Optimization of the nutritional environment for differentiation of human-induced pluripotent stem cells using design of experiments-A proof of concept.

The utilization of human-induced pluripotent stem cells (hiPSCs) in cell therapy has a tremendous potential but faces many practical challenges, including costs associated with cell culture media and growth factors. There is an immediate need to establish an optimized culture platform to direct the differentiation of hiPSCs into germ layers in a defined nutritional microenvironment to generate cost-effective and robust therapeutics. The aim of this study was to identify the optimal nutritional environment by mimicking the in vivo concentrations of three key factors (glucose, pyruvate, and oxygen) during the spontaneous differentiation of hiPSCs derived from cord blood, which greatly differ from the in vitro expansion and differentiation scenarios. Moreover, we hypothesized that the high glucose, pyruvate, and oxygen concentrations found in typical growth media could inhibit the differentiation of certain lineages. A design of experiments was used to investigate the interaction between these three variables during the spontaneous differentiation of hiPSCs. We found that lower oxygen and glucose concentrations enhance the expression of mesodermal (Brachyury, KIF1A) and ectodermal (Nestin, ß-Tubulin) markers. Our findings present a novel approach for efficient directed differentiation of hiPSCs through the manipulation of media components while simultaneously avoiding the usage of growth factors thus reducing costs.

Bioprocess considerations for T-cell therapy: Investigating the impact of agitation, dissolved oxygen, and pH on T-cell expansion and differentiation.

Adoptive T-cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing process remains a challenge. Here, a single-use 24-well microbioreactor (micro-Matrix) was assessed for its use as a high-throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion-mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions. Agitation was shown to play a fundamental role in the proliferation of T cells. A shaking speed of 200 rpm drastically improved the final viable cell concentration (VCC), while the viability was maintained above 90% throughout the cultivation. VCCs reached a maximum of 9.22 × 106 cells/ml. The distribution of CD8+ central memory T cells (TCM ), was found to be largely unaffected by the shaking speed. A clear interaction between pH and DO (p < .001) was established for the cell growth and the optimal culture conditions were identified for a combination of 200 rpm, 25% DO, and pH of 7.4. The combination of microbioreactor technology and Design of Experiment methodology provides a powerful tool to rapidly gain an understanding of the design space of the T-cell manufacturing process.

Long-Term Retinal Differentiation of Human Induced Pluripotent Stem Cells in a Continuously Perfused Microfluidic Culture Device.

Understanding how microenvironmental cues influence cellular behavior will enable development of efficient and robust pluripotent stem cell differentiation protocols. Unlike traditional cell culture dishes, microfluidic bioreactors can provide stable microenvironmental conditions by continuous medium perfusion at a controlled rate. The aim of this study is to investigate whether a microfluidic culture device could be used as a perfused platform for long-term cell culture processes such as the retinal differentiation of human induced pluripotent stem cells. The perfusion flow rate is established based on the degradation and consumption of growth factors (DKK-1, Noggin, IGF-1, and bFGF) and utilizing the Péclet number. The device's performance analyzed by qRT-PCR show improvements compared to the well-plate control as characterized by significantly higher expression of the markers Pax6, Chx10, and Crx on Day 5, Nrl on day 10, Crx, and Rhodopsin on day 21. Optimization of perfusion rate is an important operating variable in development of robust processes for differentiation cultures. Result demonstrates convective delivery of nutrients via perfusion has a significant impact upon the expression of key retinal markers. This study is the first continuously perfused long-term (21 days) retinal differentiation of hiPSCs in a microfluidic device.

A guide to manufacturing CAR T cell therapies.

In recent years, chimeric antigen receptor (CAR) modified T cells have been used as a treatment for haematological malignancies in several phase I and II trials and with Kymriah of Novartis and Yescarta of KITE Pharma, the first CAR T cell therapy products have been approved. Promising clinical outcomes have yet been tempered by the fact that many therapies may be prohibitively expensive to manufacture. The process is not yet defined, far from being standardised and often requires extensive manual handling steps. For academia, big pharma and contract manufacturers it is difficult to obtain an overview over the process strategies and their respective advantages and disadvantages. This review details current production processes being used for CAR T cells with a particular focus on efficacy, reproducibility, manufacturing costs and release testing. By undertaking a systematic analysis of the manufacture of CAR T cells from reported clinical trial data to date, we have been able to quantify recent trends and track the uptake of new process technology. Delivering new processing options will be key to the success of the CAR-T cells ensuring that excessive manufacturing costs do not disrupt the delivery of exciting new therapies to the wide possible patient cohort.

