Microfabricated Devices for Adherent Stem Cell Culture

This thesis details the development of a system of microfabricated devices for the adherent culture of stem cells. The multipotency and self renewal of stem cells make them a potentially abundant source of valuable human cells, for both drug screening and regenerative medicine. However, processing stem cells is challenging due to the complexity of whole cell products, the number of process parameters, and the typical use of adherent culture. It is hypothesised that a microfabricated adherent culture system could facilitate process development with minimal use of resources. Furthermore, microfluidic systems offer advantages in spatial and temporal control over the microenvironment that would benefit process development. An existing prototype culture system is critically evaluated by: assessing the design, modelling fluid flow and dissolved oxygen, and successfully co-culturing human embryonic stem cells, on inactivated mouse embryonic fibroblasts, under perfused conditions. The utilisation of reversible seals facilitates the use of standard tissue-culture polystyrene culture surfaces and manual seeding techniques. The evaluation of the prototype system is used to inform improvements to the design, making it easier to use, increasing the robustness, allowing monitoring of whole culture chambers by microscopy, and improving control over mean pericellular dissolved oxygen. Modelling shows the improved culture system also achieves more uniform distribution of both pericellular dissolved oxygen and fluid velocity. The improved culture system shows similar mouse embryonic stem cell seeding behaviour to tissue culture flasks, but, with medium perfused at 300 μl.h 1, mouse embryonic stem cells reach full confluency in less than 48 h, compared with 72 hours for cells maintained statically in flasks. There is also inconclusive data suggesting that the growth rate is limited by pericellular dissolved oxygen and is, therefore, increased and made more uniform by the inclusion of a gas permeable lid system. The reliability, ease of use, comparability with traditional culture systems, and control over process parameters of the improved system should make it a useful tool for stem cell process development

Robust, microfabricated culture devices with improved control over the soluble microenvironment for the culture of embryonic stem cells.

The commercial use of stem cells continues to be constrained by the difficulty and high cost of developing efficient and reliable production protocols. The use of microfabricated systems combines good control over the cellular microenvironment with reduced use of resources in process optimization. Our previously reported microfabricated culture device was shown suitable for the culture of embryonic stem cells but required improvements to robustness, ease of use and dissolved gas control. In this report we describe a number of improvements to the design of the microfabricated system to significantly improve the control over shear stress and soluble factors, particularly dissolved oxygen. These control improvements are investigated by finite element modeling. Design improvements also make the system easier to use and improve the robustness. The culture device could be applied to the optimization of pluripotent stem cell growth and differentiation, as well as the development of monitoring and control strategies and improved culture systems at various scales

Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies

Automated and online characterization of adherent cell culture growth in a microfabricated bioreactor.

Adherent cell lines are widely used across all fields of biology, including drug discovery, toxicity studies, and regenerative medicine. However, adherent cell processes are often limited by a lack of advances in cell culture systems. While suspension culture processes benefit from decades of development of instrumented bioreactors, adherent cultures are typically performed in static, noninstrumented flasks and well-plates. We previously described a microfabricated bioreactor that enables a high degree of control on the microenvironment of the cells while remaining compatible with standard cell culture protocols. In this report, we describe its integration with automated image-processing capabilities, allowing the continuous monitoring of key cell culture characteristics. A machine learning-based algorithm enabled the specific detection of one cell type within a co-culture setting, such as human embryonic stem cells against the background of fibroblast cells. In addition, the algorithm did not confuse image artifacts resulting from microfabrication, such as scratches on surfaces, or dust particles, with cellular features. We demonstrate how the automation of flow control, environmental control, and image acquisition can be employed to image the whole culture area and obtain time-course data of mouse embryonic stem cell cultures, for example, for confluency

Microfabricated modular scale-down device for regenerative medicine process development.

The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development

Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images

The quantitative determination of key adherent cell culture characteristics such as confluency, morphology and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post-hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (< 1s per 1208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license