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Nature Biomedical Engineering Jun 2024The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring...
The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.
PubMed: 38834752
DOI: 10.1038/s41551-024-01219-1 -
Scientific Reports Jun 2024Snakebite envenomation is a major public health issue which causes severe morbidity and mortality, affecting millions of people annually. Of a diverse range of clinical...
Snakebite envenomation is a major public health issue which causes severe morbidity and mortality, affecting millions of people annually. Of a diverse range of clinical manifestations, local and systemic haemorrhage are of particular relevance, as this may result in ischemia, organ failure and even cardiovascular shock. Thus far, in vitro studies have failed to recapitulate the haemorrhagic effects observed in vivo. Here, we present an organ-on-a-chip approach to investigate the effects of four different snake venoms on a perfused microfluidic blood vessel model. We assess the effect of the venoms of four snake species on epithelial barrier function, cell viability, and contraction/delamination. Our findings reveal two different mechanisms by which the microvasculature is being affected, either by disruption of the endothelial cell membrane or by delamination of the endothelial cell monolayer from its matrix. The use of our blood vessel model may shed light on the key mechanisms by which tissue-damaging venoms exert their effects on the capillary vessels, which could be helpful for the development of effective treatments against snakebites.
Topics: Animals; Snake Venoms; Lab-On-A-Chip Devices; Humans; Endothelial Cells; Hemorrhage; Cell Survival; Snake Bites; Human Umbilical Vein Endothelial Cells; Microphysiological Systems
PubMed: 38834598
DOI: 10.1038/s41598-024-60282-5 -
Molecular Therapy. Methods & Clinical... Jun 2024Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies...
Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 × 10 cells cm providing similar specific productivities of infectious LV. Seeding at 3 × 10 cells cm was selected, and the quasi-perfusion was modulated to minimize inhibitory metabolite accumulation and vector exposure at 37°C. Similar specific productivities of infectious LV and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimize downstream processing volumes. The optimized process was scaled 50-fold to 1,264 cm flasks, achieving similar LV titers. However, scaling up beyond this to a 6,320 cm multilayer flask reduced titers, possibly from suboptimal gas exchange. Across three independent processes in 25 cm to 6,320 cm flasks, reproducibility was high with a coefficient of variation of 7.7% ± 2.9% and 11.9% ± 3.0% for infectious and physical LV titers, respectively. The optimized flask process was successfully transferred to the iCELLis Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 × 10 TU.
PubMed: 38827249
DOI: 10.1016/j.omtm.2024.101264 -
STAR Protocols Jun 2024Microfluidic single-cell cultivation (MSCC) is a powerful tool for investigating the cellular behavior of various cell types at the single-cell level. Here, we present a...
Microfluidic single-cell cultivation (MSCC) is a powerful tool for investigating the cellular behavior of various cell types at the single-cell level. Here, we present a protocol specifically developed for the reliable and reproducible MSCC of industrially relevant Chinese hamster ovary (CHO) suspension cell lines. We summarize critical experimental steps from the initial seed train up to the final MSCC experiment, with a special focus on pre-culture management and medium preparation, device inoculation, and the establishment of a constant medium perfusion.
Topics: Animals; CHO Cells; Cricetulus; Cell Culture Techniques; Single-Cell Analysis; Cricetinae; Microfluidics; Microfluidic Analytical Techniques
PubMed: 38824641
DOI: 10.1016/j.xpro.2024.103106 -
Journal of Neuroscience Methods Aug 2024Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is...
BACKGROUND
Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met.
NEW METHOD
This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo.
RESULTS
We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture.
COMPARISON WITH EXISTING METHODS
Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation.
CONCLUSIONS
This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.
Topics: Animals; Organoids; Retina; Exosomes; Perfusion; Mice; Mice, Inbred C57BL; Tissue Culture Techniques; Cell Survival
PubMed: 38823594
DOI: 10.1016/j.jneumeth.2024.110181 -
Talanta May 2024Combination drug therapy represents an effective strategy for treating certain drug-resistant and intractable cancer cases. However, determining the optimal combination...