Transfection in perfused microfluidic cell culture devices: A case study.

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.

An integrated experimental and economic evaluation of cell therapy affinity purification technologies.

AIM: To present an integrated techno-economic analysis assessing the feasibility of affinity purification technologies using the manufacture of induced pluripotent stem cell-derived progenitor photoreceptors for retinal dystrophies as a case study. MATERIALS & METHODS: Sort purity, progenitor yield and viable cell recovery were investigated for three cell sorting techniques: fluorescent-activated cell sorting (FACS); magnetic-activated cell sorting (MACS); and a novel technology SpheriTech beads. Experimentally derived metrics were incorporated into an advanced bioprocess economics tool to determine cost of goods per dose for each technology. RESULTS & CONCLUSION: Technical and bioprocess benefits were noted with SpheriTech beads which, unlike FACS and MACS, require no cell labeling. This simplifies the bioprocess, reduces cell loss and leaves target cells label free. The economic tool predicted cost drivers and a critical dose (7 × 107 cells per dose) shifting the most cost-effective technology from FACS to MACS. Process optimization is required for SpheriTech to compete economically.

A novel filtration system for point of care washing of cellular therapy products.

The cell therapy industry would greatly benefit from a simple point of care solution to remove dimethylsulphoxide (DMSO) from small-volume thawed cell suspensions before injection. A novel dead-end filtration device has been designed and validated, which takes advantage of the higher density of thawed cell suspensions to remove the DMSO and protein impurities from the cell suspension without fouling the filter membrane. The filter was designed to avoid fluid circuits and minimize the surface area that is contacted by the cell suspension, thus reducing cell losses by design. The filtration process was established through optimization of the fluid flow configuration, backflush cycles and filter geometry. Overall, this novel filtration device allows for a 1 ml of thawed cryopreserved cell suspensions, containing 107 cells of a fetal lung fibroblast cell line (MRC-5), to be washed in less than 30 min. More than 95% of the DMSO and up to 94% of the albumin-fluorescein-isothiocyanate content can be removed while the viable cell recovery is higher than 80%. It is also demonstrated that this system can be used for bone marrow-derived human mesenchymal stem cells with more than 73% cell recovery and 85% DMSO reduction. This is the first time that a dead end (normal) filtration process has been used to successfully wash high-density human cell suspensions. In practice, this novel solid-liquid separation technology fills the need for small-volume washing in closed processing systems for cellular therapies. Copyright © 2016 John Wiley & Sons, Ltd.

Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures.

OBJECTIVE: To develop a microwell suspension platform for the adaption of attached stem cell differentiation protocols into mixed suspension culture. RESULTS: We adapted an adherent protocol for the retinal differentiation of human induced pluripotent stem cells (hiPSCs) using a two-step protocol. Establishing the optimum embryoid body (EB) starting size and shaking speed resulted in the translation of the original adherent process into suspension culture. Embryoid bodies expanded in size as the culture progressed resulting in the expression of characteristic markers of early (Rx, Six and Otx2) and late (Crx, Nrl and Rhodopsin) retinal differentiation. The new process also eliminated the use of matrigel, an animal-derived extracellular matrix coating. CONCLUSIONS: Shaking microwells offer a fast and cost-effective method for proof-of-concept studies to establish whether pluripotent stem cell differentiation processes can be translated into mixed suspension culture.

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.

The effect of Young's modulus on the neuronal differentiation of mouse embryonic stem cells.

