemic heart disease, respiratory distress and etc. Consequently, various approaches to overcome these diseases have been proposed, including transfected cells expressing angiogenic peptides, local delivery of angiogenic peptides and many others. Nonetheless, those approaches are less preferred as they deal with one or few angiogenic factors at a time which is different from the physiological conditions where the angiogenesis is regulated by at least 20 angiogenic growth factors such as VEGF, placental growth factor, interleukin-8 and etc while many others are yet to be identified.

The main goal of this project is to study the angiogenic effect of PHi (prolyl hydroxylase inhibitors) on HUVEC cells in a defined way where the concentration gradient of growth factors can be established while the angiogenesis could be observed in a 3D scaffold which is a step closer to the in vivo condition.

8. Three-Dimensional Microfluidic Bioassay for Early Differentiation of Murine Embryonic Stem Cells

(Roger Kamm (MIT/SMART-BioSyM) and Young Kum Park (SMART-BioSyM)

Embryonic stem cells cultured in a three-dimensional collagen gel scaffold within a microfluidic device were investigated by live confocal microscopic imaging and IMARIS analysis. Using an efficient three-dimensional microfluidic bioassay, we qualitatively and quantitatively determine the differentiation ratio of embryonic stem cells based on the expression of a vascular progenitor cell marker, Flk-1.

9. In Vitro Preclinical Microfluidic Screening for Drug-Like Molecules as Cardiomyocyte Differentiation Agents

(Roger Kamm (MIT/SMART-BioSyM), Mayasari Lim (NTU), Young Kum Park (SMART-BioSyM), Ong L.Sharon (SMART-BioSyM), Se Young Yang (MIT) and Devin Niel (MIT)

There is increasing importance in the study of screening drug-like small molecules to induce cardiomyocyte differentiation from ES cells by way of a robust and dependable bioassay. However, an efficient and cost-effective in vitro screening for this investigation has not been successfully developed yet. Here, we focus on the development of an ES cell-based microfluidic device with multi-parametric control, high-throughput, and time-lapse imaging for screening drug-like target molecules on cardiomyocyte differentiation.

10. Mesenchymal Stem Cells paracrine contributions on vascular formation and stabilization

(Jerry Chan (NUS-Duke/KKH), Roger Kamm (MIT/SMART-BioSyM) and Liu Yuchun (NUS)

MSC paracrine activity stimulates repair at the wound site through release of angiogenic factors. Here, we aim to investigate the effect of these factors present in MSC conditioned media on EC behaviour in vitro in a microfluidic platform, identify the critical factors that stimulate angiogenesis, followed by ischemic rescue upon bolus delivery of MSC conditioned media in vivo in an ischaemic animal study model. 

Here we (a) investigate the effect of MSC conditioned media (MSCCM) culture on EC growth, proliferation and survival in vitro; (b) identify the angiogenic proteins present in MSCCM through a proteomic approach.; (c) study the migratory behaviour of EC as well as vascular stability/(disintegration) upon MSCCM  treatment in the microfluidic devices over extended durations under real-time imaging; (d) isolate angiogenic proteins for use as treatment groups in microfluidic devices to study their involvement in vascular formation/stabilization and thus, identify critical angiogenic factors; (e) investigate the effect of in vivo injection of bolus MSCCM/ isolated factors on limb recovery in a hindlimb ischaemic animal model.