Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks

Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck. KEY POINTS: • hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF • Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days • The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics

One, two, or three stars? An investigation of an unusual eclipsing binary candidate undergoing dramatic period changes

We report our investigation of 1SWASP J234401.81-212229.1, a variable with a 18 461.6 s period. After identification in a 2011 search of the SuperWASP archive for main-sequence eclipsing binary candidates near the distribution's short-period limit of ~0.20 d, it was measured to be undergoing rapid period decrease in our earlier work, though later observations supported a cyclic variation in period length. Spectroscopic data obtained in 2012 with the Southern African Large Telescope did not, however, support the interpretation of the object as a normal eclipsing binary. Here, we consider three possible explanations consistent with the data: a single-star oblique rotator model in which variability results from stable cool spots on opposite magnetic poles; a two-star model in which the secondary is a brown dwarf; and a three-star model involving a low-mass eclipsing binary in a hierarchical triple system. We conclude that the latter is the most likely model

The Rigidly Rotating Magnetosphere of Sigma Ori E

We attempt to characterize the observed variability of the magnetic helium-strong star sigma Ori E in terms of a recently developed rigidly rotating magnetosphere model. This model predicts the accumulation of circumstellar plasma in two co-rotating clouds, situated in magnetohydrostatic equilibrium at the intersection between magnetic and rotational equators. We find that the model can reproduce well the periodic modulations observed in the star's light curve, H alpha emission-line profile, and longitudinal field strength, confirming that it furnishes an essentially correct, quantitative description of the star's magnetically controlled circumstellar environment.Comment: 4 pages, 3 figures, accepted by Ap

Fundamental parameters of Cepheids. V. Additional photometry and radial velocity for southern Cepheids

I present photometric and radial velocity data for Galactic Cepheids, most of them being in the southern hemisphere. There are 1250 Geneva 7-color photometric measurements for 62 Cepheids, the average uncertainty per measurement is better than 0.01 mag. A total of 832 velocity measurements have been obtained with the CORAVEL radial velocity spectrograph for 46 Cepheids. The average accuracy of the radial velocity data is 0.38 km/s. There are 33 stars with both photometry and radial velocity data. I discuss the possible binarity or period change that these new data reveal. I also present reddenings for all Cepheids with photometry. The data are available electronically.Comment: To appear in ApJS. Data available electronically at ftp://cfa-ftp.harvard.edu/pub/dbersier

Continuous production of lentiviral vectors using a fixed-bed bioreactor

Lentiviral vectors (LVs) have emerged as indispensable tools for mediating stable transfer of large transgenes in mammalian cells, which has resulted in the widespread application of LVs for the manufacture of gene-modified cell therapies, particularly chimeric antigen receptor T-cell (CAR-T-cell) therapies. The typical manufacture of LVs through chemical transfection of adherent embryonic kidney 293T-cells with plasmid DNA is highly versatile and enables manufacturing to pivot to produce vectors with alternative transgenes and envelope proteins. However, transient transfection presents several challenges, including batch-to-batch variability, limited scalability and high costs attributed to the plasmid DNA and transfection reagent. Therefore, the transition towards continuous LV production using producer cell lines would allow for cost effective and scalable manufacturing along with enhanced safety and reproducibility. Specifically, continuous production using stable producer cell lines could extend vector expression by counteracting the limited post-induction expression time and eliminating the requirement for removal of the inducing agent during downstream processing. In this study, a continuous LV manufacturing process was established using a stable producer cell line, which constitutively expresses third-generation LVs pseudotyped the RDpro envelope protein (WinPacRDpro). Production of LVs in typical 2D culture vessels was performed, however, these systems lack the control of culture parameters, such as pH and dissolved oxygen, and require laborious handling, which introduce risks of contamination. Therefore, the transition to LV production in bioreactors is a necessary step to achieve sufficient LV supply, e.g. for late-stage clinical trials. This work demonstrates the establishment of a quasi-perfusion process using repetitive batch strategy with medium exchange, first, in 2D culture vessels and then, to an iCELLis fixed-bed bioreactor to continuously produce RDpro-pseudotyped LVs. This initial work using a continuous process-mimic in a fixed-bed bioreactor enables cell expansion in a controlled environment and the bioreactor characterization was used to gain insights for the development of a perfusion strategy. Growth, substrate utilization, lactate generation and LV production were evaluated under different medium exchange strategies. Finally, the established bioreactor process was then transformed to perfusion culture with studies aimed at understanding the impact of perfusion conditions on LV production