There is substantial evidence that cells produce a diverse response to changes in ECM stiffness depending on their identity. Our aim was to understand how stiffness impacts neuronal differentiation of embryonic stem cells (ESC's), and how this varies at three specific stages of the differentiation process. In this investigation, three effects of stiffness on cells were considered; attachment, expansion and phenotypic changes during differentiation. Stiffness was varied from 2 kPa to 18 kPa to finally 35 kPa. Attachment was found to decrease with increasing stiffness for both ESC's (with a 95% decrease on 35 kPa compared to 2 kPa) and neural precursors (with a 83% decrease on 35 kPa). The attachment of immature neurons was unaffected by stiffness. Expansion was independent of stiffness for all cell types, implying that the proliferation of cells during this differentiation process was independent of Young's modulus. Stiffness had no effect upon phenotypic changes during differentiation for mESC's and neural precursors. 2 kPa increased the proportion of cells that differentiated from immature into mature neurons. Taken together our findings imply that the impact of Young's modulus on attachment diminishes as neuronal cells become more mature. Conversely, the impact of Young's modulus on changes in phenotype increased as cells became more mature.

The differential effects of 2% oxygen preconditioning on the subsequent differentiation of mouse and human pluripotent stem cells.

A major challenge facing the development of effective cell therapies is the efficient differentiation of pluripotent stem cells (PSCs) into pure populations. Lowering oxygen tension to physiological levels can affect both the expansion and differentiation stages. However, to date, there are no studies investigating the knock-on effect of culturing PSCs under low oxygen conditions on subsequent lineage commitment at ambient oxygen levels. PSCs were passaged three times at 2% O2 before allowing cells to spontaneously differentiate as embryoid bodies (EBs) in high oxygen (20% O2) conditions. Maintenance of mouse PSCs in low oxygen was associated with a significant increase in the expression of early differentiation markers FGF5 and Eomes, while conversely we observed decreased expression of these genes in human PSCs. Low oxygen preconditioning primed mouse PSCs for their subsequent differentiation into mesodermal and endodermal lineages, as confirmed by increased gene expression of Eomes, Goosecoid, Brachyury, AFP, Sox17, FoxA2, and protein expression of Brachyury, Eomes, Sox17, FoxA2, relative to high oxygen cultures. The effects extended to the subsequent formation of more mature mesodermal lineages. We observed significant upregulation of cardiomyocyte marker Nkx2.5, and critically a decrease in the number of contaminant pluripotent cells after 12 days using a directed cardiomyocyte protocol. However, the impact of low oxygen preconditioning was to prime human cells for ectodermal lineage commitment during subsequent EB differentiation, with significant upregulation of Nestin and ß3-tubulin. Our research demonstrates the importance of oxygen tension control during cell maintenance on the subsequent differentiation of both mouse and human PSCs, and highlights the differential effects.

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.

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 to be 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.

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 (<1 s per 1,208 × 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.

Reproducible culture and differentiation of mouse embryonic stem cells using an automated microwell platform.

The use of embryonic stem cells (ESCs) and their progeny in high throughput drug discovery and regenerative medicine will require production at scale of well characterized cells at an appropriate level of purity. The adoption of automated bioprocessing techniques offers the possibility to overcome the lack of consistency and high failure rates seen with current manual protocols. To build the case for increased use of automation this work addresses the key question: "can an automated system match the quality of a highly skilled and experienced person working manually?" To answer this we first describe an integrated automation platform designed for the 'hands-free' culture and differentiation of ESCs in microwell formats. Next we outline a framework for the systematic investigation and optimization of key bioprocess variables for the rapid establishment of validatable Standard Operating Procedures (SOPs). Finally the experimental comparison between manual and automated bioprocessing is exemplified by expansion of the murine Oct-4-GiP ESC line over eight sequential passages with their subsequent directed differentiation into neural precursors. Our results show that ESCs can be effectively maintained and differentiated in a highly reproducible manner by the automated system described. Statistical analysis of the results for cell growth over single and multiple passages shows up to a 3-fold improvement in the consistency of cell growth kinetics with automated passaging. The quality of the cells produced was evaluated using a panel of biological markers including cell growth rate and viability, nutrient and metabolite profiles, changes in gene expression and immunocytochemistry. Automated processing of the ESCs had no measurable negative effect on either their pluripotency or their ability to differentiate into the three embryonic germ layers. Equally important is that over a 6-month period of culture without antibiotics in the medium, we have not had any cases of culture contamination. This study thus confirms the benefits of adopting automated bioprocess routes to produce cells for therapy and for use in basic discovery research.

