Development and characterisation of a cytokine supplemented serum free medium formulation for bone marrow derived mesenchymal stromal cells

Eng D ThesisBackground: The discovery and subsequent characterisation of adult human-derived stem cells appears poised to help revolutionise the fields of regenerative medicine and tissue engineering, offering clinicians the opportunity to develop fully functional replacement tissues, alongside holding significant promise as next generation gene or protein delivery vehicles. Despite their apparent potential however, current stem cell-based therapeutics typically rely on delivering massive doses of cells to sites of injury in order to help ensure adequate cell survival in the highly detrimental microenvironments presented by damaged and degrading biological material. In order to produce the cell numbers required for these types of treatments, relatively modest donor cell populations are subjected to extended periods of in vitro expansion, within highly regulated GMP culture conditions. One crucially important aspect of this manufacturing framework is the requirement for xeno-free expansion systems, including the use of serum-free culture medium. Unfortunately, whilst a number of functional serum-free media formulations are commercially available at the present time, their proprietary nature makes them both highly expensive and wholly unsuitable for use in academic research. Aims: The goal of this project is to begin development of a characterised cytokinesupplemented serum-free medium formulation using a design of experiments-based methodology. Methods: Primary bone marrow-derived mesenchymal stromal cells (BM-MSCs) were isolated, characterised and used to screen a series of selected cytokines and growth supplements for their ability to successfully support cell proliferation and continued survival in the absence of serum. Cells cultured in the resultant serum-free formulation were then compared to those grown in conventional medium in regard to genetic, metabolic and morphological factors. In addition, the impact of batch-to-batch variability on BM-MSC growth and metabolic activity was assessed as a means of determining the potential impact of raw material variation on cell quality and any related manufacturing processes. Finally, a number of different extracellular matrix proteins were also screened for the purpose of mediating cell-surface interactions in serum-free conditions. ii Results: We successfully identified a cytokine supplemented medium preparation capable of supporting the proliferation of BM-MSCs during serum-free culture. Evaluation of cells expanded in this medium provided evidence of altered secretory and genetic characteristics leading to shifted therapeutic potential. Furthermore, the identification of a combination of different extracellular matrix proteins able to enhance cell adhesion in the absence of serum served to provide the beginnings of a complete serum-free formulation. In addition, FBS batch variability was shown to have significant effects on cell proliferation and gene expression, including a number of genetic markers linked to differentiation potential and lineage specificity. Conclusions: We offer a new serum-free medium formulation for use in the expansion of primary BM-MSCs, alongside providing evidence of the impact of raw material variability on the therapeutically relevant characteristics of these cells

Delivery and use of individualised feedback in large class medical teaching

Background: Formative feedback that encourages self-directed learning in large class medical teaching is difficult to deliver. This study describes a new method, blueprinted feedback, and explores learner’s responses to assess its appropriate use within medical science teaching. Methods: Mapping summative assessment items to their relevant learning objectives creates a blueprint which can be used on completion of the assessment to automatically create a list of objectives ranked by the attainment of the individual student. Two surveys targeted medical students in years 1, 2 and 3. The behaviour-based survey was released online several times, with 215 and 22 responses from year 2, and 187, 180 and 21 responses from year 3. The attitude-based survey was interviewer-administered and released once, with 22 responses from year 2 and 3, and 20 responses from year 1. Results: 88-96% of learners viewed the blueprinted feedback report, whilst 39% used the learning objectives to guide further learning. Females were significantly more likely to revisit learning objectives than males (p = 0.012). The most common reason for not continuing learning was a ‘hurdle mentality’ of focusing learning elsewhere once a module had been assessed. Conclusions: Blueprinted feedback contains the key characteristics required for effective feedback so that with further education and support concerning its use, it could become a highly useful tool for the individual and teacher

Dry powder process for preparing uni-tape prepreg from polymer powder coated filamentary towpregs

A process for preparing uni-tape prepreg from polymer powder coated filamentary towpregs is provided. A plurality of polymer powder coated filamentary towpregs are provided. The towpregs are collimated so that each towpreg is parallel. A material is applied to each side of the towpreg to form a sandwich. The sandwich is heated to a temperature wherein the polymer flows and intimately contacts the filaments and pressure is repeatedly applied perpendicularly to the sandwich with a longitudinal oscillating action wherein the filaments move apart and the polymer wets the filaments forming a uni-tape prepreg. The uni-tape prepreg is subsequently cooled

Micromanipulation of InP lasers with optoelectronic tweezers for integration on a photonic platform

The integration of light sources on a photonic platform is a key aspect of the fabrication of self-contained photonic circuits with a small footprint that does not have a definitive solution yet. Several approaches are being actively researched for this purpose. In this work we propose optoelectronic tweezers for the manipulation and integration of light sources on a photonic platform and report the positional and angular accuracy of the micromanipulation of standard Fabry-Pérot InP semiconductor laser die. These lasers are over three orders of magnitude bigger in volume than any previously assembled with optofluidic techniques and the fact that they are industry standard lasers makes them significantly more useful than previously assembled microdisk lasers. We measure the accuracy to be 2.5 ± 1.4 µm and 1.4 ± 0.4° and conclude that optoelectronic tweezers are a promising technique for the micromanipulation and integration of optoelectronic components in general and semiconductor lasers in particular

Dynamical Mass Estimates for the Halo of M31 from Keck Spectroscopy

The last few months have seen the measurements of the radial velocities of all of the dwarf spheroidal companions to the Andromeda galaxy (M31) using the spectrographs (HIRES and LRIS) on the Keck Telescope. This paper summarises the data on the radial velocities and distances for all the companion galaxies and presents new dynamical modelling to estimate the mass of extended halo of M31. The best fit values for the total mass of M31 are between 7 and 10 x 10^{11} solar masses, depending on the details of the modelling. The mass estimate is accompanied by considerable uncertainty caused by the smallness of the dataset; for example, the upper bound on the total mass is roughly 24 x 10^{11} solar masses, while the lower bound is about 3 x 10^{11} solar masses. These values are less than the most recent estimates of the most likely mass of the Milky Way halo. Bearing in mind all the uncertainties, a fair conclusion is that the M31 halo is roughly as massive as that of the Milky Way halo. There is no dynamical evidence for the widely held belief that M31 is more massive -- it may even be less massive.Comment: In press, The Astrophysical Journal (Letters