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

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 Dimethyl Sulfoxide (DMSO) from small volume thawed cell suspensions prior to injection. We have designed and validated a novel dead-end filtration device, 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 foetal lung fibroblast cell line (MRC-5), to be washed in less than 30 minutes. 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%. We have 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

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

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

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

Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile

© 2016 The Author(s)The application of primary human hepatocytes following isolation from human tissue is well accepted to be compromised by the process of dedifferentiation. This phenomenon reduces many unique hepatocyte functions, limiting their use in drug disposition and toxicity assessment. The aetiology of dedifferentiation has not been well defined, and further understanding of the process would allow the development of novel strategies for sustaining the hepatocyte phenotype in culture or for improving protocols for maturation of hepatocytes generated from stem cells. We have therefore carried out the first proteomic comparison of primary human hepatocyte differentiation. Cells were cultured for 0, 24, 72 and 168 h as a monolayer in order to permit unrestricted hepatocyte dedifferentiation, so as to reveal the causative signalling pathways and factors in this process, by pathway analysis. A total of 3430 proteins were identified with a false detection rate of <1 %, of which 1117 were quantified at every time point. Increasing numbers of significantly differentially expressed proteins compared with the freshly isolated cells were observed at 24 h (40 proteins), 72 h (118 proteins) and 168 h (272 proteins) (p < 0.05). In particular, cytochromes P450 and mitochondrial proteins underwent major changes, confirmed by functional studies and investigated by pathway analysis. We report the key factors and pathways which underlie the loss of hepatic phenotype in vitro, particularly those driving the large-scale and selective remodelling of the mitochondrial and metabolic proteomes. In summary, these findings expand the current understanding of dedifferentiation should facilitate further development of simple and complex hepatic culture systems