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.

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. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc.

Dynamic mechanical properties of cells are becoming recognized as indicators and regulators of physiological processes such as differentiation, malignant phenotypes and mitosis. A key process in development and homeostasis is apoptosis and whilst the molecular control over this pathway is well studied, little is known about the mechanical consequences of cell death. Here, we study the caspase-dependent mechanical kinetics of single cells during early apoptosis initiated with the general protein-kinase inhibitor staurosporine. This results in internal remodelling of the cytoskeleton and nucleus which is reflected in dynamic changes in the mechanical properties of the cell. Utilizing simultaneous confocal and atomic force microscopy (AFM), we measured distinct mechanical dynamics in the instantaneous cellular Young's Modulus and longer timescale viscous deformation. This allowed us to visualize time-dependent nuclear and cytoskeletal control of force dissipation with fluorescent fusion proteins throughout the cell. This work reveals that the cell death program not only orchestrates biochemical dynamics but also controls the mechanical breakdown of the cell. Importantly, the consequences of mechanical disregulation during apoptosis may be a contributing factor to several human pathologies through the poorly timed release of dead cells and cell debris.

Embryonic stem cells (ESC) have the developmental potential to form every adult cell type, even after prolonged culture. Reproducibly culturing pluripotent populations and directing differentiation has proven technically challenging yet will underpin the provision of stem cells for both screening and therapeutic applications. This study investigated whether the variations inherent in manual handling procedures cause inconsistent proliferation and phenotypic variability. Two mouse ESC green fluorescent protein (GFP) reporter cells lines, Oct4-GiP and 46C, were used to assess Oct4 expression during expansion and Sox1 expression during directed neuroectoderm differentiation. High inoculation cell densities (ICD) had a negative impact on Oct4-GFP expression. Similarly, increasing ICD caused a drop in Sox1-GFP expression in differentiating cultures. The expansion process had an optimum ICD of 31,800 cells cm(-2) whilst the highest yield of Sox1-GFP positive cells were found at an ICD of 16,400 cells cm(-2). These results implicate variable cell density as a major cause of interindividual variability. Passaging exposes cells to dynamic and repeated changes in their micro-environment. This was associated with a rapid drop in temperature and rise in pH. Extended exposure of 1, 2 and 3 h to ambient conditions resulted in the inhibition of ESC proliferation and Oct4-GFP expression. Dissociation subjects cells to fluid flow and centrifugal forces. Repeated exposure to fluid flow in capillaries prior to cultivation reduced the proliferative capacity of undifferentiated ESCs and caused a significant drop in differentiated neuroectoderm yield. Excessive centrifugal forces up to 1,000g caused shifts in phenotype and proliferation during expansion and differentiation. These studies highlight the need for automated cultivation systems which reproducibly control cell density, fluid flow, centrifugal forces, pH and temperature for the dissociation and inoculation of ESC processes.