Combination drug therapy represents an effective strategy for treating certain drug-resistant and intractable cancer cases. However, determining the optimal combination of drugs and dosages is challenging due to clonal diversity in patients' tumors and the lack of rapid drug sensitivity evaluation methods. Microfluidic technology offers promising solutions to this issue. In this study, we propose a versatile microfluidic chip platform capable of integrating all processes, including dilution, treatment, and detection, for in vitro drug sensitivity assays. This platform innovatively incorporates several modules, including automated discrete drug logarithmic concentration generation, on-chip cell perfusion culture, and parallel drug treatments of cancer cell models. Moreover, it is compatible with microplate readers or high-content imaging systems for swift detection and automated monitoring, simplifying on-chip drug evaluation. Proof of concept is demonstrated by assessing the in vitro potency of two drugs, cisplatin, and etoposide, against the lung adenocarcinoma A549 cell line, under both single-drug and combination treatment conditions. The findings reveal that, compared to conventional microplate approaches with static cultivation, this on-chip automated perfusion bioassays yield comparable IC values with lower variation and a 50 % reduction in drug preparation time. This versatile dilution-treatment-detection microfluidic platform offers a promising tool for rapid and precise drug assessments, facilitating in vitro drug sensitivity evaluation in personalized cancer chemotherapy.
PubMed: 38823330
DOI: 10.1016/j.talanta.2024.126298 -
Fluids and Barriers of the CNS May 2024Bidirectional reciprocal motion of cerebrospinal fluid (CSF) was quantified using four-dimensional (4D) flow magnetic resonance imaging (MRI) and intravoxel incoherent...
Modeling cerebrospinal fluid dynamics across the entire intracranial space through integration of four-dimensional flow and intravoxel incoherent motion magnetic resonance imaging.
BACKGROUND
Bidirectional reciprocal motion of cerebrospinal fluid (CSF) was quantified using four-dimensional (4D) flow magnetic resonance imaging (MRI) and intravoxel incoherent motion (IVIM) MRI. To estimate various CSF motions in the entire intracranial region, we attempted to integrate the flow parameters calculated using the two MRI sequences. To elucidate how CSF dynamics deteriorate in Hakim's disease, an age-dependent chronic hydrocephalus, flow parameters were estimated from the two MRI sequences to assess CSF motion in the entire intracranial region.
METHODS
This study included 127 healthy volunteers aged ≥ 20 years and 44 patients with Hakim's disease. On 4D flow MRI for measuring CSF motion, velocity encoding was set at 5 cm/s. For the IVIM MRI analysis, the diffusion-weighted sequence was set at six b-values (i.e., 0, 50, 100, 250, 500, and 1000 s/mm), and the biexponential IVIM fitting method was adapted. The relationships between the fraction of incoherent perfusion (f) on IVIM MRI and 4D flow MRI parameters including velocity amplitude (VA), absolute maximum velocity, stroke volume, net flow volume, and reverse flow rate were comprehensively evaluated in seven locations in the ventricles and subarachnoid spaces. Furthermore, we developed a new parameter for fluid oscillation, the Fluid Oscillation Index (FOI), by integrating these two measurements. In addition, we investigated the relationship between the measurements and indices specific to Hakim's disease and the FOIs in the entire intracranial space.
RESULTS
The VA on 4D flow MRI was significantly associated with the mean f-values on IVIM MRI. Therefore, we estimated VA that could not be directly measured on 4D flow MRI from the mean f-values on IVIM MRI in the intracranial CSF space, using the following formula; e + 0.25. To quantify fluid oscillation using one integrated parameter with weighting, FOI was calculated as VA × 10 + f × 0.02. In addition, the FOIs at the left foramen of Luschka had the strongest correlations with the Evans index (Pearson's correlation coefficient: 0.78). The other indices related with Hakim's disease were significantly associated with the FOIs at the cerebral aqueduct and bilateral foramina of Luschka. FOI at the cerebral aqueduct was also elevated in healthy controls aged ≥ 60 years.
CONCLUSIONS
We estimated pulsatile CSF movements in the entire intracranial CSF space in healthy individuals and patients with Hakim's disease using FOI integrating VA from 4D flow MRI and f-values from IVIM MRI. FOI is useful for quantifying the CSF oscillation.
Topics: Humans; Cerebrospinal Fluid; Adult; Male; Female; Magnetic Resonance Imaging; Middle Aged; Young Adult; Aged; Hydrodynamics; Hydrocephalus; Brain
PubMed: 38816737
DOI: 10.1186/s12987-024-00552-6 -
British Journal of Pharmacology May 2024Pulmonary arterial hypertension (PAH) is a progressive disease in which chronic membrane potential (E) depolarisation of the pulmonary arterial smooth muscle cells...
