Development of encapuslated stem cells formed into a 3D complex transplantable device using microfluidic technology
Dr Botelho, Michael George (Principal investigator)
Stem cell, tissue engineering, Microfluidics, bone regeneration
Dentistry,Stem Cell Biology
Block Grant Earmarked for Research (104)
HKU Project Code
Seed Fund for Basic Research
Encapsulation materials have been designed for the dynamic control of the cell microenvironment for nutritional and gaseous exchanges which has been mainly performed in vitro or in animal models. However, recently there has been the design to create an extracellular matrix (ECM) for cell viability, cell stability and differentiation and proliferation using growth factors peptides incorporated into the encapsulation material. Biopolymers such as, alginate has been used for several decades in cell encapsulation for pancreatic islet cells, because of their mild, non-cytotoxic synthesis, high biocompatibility (95% cell viability), high permeability for nutrients and metabolic waste products, mechanoprotection and shielding from host immune cells. The chemical versatility of alginate polymer means that it can be chemically modified quite easily and with a diverse range of synthetic molecules and biomolecules to achieve increased strength, and better bioresponsiveness and biological resorption Recently a number of research groups have used alginates in microfluidic and mesofluidic techniques for encapsulation of biological molecules and cells. This allows the creation complex biological cell arrangements with the necessary physical and biochemical architecture for potential use as a transplantable structure. Therefore, microfluidic generated structures have potential to outperform other methods of manufacture in: 3D cell cultures for regenerative tissue engineering. Microfluidics permits management of multiple fluids in networks of capillary channels to produce droplets with complex structures and compositions mimicking the intricacy of biological systems. Microfluidic technology for cell encapsulation, allows more precise control over the cell microenvironment in its structure and composition. This allows creation of entities with a defined biological function that more accurately mimic the complexity of native cellular environments (Leclerc et al. 2003). There is considerable scope in using microfluidics for tissue engineering and regenerative medicine particularly in the manufacture of complex 3D tissue units for grafting in various tissues (Bettinger & Borenstein 2010). This will be the focus of this study- to generate an new and enhanced alginate biomatrix for the creation of capsules for MSCs using microfluidics. Critically we will orchestrate key growth factors, ECM proteins and signaling molecules in a correctly sequenced and timely manner. In addition we will create tissue engineered scaffolding for mineral nodules suitable for bone grafting to treat a variety of bone tissue traumas, lesions and fractures. From this, it is expected that cellular 3D microenvironments can be formed for the delivery and regulation of important regeneration factors both in time and space to MSCs so as to create communities of cells in a tissue like cell framework. In doing so, it is expected that bone production can be accelerated, be available in larger volumes and in a shorter time to build better quality bone forming tissue units. In other words, the isolated MSCs require systematic treatment with proper sequencing of signaling molecules that allow their selective differentiation into the desired cell type or regenerative niche that can produce the required tissues. 1) To enhance the biochemical make up of alginate used for encapsulation of MSCs to support MSCs and biological crosslinking of the alginate to improve strength After this MSCs will be encapsulated in this enhanced alginate using a microfluidic system Here, we will engineer the capsule environment to ensure the intrinsic "stemness" properties of MSCs are sustained prior to induction. We will optimize the environment to maximize stem cell viability, proliferation and stability by addition of key ECM molecules (Collagen I and RGD peptides) in the alginate as well as optimisation of cell densities. After this we will construct a new microfluidic capillary device for the automated encapsulation of MSCs (at high densities 250,000/ 50 micron capsule) inside alginate hydrogel beads using water-oil-water microscale double emulsion (Martinez et al. 2012) 2) The encapsulated MSCs will then be injected with "guest" alginate microbeads containing osteoinductive growth factors for controlled release in sequence and time. Another issue is to deliver growth factors (BMP-2, TGF-beta) to the encapsulated MSCs at the right time for a defined period and in a targeted space within the system. This will be achieved by nesting small guest microcapsules inside the host MSC capsule. Microfluidic technology is very precise in making such systems and the desired complex structures (Harink et al. 2013). It does this rapidly and in large numbers that make it ideal for therapeutic applications. 3) Establish the most effective controlled GF release system that facilitates regeneration rates of osteoid tissue and increase volumes of new bone like tissue compared with conventional encapsulated MSCs. The final part of the study will be test and validate mineralize tissue formation within the structured capsules and compare the formation properties with ordinary encapsulated MSCs and MSCs grown in normal 2D static cultures and 3D pellet cultures.