Development of a multiplexed microfluidic platform for the automated cultivation of embryonic stem cells.

We present a multiplexed platform for a microfabricated stem cell culture device. The modular platform contains all the components to control stem cell culture conditions in an automated fashion. It does not require an incubator during perfusion culture and can be mounted on the stage of an inverted fluorescence microscope for high-frequency imaging of stem cell cultures. A pressure-driven pump provides control over the medium flow rate and offers switching of the flow rates. Flow rates of the pump are characterized for different pressure settings, and a linear correlation between the applied pressure and the flow rate in the cell culture devices is shown. In addition, the pump operates with two culture medium reservoirs, thus enabling the switching of the culture medium on-the-fly during a cell culture experiment. Also, with our platform, the culture medium reservoirs are cooled to prevent medium degradation during long-term experiments. Media temperature is then adjusted to a higher controlled temperature before entering the microfabricated cell culture device. Furthermore, the temperature is regulated in the microfabricated culture devices themselves. Preliminary culture experiments are demonstrated using mouse embryonic stem cells.

Oxygen-controlled automated neural differentiation of mouse embryonic stem cells.

UNLABELLED: Automation and oxygen tension control are two tools that provide significant improvements to the reproducibility and efficiency of stem cell production processes. AIM: the aim of this study was to establish a novel automation platform capable of controlling oxygen tension during both the cell-culture and liquid-handling steps of neural differentiation processes. MATERIALS & METHODS: We built a bespoke automation platform, which enclosed a liquid-handling platform in a sterile, oxygen-controlled environment. An airtight connection was used to transfer cell culture plates to and from an automated oxygen-controlled incubator. RESULTS: Our results demonstrate that our system yielded comparable cell numbers, viabilities, metabolism profiles and differentiation efficiencies when compared with traditional manual processes. Interestingly, eliminating exposure to ambient conditions during the liquid-handling stage resulted in significant improvements in the yield of MAP2-positive neural cells, indicating that this level of control can improve differentiation processes. CONCLUSION: This article describes, for the first time, an automation platform capable of maintaining oxygen tension control during both the cell-culture and liquid-handling stages of a 2D embryonic stem cell differentiation process.

Hypoxic culture of human pluripotent stem cell lines is permissible using mouse embryonic fibroblasts.

AIM: Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate. MATERIALS & METHODS: Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A. RESULTS: Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers. CONCLUSION: The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.

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.

Hypoxia enhances the generation of retinal progenitor cells from human induced pluripotent and embryonic stem cells.

The efficient differentiation of retinal cells from human pluripotent stem cells remains a major challenge for the development of successful and cost-effective cellular therapies for various forms of blindness. Current differentiation strategies rely on exposing pluripotent stem cells to soluble growth factors that play key roles during early development (such as DKK-1, Noggin, and IGF-1) at 20% oxygen (O(2)). This O(2) tension is, however, considerably higher than O(2) levels during organogenesis and may impair the differentiation process. In this study, we examined the effect of mimicking the physiological O(2) tension (2%) on the generation of retinal progenitor cells (RPCs) from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). Both cell types were induced to differentiate into RPCs at 20% and 2% O(2). After 3 days in suspension culture as embryoid bodies (EBs), 2% O(2) caused the activation of hypoxia inducible factor responsive genes VEGF and LDHA and was accompanied by elevated expression levels of the early eye field genes Six3 and Lhx2. Twenty-one days after plating EBs in an adherent culture, we observed more RPCs co-expressing Pax6 and Chx10 at 2% O(2). Quantitative polymerase chain reaction analysis confirmed that lowering O(2) tension had caused a rise in the expression of both genes compared with 20% O(2). Our results indicate that mimicking physiological O(2) is a favorable condition for the efficient generation of RPCs from both hiPSCs and hESCs.