Microdevices and Microsystems for Cell Manipulation

Microfabricated devices and systems capable of micromanipulation are well-suited for the manipulation of cells. These technologies are capable of a variety of functions, including cell trapping, cell sorting, cell culturing, and cell surgery, often at single-cell or sub-cellular resolution. These functionalities are achieved through a variety of mechanisms, including mechanical, electrical, magnetic, optical, and thermal forces. The operations that these microdevices and microsystems enable are relevant to many areas of biomedical research, including tissue engineering, cellular therapeutics, drug discovery, and diagnostics. This Special Issue will highlight recent advances in the field of cellular manipulation. Technologies capable of parallel single-cell manipulation are of special interest

Fabrication on the microscale : a two-photon polymerized device for oocyte microinjection

Open Access funding enabled and organized by CAUL and its Member Institutions. KRD is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58–2019). KD acknowledges funding from the UK Engineering and Physical Sciences Research Council (grant EP/P030017/1). This study was funded by the Australian Research Council (ARC) Centre of Excellence for Nanoscale BioPhotonics (CE140100003).Purpose Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection. Methods The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-μm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes). Results Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection. Conclusion This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.Publisher PDFPeer reviewe

Factories of the Future

Engineering; Industrial engineering; Production engineerin

Reinventing microinjection : new microfluidic methods for cell biology

Regulatory processes are responsible for the organization, division and death of cells in multicellular organisms such as humans. Additionally, cells are highly regulated internally, able to survive and respond in vastly different micro-environments. Many types of interactions of cells with their environment can be distinguished, and need to be controlled in experiments aimed at unravelling and predicting cellular behavior in vivo. The in vivo microenvironment is mimicked by exposing cells to complex and changing environments. To describe the stochastic differences between cells and the local experimental conditions in sufficient detail and to obtain statistically relevant results, high-throughput experimentation is required. In this thesis four new research methods are developed, aimed at a deeper understanding of cellular regulation in vivo.Märzhäuser, ZF-screens BV, Eppendorf, Life Science Methods BVUBL - phd migration 201

Factories of the Future

Engineering; Industrial engineering; Production engineerin

Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo typesñYsmall molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery

Development of three-dimensional patterning strategies for osteochondral tissue engineering

Fully-realised three-dimensional patterning strategies enable the development of heterogeneous constructs which can recreate tissue architecture and cellular microenvironments over a large range of length scales. This in turn allows the development of more effective tissue models and tissue engineering therapies. The work presented in this thesis was designed to address the development of patterning methodologies and compatible biomaterial formulations. Poly(lactic-co-glycolic acid)-based (PLGA-based) microspheres were utilised for temporally-controlled protein delivery. Robust protocols were developed for the production of microspheres with two different mean sizes to provide distinct release kinetics which could be further tailored by the addition of a PLGA-poly(ethylene glycol)-PLGA (PLGA-PEG-PLGA) triblock copolymer. A semi-automated microinjection/micromanipulation (MM) system was used to precisely position individual microspheres into cell culture substrates. This approach has the potential to replicate complex interacting signal environments as seen in developmental and repair processes. Demineralised bovine bone, processed with or without a decellularisation step, was enzymatically digested to form solutions capable of gelation under physiological conditions. The resulting hydrogels outperformed collagen as in vitro culture substrates for bone-derived cells and are promising injectable scaffold materials. They were also formed into beads which could encapsulate exogenous proteins and which may be utilised in MM-based patterning strategies. Bioplotting was used to produce alginate hydrogel constructs containing highly viable cell populations. This technique was also used to deposit a PLGA-PEG microparticulate material which could be sintered under physiological conditions to achieve bone appropriate mechanical properties. PLGA-PEG/alginate dual material constructs could also be produced incorporating independent patterns of these two materials and of two cell populations and two protein signals. Bioplotting could therefore be used to produce sophisticated tissue engineering constructs for the repair of large, complex defects. Though this work focused on osteochondral applications much of the data is also more widely-applicable