Dielectric spectroscopy monitoring of a bioreactor process for hiPSC expansion and differentiation

Bioprocessing strategies using 3D cell culturing approaches, such as cell aggregates, are promising solutions to achieve efficient and scalable bioprocesses for stem cell expansion and differentiation. However, tracking viable and total cell numbers in such culture systems is not straightforward. It requires cell detachment, disaggregation or disruption, which results in measurements that are laborious, biased and with high variability. In this work, we used a commercially available capacitance probe to explore the applicability of dielectric spectroscopy for in situ monitoring of a multistep process for expansion and differentiation of human induced pluripotent stem cells (hiPSC) cultivated as cell aggregates. After 5 days of cell expansion in a bioreactor, the hepatic differentiation step was integrated by addition of different levels of specific soluble factors at various stages of the process to promote growth and generate populations successively enriched for definitive endoderm, hepatoblasts, hepatocyte progenitors and mature hepatocytes. While this differentiation procedure has been previously validated for monolayer cultures, this was the first time it was carried out in a stirred tank bioreactor operated in perfusion mode. Phenotype analysis confirmed a marked increase in key hepatic differentiation markers culminating at day 21 of differentiation. Our data shows a good correlation between total volume of the cell aggregates and permittivity measured by the probe (R2 = 0.84). However, there was a delay between changes in cell concentration and the permittivity signal. This suggests that cell expansion requires a few days to result in increased volume of the cell aggregates and that each aggregate behaves as one overall inducible dipole. The β-dispersion curve shape also appears to change over culture time and could eventually be used as an indicator for differentiation progression. Dielectric spectroscopy has been used successfully to monitor viable cell concentration in different single-cell suspension cultures, but there are few published applications to 3D cultures. Our results demonstrate the potential of dielectric spectroscopy to monitor complex bioprocesses for human stem cell aggregates in stirred cultures. Acknowledgements: Funding provided by ERA-NET/E-Rare3 programme through research project ERAdicatPH (E-Rare3/0002/2015). The authors acknowledge Dr Juan Rodriguez-Madoz (University of Navarra, Spain) and Dr Anders Aspegren (Takara Bio Europe – Cellartis AB, Sweden) for helpful discussions on hepatic differentiation of hiPSC.

Enabling PAT in insect cell bioprocesses: A monitoring toolbox for rAAV production

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Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

Precision measurement of CP\it{CP} violation in the penguin-mediated decay Bs0→ϕϕB_s^{0}\rightarrow\phi\phi

A flavor-tagged time-dependent angular analysis of the decay $B_s^{0}\rightarrow\phi\phi$ is performed using $pp$ collision data collected by the LHCb experiment at % at $\sqrt{s}=13$ TeV, the center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 6 fb^{-1}. The $\it{CP}$-violating phase and direct $\it{CP}$-violation parameter are measured to be $\phi_{s\bar{s}s} = -0.042 \pm 0.075 \pm 0.009$ rad and $|\lambda|=1.004\pm 0.030 \pm 0.009$, respectively, assuming the same values for all polarization states of the $\phi\phi$ system. In these results, the first uncertainties are statistical and the second systematic. These parameters are also determined separately for each polarization state, showing no evidence for polarization dependence. The results are combined with previous LHCb measurements using $pp$ collisions at center-of-mass energies of 7 and 8 TeV, yielding $\phi_{s\bar{s}s} = -0.074 \pm 0.069$ rad and $|lambda|=1.009 \pm 0.030$. This is the most precise study of time-dependent $\it{CP}$ violation in a penguin-dominated $B$ meson decay. The results are consistent with $\it{CP}$ symmetry and with the Standard Model predictions.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2023-001.html (LHCb public pages

Measurement of the Λb0→Λ(1520)μ+μ−\Lambda_{b}^{0}\to \Lambda(1520) \mu^{+}\mu^{-} differential branching fraction

The branching fraction of the rare decay $\Lambda_{b}^{0}\to \Lambda(1520) \mu^{+}\mu^{-}$ is measured for the first time, in the squared dimuon mass intervals, $q^2$, excluding the $J/\psi$ and $\psi(2S)$ regions. The data sample analyzed was collected by the LHCb experiment at center-of-mass energies of 7, 8, and 13 TeV, corresponding to a total integrated luminosity of $9\ \mathrm{fb}^{-1}$. The result in the highest$q^{2}$interval,$q^{2} >15.0\ \mathrm{GeV}^2/c^4$, where theoretical predictions have the smallest model dependence, agrees with the predictions.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-050.html (LHCb public pages