BACKGROUND AND PURPOSE
Pulmonary arterial hypertension (PAH) is a progressive disease in which chronic membrane potential (E) depolarisation of the pulmonary arterial smooth muscle cells (PASMCs) causes calcium overload, a key pathological alteration. Under resting conditions, the negative E is mainly set by two pore domain potassium (K) channels, of which the TASK-1 has been extensively investigated.
EXPERIMENTAL APPROACH
Ion channel currents and membrane potential of primary cultured human(h) PASMCs were measured using the voltage- and current clamp methods. Intracellular [Ca] was monitored using fluorescent microscopy. Pulmonary BP and vascular tone measurements were also performed ex vivo using a rat PAH model.
KEY RESULTS
TREK-1 was the most abundantly expressed K in hPASMCs of healthy donors and idiopathic(I) PAH patients. Background K-current was similar in hPASMCs for both groups and significantly enhanced by the TREK activator ML-335. In donor hPASMCs, siRNA silencing or pharmacological inhibition of TREK-1 caused depolarisation, reminiscent of the electrophysiological phenotype of idiopathic PAH. ML-335 hyperpolarised donor hPASMCs and normalised the E of IPAH hPASMCs. A close link was found between TREK-1 activity and intracellular Ca-signalling using a channel activator, ML-335, and an inhibitor, spadin. In the rat, ML-335 relaxed isolated pre-constricted pulmonary arteries and significantly decreased pulmonary arterial pressure in the isolated perfused lung.
CONCLUSIONS AND IMPLICATIONS
These data suggest that TREK-1is a key factor in E setting and Ca homeostasis of hPASMC, and therefore, essential for maintenance of a low resting pulmonary vascular tone. Thus TREK-1 may represent a new therapeutic target for PAH.
PubMed: 38807478
DOI: 10.1111/bph.16426 -
Medical Science Monitor : International... May 2024BACKGROUND Cold and heat therapies for recovery in sports are commonly used, including in the mixed martial arts (MMA). The Game Ready (GR) device can be used for local... (Comparative Study)
Comparative Study
BACKGROUND Cold and heat therapies for recovery in sports are commonly used, including in the mixed martial arts (MMA). The Game Ready (GR) device can be used for local monotherapy with either heat or cold and for contrast therapy. This study aimed to compare the effects of duration of cold and heat compression on biomechanical changes in the forearm muscles of 20 healthy mixed martial arts athletes. MATERIAL AND METHODS Twenty MMA volunteers (26.5±4.5 years old) underwent 3 different phases of the GR: (1) stimulation time 10 min (eGR-10, GR experimental group), (2) 10 min (cGR-10, sham control group) and (3) 20 min (eGR-20, GR experimental group). The following outcomes were assessed: muscle tone (T), stiffness (S), flexibility (E), pressure pain threshold (PPT), microvascular response (PU), and maximum isometric strength (Fmax). All measurements were performed before GR (rest) and after GR stimulation (post). RESULTS Both eGR-10 and eGR-20 significantly improved outcomes T (p<0.001), S (p<0.001), E (p=0.001, and p<0.001, respectively), PPT (p<0.001), PU (p<0.001), and Fmax (p<0.001). Notably, eGR-20 exhibited superior improvements in PU, Fmax, and PPT, with larger effect sizes (p<0.001). While eGR-10 demonstrated more pronounced reductions in T and S (p<0.001), these results underscore the potential for tailored GR therapy durations to optimize specific recovery goals for MMA athletes. CONCLUSIONS GR stimulation affects muscle biomechanical changes, pain threshold, muscle strength, and tissue perfusion. The study results suggest that 10 min of GR stimulation is sufficient to achieve changes that can be used to optimize recovery for MMA athletes.
Topics: Humans; Male; Adult; Muscle, Skeletal; Martial Arts; Forearm; Biomechanical Phenomena; Athletes; Hot Temperature; Female; Young Adult; Cold Temperature; Pain Threshold; Muscle Strength
PubMed: 38805404
DOI: 10.12659/MSM.944149 -
Research Square May 2024Fetal membrane(amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic...
Fetal membrane(amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. An amnion membrane (AM) organ-on-chip (OOC) was utilized to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 hours flow culture to mimic fluid motion. Static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control for pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to CK-18 ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a flow culture environment is not necessary to mimic any physiologic cellular conditions of fetal membrane-derived cells.
PubMed: 38798515
DOI: 10.21203/rs.3.rs-4372328/v1