Background
Peristaltic pumps are commonly used in unit operations for the production of cellular therapeutics, with some geared toward shear-sensitive applications. However, cells can experience high ...shear stress when pumped through peristaltic pumps due to fluid path occlusion, resulting in reduced cell viability.
An innovative pump technology, the hybrid piston/diaphragm (HPD) pump, is an alternative to the peristaltic pump. The HPD pump may offer advantages for cell therapy unit operations due to its increased control capability and absence of occlusion to the fluid path, resulting in reduced shear stress.
An initial study was performed to measure cell viability effects of the two pumps at varying circulation times.
Aims: The purpose of the previous study was to compare the impact on cell viability of the HPD pump to the convex roller peristaltic (CRP) pump as a first consideration of this pump for cell therapy applications. The purpose for this study will remain the same, however, it incorporates the most used peristaltic pumps and Jurkat T-lymphocytes suspended in relevant solutions, representing real-world conditions for cell therapy processes.
Methods
While the initial study involved NIH-3T3 Mouse Fibroblast cells suspended in growth media, the follow-on study uses T cells in an industry-accepted suspension. The cell suspension is pumped through a tubing circuit, circulating for 5, 20, and 60 minutes in triplicate. Cell viability is determined using a hemacytometer and Presto Blue to determine cell viability as well as impact or disruption to the cell membrane. The cells are imaged using light microscopy 2 hours and 2 days post-circulation.
This follow-on study will add data from two additional commercially available pumps and be presented at ISCT2024.
The previous study served as a first step to show the potential of the HPD pump as an innovation in cell therapy unit operations. HPD pumps offer several advantages over the CRP pump such as true positive displacement pumps with highly adjustable pumping parameters. This study will compare two flat roller peristaltic pumps frequently seen in cell therapy processes to the HPD pump. Additionally, this study will look at the impact on cell viability for a more relevant cell line suspended in an appropriate process solution.
Dental avulsion is considered as one of the most
severe types of traumatic tooth injuries because it causes damage to several
structures and results in the complete displacement of the tooth from its
...socket in the alveolar bone. The ideal situation is to replant the tooth
immediately after avulsion because the extra oral time is an important
determinant for the success of the treatment and for a good prognosis. The aim
of this systematic review was to identify the recommended natural storage m to
store and transport avulsed tooth based on the survival capacity of periodontal
ligament cells.This paper reviews the different storage media that
have been evaluated for avulsed teeth based on full-length research papers
retrieved from PubMed/Medline, Lilacs, BBO and SciELO electronic databases
using the key words ‘storage medium’, ‘avulsion’, ‘tooth avulsion’,
‘replantation’, ‘tooth replantation’, ‘milk’ and ‘propolis’. Based on the
application of inclusion and exclusion criteria, About 14 papers have been
selected and critically reviewed with respect to the characteristics, efficacy
and ease of access of the storage media. The review of this study shows and
includes a wide array of wet storage media that have been evaluated in
laboratory-based studies on PDL cells found on adult permanent teeth.
Among the natural products other than milk,
propolis, coconut water, green tea extract, egg white, green tea extract,
Alovera gel, pomegranate juice, salvia officinalis followed by dragon blood sap
(Croton Lechleri) were recommended based
on the cell viability and its longevity. In an emergency, it is important for
dentists to consider the circumstances of the accident, the location and
suggest an appropriate transport media.
Cell Viability Under Anoxic Conditions WAGNER, Stephanie; POHL, Jakob; HACKBARTH, Steffen
Photodiagnosis and photodynamic therapy,
March 2023, 2023-03-00, Letnik:
41
Journal Article
Recenzirano
PDT relies on the availability of oxygen to generate cytotoxic ROS and singlet oxygen in particular. Yet, after photosensitizer illumination, the oxygen in the direct vicinity is consumed very ...quickly through reactions with biological material, especially in the hypoxic environment caused by the increased metabolism of tumour cells. 1 Considering these facts, the illumination regimes currently used in clinical PDT should be investigated for the extent to which they promote or even hinder generation of singlet oxygen in tumour tissue. Assuming the latter, the question arises of whether illumination induced anoxia might lead to reliable cell death. For this purpose, the correlation between the oxygen available to the cells and their short-, medium- and long-term survival rates was examined.
