Defining cell culture dynamics in response to growth factor provision for efficient optimization of cell based therapies

The design of processes to manufacture Cell Based Therapies (CBTs) is particularly challenging due to the dynamic cell culture environment and the complex response of the cell product. An approach to process design needs to achieve a balance between resource spent and process knowledge gained to meet manufacturing goals around cost, quality and risk. In order to address this, we developed a parsimonious mathematical modelling framework designed to allow representation of common mechanisms in cell culture, and associated process operations, that drive cell culture outcomes(1). We propose that this framework can be applied to generate widely applicable ‘template models’ and experimental strategies for process optimization. In this case we have demonstrated the application to define provision rates of elements that support cell growth in a hematopoietic culture system (erythroblasts). Building on a model that defined the influence of environmental change (incorporated as a single non-specific variable linked to cell growth and density) on population growth(2), we have defined the cell culture dynamics in response to specific elements such as growth factor provision. We have shown how achieving the correct combination of delivery rates of growth factors with other factors (e.g. metabolic) can ensure that each is used with maximal efficiency, significantly driving down volume use (more than 3-fold) and process supplementation costs. The models also define the corollary of such intensification in terms of increasingly demanding process control with respect to volume handling and process timing; however, an advantage of the level of intensification achieved is that the system oxygen demand increases to a level that oxygen input rates can be used to identify cell growth-inhibition in advance of reduced viability. This work demonstrates that models that appropriately represent common process dynamics will be a key tool to achieve the risk and cost reduction required for CBT manufacture. (1) AJ Stacey, EA Cheeseman, KE Glen, RLL Moore, RJ Thomas. Experimentally integrated dynamic modelling for intuitive optimization of cell-based processes and manufacture. Biochem Eng J. 2018. 132: 130-138 (2) KE Glen, EA Cheeseman, AJ Stacey, RJ Thomas. A mechanistic model of erythroblast growth inhibition providing a framework for optimization of cell therapy manufacturing. Biochem Eng J. 2018. 133:28-38

Orientation and structure of the Ndc80 complex on the microtubule lattice

The four-subunit Ndc80 complex, comprised of Ndc80/Nuf2 and Spc24/Spc25 dimers, directly connects kinetochores to spindle microtubules. The complex is anchored to the kinetochore at the Spc24/25 end, and the Ndc80/Nuf2 dimer projects outward to bind to microtubules. Here, we use cryoelectron microscopy and helical image analysis to visualize the interaction of the Ndc80/Nuf2 dimer with microtubules. Our results, when combined with crystallography data, suggest that the globular domain of the Ndc80 subunit binds strongly at the interface between tubulin dimers and weakly at the adjacent intradimer interface along the protofilament axis. Such a binding mode, in which the Ndc80 complex interacts with sequential α/β-tubulin heterodimers, may be important for stabilizing kinetochore-bound microtubules. Additionally, we define the binding of the Ndc80 complex relative to microtubule polarity, which reveals that the microtubule interaction surface is at a considerable distance from the opposite kinetochore-anchored end; this binding geometry may facilitate polymerization and depolymerization at kinetochore-attached microtubule ends

Application of quality by design tools to upstream processing of platelet precursor cells to enable in vitro manufacture of blood products

