Advanced 3D cell culture techniques in micro-bioreactors, Part II: Systems and applications

In this second part of our systematic review on the research area of 3D cell culture in micro-bioreactors we give a detailed description of the published work with regard to the existing micro-bioreactor types and their applications, and highlight important results gathered with the respective systems. As an interesting detail, we found that micro-bioreactors have already been used in SARS-CoV research prior to the SARS-CoV2 pandemic. As our literature research revealed a variety of 3D cell culture configurations in the examined bioreactor systems, we defined in review part one “complexity levels” by means of the corresponding 3D cell culture techniques applied in the systems. The definition of the complexity is thereby based on the knowledge that the spatial distribution of cell-extracellular matrix interactions and the spatial distribution of homologous and heterologous cell–cell contacts play an important role in modulating cell functions. Because at least one of these parameters can be assigned to the 3D cell culture techniques discussed in the present review, we structured the studies according to the complexity levels applied in the MBR systems

Design and fabrication of microfluidic valves using poly(N-isopropylacrylamide)

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.In title on title page, "N" appears as italic.Includes bibliographical references (leaves [181]-185).A compact printable microfluidic valve composed of poly(N-isopropylacrylamide) has been designed, fabricated, and tested. The design of the valve consists of filling microwells with poly(NIPAAm) and bonding PDMS channels above them. This filling is achieved using thermal inkjet printing of a prepolymer solution and subsequent polymerization using UV irradiation. When the gel is swollen, it blocks flow from passing through the channel. Upon heating, the gel shrinks and allows flow in the channel. Poly(NIPAAm) is a thermosensitive hydrogel that exhibits an inverse temperature expansion behavior. When the temperature of the swollen gel is raised above a lower critical solution temperature (LCST) of approximately 32°C, the gel becomes hydrophobic. This change in hydrophobicity results in expulsion of the water molecules from within the hydrogel network, thus resulting in shrinking of the gel. By adding magnetic nanoparticles to the hydrogel and exposing it to an external magnetic field, volumetric change of the hydrogel can be locally and externally induced. External heating of the magnetic nanoparticles, however, is not included in this thesis. In order to ensure shrinkage that is predictable in favor of flow control, microanchor structures have been designed, modeled, and fabricated at the bottom of the microwells. These microanchors hold the poly(NIPAAm) at the bottom of the plug such that the shrinkage of the gel always acts to open the flow channel at the top yielding a minimum pressure drop. Design decisions were made using the principles of Axiomatic Design in order to minimize the response time and pressure drops in the valve. Modeling of the underlying mechanisms is described along with the application of these models to the final device. Results of fabrication suggest the feasibility while also eliciting possible improvements to the fabrication process.(cont.) Profilometry measurements of the swollen and shrunken valves reveal flow control operation as intended. Additionally, design and modeling of magnetic heating using mixed-in nanoparticles is presented. A fabrication plan designed to include this mechanism is proposed.by Nathan Edward Reticker-Flynn.S.M

Design and Fabrication of Cell-laden Gelatin Methacrylated Hydrogel Scaffold for Improving Biotransportation

One of the main goals of Tissue Engineering (TE), which has been developed rapidly over the recent years, is to re-create organs or tissues in vitro or in vivo with mimicked the anatomy and functions of body systems. Nowadays, replacing damaged tissues or organs has been a main focus in this field for addressing a significant shortage of donor tissues. Vascularisation plays a crucial role in supplying cells and tissue with essential oxygen and nutrients and removing waste products from the engineered tissue constructs. Any issue in nutrient perfusion and mass transport could significantly restrict construct development to dimensions smaller than clinically useful size, thus limiting the ability for in vivo integration. The main objectives of this study are to develop a novel framework for computational design using topology optimisation and microfabrication of 3D scaffolds using gelatin-based hydrogels (GelMa), allowing artificial vascularisation in vitro for testing if the framework is valid through the investigation into cellular viability inside the construct. In this thesis, computational models were first generated to simulate oxygen transport through solving the diffusion equation. The diffusion models are then used to optimise scaffold topology. By means of microfabrication technologies, hydrogel-based constructs were fabricated to prototype the sophisticated scaffolds. Cellular viability study was also performed to validate computational simulations and design. The results showed a higher cellular survival rate in optimally patterned constructs than the control. In summary, the work presented here is not only technically simple and cost-effective, but also establishes an effective approach to the design and fabrication of a vascularised biodegradable and scaffold-free constructs. The proposed methodology will be of considerable implication for engineering bulk tissue constructs which require sufficient ongoing vascularization in the future