Using the adherent cell line FaDu as a tumour model, the atmospheric oxygen concentration inside an incubator is adjusted with a gas composition system developed in the group. To determine both the oxygen concentration actually available to the cells and their consumption 2, a simulation of oxygen diffusion in culture medium 3, 4 was derived. The survival rate of the cells was checked with fluorescence-based vitality assays both during and after exposure to anoxia.
Using oxygen consumption achieved through illumination of a photosensitizer in solution in the presence of extracellular quenchers as a first measurement, the determined results compared well with the numerical simulation.
In the series of experiments, it became clear that even at normal atmospheric pressure, significantly less oxygen than expected is available, especially for adherent cells. Accordingly, every system above anoxia is in a highly dynamic state in which the oxygen concentration at the level of the cells differs significantly from the oxygen concentration above the medium. For this reason, the simulation of this complex system, which considers cell respiration and growth as well as oxygen diffusion through the culture medium, was compared with first results of the viability assays.
Although the current experiments included only two cell lines, the model provides deep insights into the role of anoxia in PDT. Furthermore, the model can be easily adapted to the particular metabolisms of different tumours. Thus, it is an important tool to inform future case-based decisions about the potential targeted use of localized anoxia as an alternative therapy.
Maintenance of protein homeostasis and organelle integrity and function is critical for cellular homeostasis and cell viability. Autophagy is the principal mechanism that mediates the delivery of ...various cellular cargoes to lysosomes for degradation and recycling. A myriad of studies demonstrate important protective roles for autophagy against disease. However, in cancer, seemingly opposing roles of autophagy are observed in the prevention of early tumour development versus the maintenance and metabolic adaptation of established and metastasizing tumours. Recent studies have addressed not only the tumour cell intrinsic functions of autophagy, but also the roles of autophagy in the tumour microenvironment and associated immune cells. In addition, various autophagy-related pathways have been described, which are distinct from classical autophagy, that utilize parts of the autophagic machinery and can potentially contribute to malignant disease. Growing evidence on how autophagy and related processes affect cancer development and progression has helped guide efforts to design anticancer treatments based on inhibition or promotion of autophagy. In this Review, we discuss and dissect these different functions of autophagy and autophagy-related processes during tumour development, maintenance and progression. We outline recent findings regarding the role of these processes in both the tumour cells and the tumour microenvironment and describe advances in therapy aimed at autophagy processes in cancer.
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•Hydrothermal synthesis of reduced graphene oxide (RGO) nanosheets using soluble starch as a reducing agent.•Different concentrations of soluble starch hydrothermally yielded SRGO ...nanosheets under basic conditions.•X-ray photoelectron spectroscopy showed the reduction of GO to high C/O ratios.•SRGOs were compatible with human fibroblasts and red blood cells.•Biofilm formation and the viability of S. aureus in biofilms in the presence of SRGOs were evaluated.
Graphene-based nanomaterials (GBNs) have many applications as biomedical materials in tissue engineering and regenerative medicine. We report on the preparation of starch-(functionalized) reduced graphene oxide nanosheets (SRGO) using soluble starch as a reducing agent in a hydrothermal method, and their in vitro interactions with human skin fibroblasts and red blood cells (RBCs). Our results indicate that soluble SRGO nanosheets were prepared using graphene oxide (GO) as a raw material. SRGO-1 and -10, which were prepared using different concentrations of soluble starch after hydrothermal treatment, were characterized by ultraviolet-visible spectroscopy and showed a peak shift at 260 nm corresponding to the deoxygenation of GO and restoration of the conjugated aromatic structure. Dynamic light scattering and zeta potential measurements were used to determine Z-average sizes and surface charges of GO and SRGOs. X-ray diffractometry, attenuated total reflectance Fourier-transform infrared spectroscopy, and Raman spectroscopy revealed the progressive elimination of labile oxygen functional groups during hydrothermal treatment and restoration of the π-conjugated network. X-ray photoelectron spectroscopy showed de-oxidation of SRGOs, which had high carbon to oxygen ratios (C/O) as compared with GO. Interactions of SRGO-1 and -10 with skin fibroblasts showed excellent biocompatibility even at a concentration of 200 μg/ml with cell viabilities up to 88% and 90%, respectively, whereas notable cytotoxicity was observed for GO at 20 μg/ml. Similarly, SRGO-1 and -10 did not exhibit toxicity to RBCs compared to GO. Biofilm formation and metabolic activities of biofilm by the bacterium Staphylococcus aureus were also evaluated using a crystal violet and a tetrazolium reduction assay, respectively. The described hydrothermal method used to synthesize SRGO provides a cheap, facile, and environmentally friendly means of producing water-dispersible, biocompatible and hemocompatible reduced GOs for the fabrication of novel GBNs for various biomedical applications.