Precision measurement of CP violation in the penguin-mediated decay Bs0→ϕϕ

A flavor-tagged time-dependent angular analysis of the decay B 0 s → ϕ ϕ is performed using p p collision data collected by the LHCb experiment at the center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 6     fb − 1 . The C P -violating phase and direct C P -violation parameter are measured to be ϕ s ¯ s s s = − 0.042 ± 0.075 ± 0.009     rad and | λ | = 1.004 ± 0.030 ± 0.009 , respectively, assuming the same values for all polarization states of the ϕ ϕ system. In these results, the first uncertainties are statistical and the second systematic. These parameters are also determined separately for each polarization state, showing no evidence for polarization dependence. The results are combined with previous LHCb measurements using p p collisions at center-of-mass energies of 7 and 8 TeV, yielding ϕ s ¯ s s s = − 0.074 ± 0.069     rad and | λ | = 1.009 ± 0.030 . This is the most precise study of time-dependent C P violation in a penguin-dominated B meson decay. The results are consistent with C P symmetry and with the standard model predictions

Modification of χc1(3872) and ψ(2S) production in pPb collisions at √sNN = 8.16 TeV

The LHCb Collaboration measures production of the exotic hadron χc1(3872) in proton-nucleus collisions for the first time. Comparison with the charmonium state ψ(2S) suggests that the exotic χc1(3872) experiences different dynamics in the nuclear medium than conventional hadrons, and comparison with data from proton-proton collisions indicates that the presence of the nucleus may modify χc1(3872) production rates. This is the first measurement of the nuclear modification factor of an exotic hadron

Improved measurement of CP violation parameters in Bs0→J/ψK+K− decays in the vicinity of the ϕ(1020) resonance

The decay-time-dependent C P asymmetry in B 0 s → J / ψ ( → μ + μ − ) K + K − decays is measured using proton-proton collision data, corresponding to an integrated luminosity of 6     fb − 1 , collected with the LHCb detector at a center-of-mass energy of 13 TeV. Using a sample of approximately 349 000 B 0 s signal decays with an invariant K + K − mass in the vicinity of the ϕ ( 1020 ) resonance, the C P -violating phase ϕ s is measured, along with the difference in decay widths of the light and heavy mass eigenstates of the B 0 s − ¯ B 0 s system, Δ Γ s , and the difference of the average B 0 s and B 0 meson decay widths, Γ s − Γ d . The values obtained are ϕ s = − 0.039 ± 0.022 ± 0.006     rad , Δ Γ s = 0.0845 ± 0.0044 ± 0.0024     ps − 1 , and Γ s − Γ d = − 0.005 6 + 0.0013 − 0.0015 ± 0.0014     ps − 1 , where the first uncertainty is statistical and the second systematic. These are the most precise single measurements to date and are consistent with expectations based on the Standard Model and with the previous LHCb analyses of this decay. These results are combined with previous independent LHCb measurements. The phase ϕ s is also measured independently for each polarization state of the K + K − system and shows no evidence for polarization dependence

Amplitude analysis of the B0→K*0μ+μ− decay

An amplitude analysis of the B 0 → K * 0 μ + μ − decay is presented using a dataset corresponding to an integrated luminosity of 4.7     fb − 1 of p p collision data collected with the LHCb experiment. For the first time, the coefficients associated to short-distance physics effects, sensitive to processes beyond the standard model, are extracted directly from the data through a q 2 -unbinned amplitude analysis, where q 2 is the μ + μ − invariant mass squared. Long-distance contributions, which originate from nonfactorizable QCD processes, are systematically investigated, and the most accurate assessment to date of their impact on the physical observables is obtained. The pattern of measured corrections to the short-distance couplings is found to be consistent with previous analyses of b - to s -quark transitions, with the largest discrepancy from the standard model predictions found to be at the level of 1.8 standard deviations. The global significance of the observed differences in the decay is 1.4 standard deviations