Annually 4.5 million platelet units are transfused in Europe and the United States. These are obtained solely from allogeneic donations and have a shelf life of 5-7 days. To address the corresponding supply challenge, Moreau et al.1 devised a novel process for producing megakaryocytes (MKs, the platelet precursor cell) in vitro. A transcription-factor driven, forward-programming (FOP) approach converts human pluripotent stem cells into MKs. This strategy has the unique advantage of generating high yields of pure MKs in chemically defined medium which could lead to the production of a consistent, reliable supply of platelets which overcomes the logistical, financial and biosafety challenges for health organisations worldwide. Here we follow a Quality by Design (QbD) approach to enable improvements to the upstream processing of FOPMKs. Firstly, we created a process flow diagram for production of in vitro platelets for transfusion, which segregated processes into individual unit operations for control and optimisation. Next, we developed a Quality Target Product Profile (QTPP) and identified Critical Quality Attributes (CQAs) for each stage. We conducted a range of experiments utilising Design of Experiments (DOE) and mechanistic modelling2 tools to link Critical Process Parameters (CPPs) to CQAs. For adherent culture, we identified a productivity limit related to surface area available for growth and a cell loss phase which was dependent on cell seeding density, RhoK inhibitor usage and seed density. Using suspension cultures of FOPMK. We noted that TPO and Doxycycline concentration were CPPs as these impacted cell net growth rate and phenotype trajectory. Furthermore, we noted that medium exhaustion led to a 30% loss of viable cells over 8 hours. Proof of concept studies also showed that FOPMKs can be cultured in scaled-down suspension systems (ambr-15 and spinner flask culture) whilst retaining CQAs. 1. Moreau, T. et al. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming. Nat. Commun. 7, 1–15 (2016). 2. Stacey, A. J., Cheeseman, E. A., Glen, K. E., Moore, R. L. L. & Thomas, R. J. Experimentally integrated dynamic modelling for intuitive optimisation of cell-based processes and manufacture. Biochem. Eng. J. 132, 130–138 (2018)

ENU Mutagenesis Reveals a Novel Phenotype of Reduced Limb Strength in Mice Lacking Fibrillin 2

Background: Fibrillins 1 (FBN1) and 2 (FBN2) are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA) result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.Methodology/Principal Findings: As part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2(fp), identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.Conclusions: These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further

Structural comparison of the Caenorhabditis elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior

During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore–microtubule interactions remains poorly understood. The conserved four-subunit Ndc80 complex plays an essential and direct role in generating dynamic kinetochore–microtubule attachments. Here we compare the binding of the Caenorhabditis elegans and human Ndc80 complexes to microtubules at high resolution using cryo–electron microscopy reconstructions. Despite the conserved roles of the Ndc80 complex in diverse organisms, we find that the attachment mode of these complexes for microtubules is distinct. The human Ndc80 complex binds every tubulin monomer along the microtubule protofilament, whereas the C. elegans Ndc80 complex binds more tightly to β-tubulin. In addition, the C. elegans Ndc80 complex tilts more toward the adjacent protofilament. These structural differences in the Ndc80 complex between different species may play significant roles in the nature of kinetochore–microtubule interactions.National Institutes of Health (U.S.) (Grant GM088313

Structural comparison of the Caenorhabditis elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior

During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore–microtubule interactions remains poorly understood. The conserved four-subunit Ndc80 complex plays an essential and direct role in generating dynamic kinetochore–microtubule attachments. Here we compare the binding of the Caenorhabditis elegans and human Ndc80 complexes to microtubules at high resolution using cryo–electron microscopy reconstructions. Despite the conserved roles of the Ndc80 complex in diverse organisms, we find that the attachment mode of these complexes for microtubules is distinct. The human Ndc80 complex binds every tubulin monomer along the microtubule protofilament, whereas the C. elegans Ndc80 complex binds more tightly to β-tubulin. In addition, the C. elegans Ndc80 complex tilts more toward the adjacent protofilament. These structural differences in the Ndc80 complex between different species may play significant roles in the nature of kinetochore–microtubule interactions.National Institutes of Health (U.S.) (Grant GM088313

The job in the joint: The impact of generation and gender on work stress in prison

Most of the existing literature on correctional officer work stress examined factors such as leadership, job environment, stress and job satisfaction. This study surveyed correctional officers and examined the impact of gender and generation on work stress. Results indicated that gender is a significant factor regarding work-related stress, more than any other demographic variable, although generation had a small impact on job stress. Generation and gender explained a miniscule portion of work stress variance. Further studies are needed to assess the overall relationship among demographic factors, non-static officer attributes, and job stress.