Measuring the number of viable cells in a sample is critical to characterizing cell-based products. Many image-based viability assays are semi-automated to reduce user bias and improve assay ...precision and throughput; however, comparability of viability results between instruments and across sites can be challenging due to differences in sample preparation, image acquisition and image analysis. Setting gates around live/dead populations manually from sample to sample can be subjective and a significant source of variability. Here, we demonstrate the use of calibration beads with assigned ERF (equivalent reference fluorophore) values and fixed cell control materials to transfer viability gates across assay conditions and between instruments, sites, and studies.
Jurkat cells are fixed with MBS (M-maleimidobenzoyl-N-hydroxysulfosuccinimide ester) and stained with Alexa Fluor 680 C2 Maleimide and stably stored at 4oC. MBS fixed cells are spiked into overgrown (low viability) Jurkat test samples. For viability testing, the mixed sample is stained with Acridine Orange (AO)/DAPI solution and imaged on the Nucleocounter NC-3000 or a widefield Nikon microscope. ERF beads are imaged to serve as a fluorescent scale. Images are processed via NC-3000 software or Image J. Gate transfer is achieved by converting mean fluorescence intensity (MFI) to ERF using the ERF bead calibration curve and converting the fixed cell control material MFI to ERF to generate an Equivalent Reference Cell (ERC) Intensity scale. On the ERC scale, gates can be transferred between studies across multiple instruments when the fixed cell control material is spiked into the test sample.
Fixed cells were fabricated with stability of optical properties when stored at 4oC. Use of fixed cells and ERF beads enabled gate transfer for AO/DAPI viability across imaging modes (Fig 1) and across assays utilizing different dye concentrations or illumination strategies. By controlling gate transfer, variability between assays was reduced. By eliminating gating as a source of variability, other biological differences between samples were observed, such as changes in sample properties that impacted comparability of results. Strategies using appropriate control materials to systematically reduce bias and variability in image-based measurements such as cell viability will improve measurement assurance for critical assays.
The identification of commercial products does not imply endorsement or recommendation by NIST.
Nanoplastics in the environment lead to the human exposure to these particles. However, the consequences of this exposure are not yet fully understood. Here, the cytotoxicity of polystyrene ...nanoparticles (PS-NPs) with a uniform size (50 nm) but distinct surface functionalization (pristine polystyrene, PS; carboxy and amino functionalized, PS-COOH and PS-NH2, respectively), and at an exposure dosage of 10, 50 and 100 μg/mL, were assessed in the human hepatocellular carcinoma (HepG2) cell line. Although all PS-NPs could be internalized by the HepG2 cells, according to the fluorescent intensities, more of PS-COOH and PS-NH2 than PS, accumulated in the cells. The cell viability was significantly affected in a positively dose-related manner. Functionalized PS-NPs exhibited greater inhibition of cell viability than PS, and the viability inhibition peaked (46%) at 100 μg/mL of PS-NH2 exposure. Superoxide dismutase (SOD) activity was maximum when HepG2 cells were exposed to 10 μg/mL of PS-COOH (1.8 folds higher than that without PS-COOH exposure). The glutathione (GSH) content was maximum when the cells were treated with 50 μg/mL of PS (3.75 fold increase compared to untreated cells). Although the difference in inhibition of cell viability was not significant between PS-NH2 and PS-COOH exposure, 100 μg/mL of PS-NH2 exposure caused the most severe oxidative stress due to dramatically increased accumulation of malondialdehyde (MDA); however, a decrease in the antioxidants levels as the SOD activity and GSH content were also found. The results demonstrated that the cellular oxidative damage occurred and that the antioxidation enzymes may not be able to maintain the balance between the generation of oxidant species and the antioxidant defense. Consequently, 100 μg/mL of PS-NH2 exposure triggered the destruction of antioxidant structures. This study defines the cytotoxic effects of PS-NPs on HepG2 cells and emphasizes the significance of investigating the cytotoxic outcomes of nanoplastics in humans.