Continuous manufacturing of lentiviral vectors

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The Shape and Scale of Galactic Rotation from Cepheid Kinematics

A catalog of Cepheid variables is used to probe the kinematics of the Galactic disk. Radial velocities are measured for eight distant Cepheids toward l = 300; these new Cepheids provide a particularly good constraint on the distance to the Galactic center, R_0. We model the disk with both an axisymmetric rotation curve and one with a weak elliptical component, and find evidence for an ellipticity of 0.043 +/- 0.016 near the Sun. Using these models, we derive R_0 = 7.66 +/- 0.32 kpc and v_circ = 237 +/- 12 km/s. The distance to the Galactic center agrees well with recent determinations from the distribution of RR Lyrae variables, and disfavors most models with large ellipticities at the solar orbit.Comment: 36 pages, LaTeX, 10 figure

Detection of Magnetic Massive Stars in the Open Cluster NGC 3766

A growing number of observations indicate that magnetic fields are present among a small fraction of massive O- and B-type stars, yet the origin of these fields remains unclear. Here we present the results of a VLT/FORS1 spectropolarimetric survey of 15 B-type members of the open cluster NGC 3766. We have detected two magnetic B stars in the cluster, including one with a large field of nearly 2 kG, and we find marginal detections of two additional stars. There is no correlation between the observed longitudinal field strengths and the projected rotational velocity, suggesting that a dynamo origin for the fields is unlikely. We also use the Oblique Dipole Rotator model to simulate populations of magnetic stars with uniform or slightly varying magnetic flux on the ZAMS. None of the models successfully reproduces our observed range in B_l and the expected number of field detections, and we rule out a purely fossil origin for the observed fields.Comment: 6 pages ApJ emulate style; accepted to Ap

Microlensing of circumstellar envelopes III. Line profiles from stellar winds in homologous expansion

This paper examines line profile evolution due to the linear expansion of circumstellar material obsverved during a microlensing event. This work extends our previous papers on emission line profile evolution from radial and azimuthal flow during point mass lens events and fold caustic crossings. Both "flavours" of microlensing were shown to provide effective diagnostics of bulk motion in circumstellar envelopes. In this work a different genre of flow is studied, namely linear homologous expansion, for both point mass lenses and fold caustic crossings. Linear expansion is of particular relevance to the effects of microlensing on supernovae at cosmological distances. We derive line profiles and equivalent widths for the illustrative cases of pure resonance and pure recombination lines, modelled under the Sobolev approximation. The efficacy of microlensing as a diagnostic probe of the stellar environs is demonstrated and discussed

A dominant magnetic dipole for the evolved Ap star candidate EK Eridani

EK Eri is one of the most slowly rotating active giants known, and has been proposed to be the descendant of a strongly magnetic Ap star. We have performed a spectropolarimetric study of EK Eri over 4 photometric periods with the aim of inferring the topology of its magnetic field. We used the NARVAL spectropolarimeter at the Bernard Lyot telescope at the Pic du Midi Observatory, along with the least-squares deconvolution method, to extract high signal-to-noise ratio Stokes V profiles from a timeseries of 28 polarisation spectra. We have derived the surface-averaged longitudinal magnetic field Bl. We fit the Stokes V profiles with a model of the large-scale magnetic field and obtained Zeeman Doppler images of the surface magnetic strength and geometry. Bl variations of up to about 80 G are observed without any reversal of its sign, and which are in phase with photometric ephemeris. The activity indicators are shown to vary smoothly on a timescale compatible with the rotational period inferred from photometry (308.8 d.), however large deviations can occur from one rotation to another. The surface magnetic field variations of EK Eri appear to be dominated by a strong magnetic spot (of negative polarity) which is phased with the dark (cool) photometric spot. Our modeling shows that the large-scale magnetic field of EK Eri is strongly poloidal. For a rotational axis inclination of i = 60{\deg}, we obtain a model that is almost purely dipolar. In the dipolar model, the strong magnetic/photometric spot corresponds to the negative pole of the dipole, which could be the remnant of that of an Ap star progenitor of EK Eri. Our observations and modeling conceptually support this hypothesis, suggesting an explanation of the outstanding magnetic properties of EK Eri as the result of interaction between deep convection and the remnant of an Ap star magnetic dipole.Comment: 8 pages, 6 figures, accepted for publication in Astronomy & Astrophysic