Towards rapid 3D direct manufacture of biomechanical microstructures

The field of stereolithography has developed rapidly over the last 20 years, and commercially available systems currently have sufficient resolution for use in microengineering applications. However, they have not as yet been fully exploited in this field. This thesis investigates the possible microengineering applications of microstereolithography systems, specifically in the areas of active microfluidic devices and microneedles. The fields of micropumps and microvalves, stereolithography and microneedles are reviewed, and a variety of test builds were fabricated using the EnvisionTEC Perfactory Mini Multi-Lens stereolithography system in order to define its capabilities. A number of microneedle geometries were considered. This number was narrowed down using finite element modelling, before another simulation was used to optimise these structures. 9 × 9 arrays of 400 μm tall, 300 μm base diameter microneedles were subjected to mechanical testing. Per needle failure forces of 0.263 and 0.243 N were recorded for the selected geometries, stepped cone and inverted trumpet. The 90 μm needle tips were subjected to between 30 and 32 MPa of pressure at their failure point - more than 10 times the required pressure to puncture average human skin. A range of monolithic micropumps were produced with integrated 4 mm diameter single-layer 70 μm-thick membranes used as the basis for a reciprocating displacement operating principle. The membranes were tested using an oscillating pneumatic actuation, and were found reliable (>1,000,000 cycles) up to 2.0 PSIG. Pneumatic single-membrane nozzle/diffuser rectified devices produced flow rates of up to 1,000 μl/min with backpressures of up to 375 Pa. Another device rectified using active membrane valves was found to self-prime, and produced backpressures of up to 4.9 kPa. These devices and structures show great promise for inclusion in complex, fully integrated and active microfluidic systems fabricated using microstereolithography alone, with implications for both cost of manufacture and lead time

DEVELOPMENT OF INNOVATIVE MULTICOMPARTMENT MICROFLUIDIC PLATFORMS TO INVESTIGATE TRAUMATIC AXONAL INJURY

Compartmentalization of cell body from the axon of a neuron is an important aspect in studying the influence of microenvironments. Microenvironment is an integral part of neuronal studies involving traumatic axonal injuries (TAI). While TAI is one of the possible outcomes of various forms of traumatic insults to the central nervous system (CNS) and peripheral nervous system (PNS), many of the mechanistic details are still unknown, it can be agreed that the level of injury often dictates the functional deficit. This motivates the question, what is occurring at both the morphological and biomolecular scale in CNS and PNS axons during and throughout the recovery phase after injury? And, are there any treatment strategies that could be applied to improve the recovery and regeneration of the axons subject to TAI? Motivated by this, I propose to develop novel microfluidic platforms as a part of my master’s thesis to allow unprecedented, physiologically relevant focal and graded mechanical injury and observation to both CNS and PNS axons. My research for this thesis can be broadly classified into two fold. 1) I examined the regenerative effects of the members of the Glial cell line-derived neurotrophic factor (GDNF), a family of neurotrophic factors after axotomy. This work resulted in the discovery of the fact that GDNF is the most potent neurotrophic factor among the family of growth factors for axon regeneration in dorsal root ganglion (DRG) neurons after in vitro axotomy. It was also found that GDNF locally applied to cell body better promotes axonal regeneration in comparison to when applied locally to axons. 2) Development and refinement of existing axon injury microplatform (AIM) to closely mimic physiological conditions during traumatic injury in CNS neurons. This work was my attempt in improving already existing microfluidic compression platform. I successfully developed a displacement control injury device and demonstrated displacement control as a proof of principle. Further development of these microfluidic platforms will significantly contribute to the field of basic neuroscience, neurobiology, and biomedical engineering