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•PS-NPs reduced the cell viability of HepG2 cells.•Reduced of cell viability showed positively dosage-related trend.•PS-NPs with size of 50 nm can be rapidly internalized by HepG2 cells.•Functionalized PS-NPs exhibited higher negative impact on cell viability than PS.•PS-NH2 of 100 μg/mL caused the destruction of antioxidant capabilities.
The therapeutic efficacy of natural killer (NK) cells‐based immunotherapy is greatly related with the survival of transplanted NK cells. However, no effective strategy was reported to monitor NK cell ...viability in adoptive immunotherapy in vivo. Herein, we develop a ratiometric NIR‐II fluorescence imaging strategy to quantitively track and visualize the adoptive NK cell viability in vivo in real‐time. The nanoprobe consists of lanthanide‐based down‐conversion nanoparticles (DCNP) coated with IR786s, a reactive oxygen species (ROS) sensitive to NIR dye, which was directly labeled with NK cells. Upon cell death, the excessive ROS generation occurred within NK cells, along with IR786s degradation, turning on NIR‐II fluorescent signal at 1550 nm of DCNP under 808‐nm excitation, while the fluorescent signal at 1550 nm of DCNP under 980‐nm excitation was stable. Such an intracellular ROS‐induced ratiometric NIR‐II fluorescent signal was validated to correlate well with NK cell viability in vivo. Using this nanoreporter, we further demonstrated that co‐treatment with IL‐2, IL‐15, and IL‐21 could improve NK cell viability in vivo, achieving enhanced immunotherapy for orthotopic hepatocellular carcinoma. Overall, this strategy allows for longitudinal and quantitative tracking of NK cell viability in NK cell‐based immunotherapy.
Using a ROS‐sensitive NIR‐II fluorescence probe to label NK cells was reported for tracking cell viability in adoptive NK cell‐based immunotherapy. Intracellular ROS generation of dying NK cells, caused by exogenous physical or chemical factors, such as hypoxia, starvation, and oxidative stress in the tumor microenvironment, results in a ratiometric NIR‐II fluorescence signal in vivo, which holds considerable potential for adoptive NK cell‐based immunotherapy.
Cancer drug development has been riddled with high attrition rates, in part, due to poor reproducibility of preclinical models for drug discovery. Poor experimental design and lack of scientific ...transparency may cause experimental biases that in turn affect data quality, robustness and reproducibility. Here, we pinpoint sources of experimental variability in conventional 2D cell-based cancer drug screens to determine the effect of confounders on cell viability for MCF7 and HCC38 breast cancer cell lines treated with platinum agents (cisplatin and carboplatin) and a proteasome inhibitor (bortezomib). Variance component analysis demonstrated that variations in cell viability were primarily associated with the choice of pharmaceutical drug and cell line, and less likely to be due to the type of growth medium or assay incubation time. Furthermore, careful consideration should be given to different methods of storing diluted pharmaceutical drugs and use of DMSO controls due to the potential risk of evaporation and the subsequent effect on dose-response curves. Optimization of experimental parameters not only improved data quality substantially but also resulted in reproducible results for bortezomib- and cisplatin-treated HCC38, MCF7, MCF-10A, and MDA-MB-436 cells. Taken together, these findings indicate that replicability (the same analyst re-performs the same experiment multiple times) and reproducibility (different analysts perform the same experiment using different experimental conditions) for cell-based drug screens can be improved by identifying potential confounders and subsequent optimization of experimental parameters for each cell line.
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