Transient Pores Research Articles

Genetically-encoded BRET probes shed light on ligand bias–induced variable ion selectivity in TRPV1 and P2X5/7

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Numerical study of opposed zero-net-mass-flow jet-induced erythrocyte mechanoporation

With the advantages of biosafety and efficiency, increasing attention has been paid to the devices for gene and macromolecular drug delivery based on mechanoporation. The transient pore formation on the cell membrane allows cargo transportation when the membrane areal strain is beyond the critical pore value and below the lysis tension threshold. Based on this principle, we propose a method to apply the proper fluid stress on cells moving in a microchannel under the action of zero-net-mass-flux (ZNMF) jets. In this study, an immersed finite element method (IFEM) is adopted to simulate the interaction between the cells and the fluid fields so as to investigate the cell movement and deformation in this mechanoporation system. To evaluate the efficiency of the cargo delivery, a pore integral is defined as the mean pore rate when the cell passes through the jet region. By analyzing the effects of the parameters, including the pressure gradient along the microchannel, the jet amplitude, and the jet frequency, on the pore integrals, a group of optimized parameters for cargo delivery efficiency are obtained. Additionally, the stability and safety of this system are analyzed in detail. These results are helpful in designing the mechanoporation devices and improving their efficiency of drug delivery.

Electroporation in Clinical Applications—The Potential of Gene Electrotransfer and Electrochemotherapy

Electroporation (EP) allows for the transport of molecules into the cytoplasm with significant effectiveness by forming transient pores in the cell membrane using electric pulses. This can be used for cellular transport (RE—reversible electroporation) or ablation (IRE—irreversible electroporation). The first of described options fortifies medicine with novel possibilities: electrochemotherapy (ECT), which creates promising perspectives for cancer treatment, and gene electrotransfer (GET), a powerful method of DNA delivery as well as immunogen electrotransfer. The review constitutes a comprehensive explanation of the mechanism of EP in the case of GET, its present and prospective employment in medicine, including gene delivery, vaccinations, therapy, and transfection, are also presented.

Acoustically driven shear mechanoporation for efficient intracellular delivery and transfection

Delivery of large and structurally complex molecules into cells is useful in numerous biomedical applications. Furthermore, this capability is critical to the emerging area of cell-based therapeutics. Extant in vivo (viral) and in vitro (chemical, electrical) methods are often inadequate for protein, nucleic acid, and synthetic nanomaterial delivery, leading researchers to develop alternative physical approaches. Over the past two decades, our lab has established a micromachined ultrasonic droplet generator that uses geometric focusing of acoustic waves to break the liquid surface and form extremely fine droplets. More recently, we have found that these focused mechanical forces generated on a microsecond time scale can be applied to reversibly disrupt the membranes of cells. Acoustic shear poration (ASP) relies on cell ejection from microscale orifices only slightly larger than the cells to open transient pores large enough for passage of small to large macromolecules at greater than 75% efficiency. I will discuss the principles of device operation, the connection between operating parameters and treatment outcomes, and the limitations of diffusive delivery following mechanoporation. I will also introduce a two-stage approach that combines mechanoporation and an electrophoretic action to improve transfection and delivery of complex assemblies that carry multiple payloads.

Landscape of Cellular Bioeffects Triggered by Ultrasound-Induced Sonoporation.

Sonoporation is the process of transient pore formation in the cell membrane triggered by ultrasound (US). Numerous studies have provided us with firm evidence that sonoporation may assist cancer treatment through effective drug and gene delivery. However, there is a massive gap in the body of literature on the issue of understanding the complexity of biophysical and biochemical sonoporation-induced cellular effects. This study provides a detailed explanation of the US-triggered bioeffects, in particular, cell compartments and the internal environment of the cell, as well as the further consequences on cell reproduction and growth. Moreover, a detailed biophysical insight into US-provoked pore formation is presented. This study is expected to review the knowledge of cellular effects initiated by US-induced sonoporation and summarize the attempts at clinical implementation.

Assembly of transmembrane pores from mirror-image peptides

Tailored transmembrane alpha-helical pores with desired structural and functional versatility have promising applications in nanobiotechnology. Herein, we present a transmembrane pore DpPorA, based on the natural pore PorACj, built from D-amino acid α-helical peptides. Using single-channel current recordings, we show that DpPorA peptides self-assemble into uniform cation-selective pores in lipid membranes and exhibit properties distinct from their L-amino acid counterparts. DpPorA shows resistance to protease and acts as a functional nanopore sensor to detect cyclic sugars, polypeptides, and polymers. Fluorescence imaging reveals that DpPorA forms well-defined pores in giant unilamellar vesicles facilitating the transport of hydrophilic molecules. A second D-amino acid peptide based on the polysaccharide transporter Wza forms transient pores confirming sequence specificity in stable, functional pore formation. Finally, molecular dynamics simulations reveal the specific alpha-helical packing and surface charge conformation of the D-pores consistent with experimental observations. Our findings will aid the design of sophisticated pores for single-molecule sensing related technologies.

1295TiP Investigation of calcium electroporation (CaEP) therapy in malignant cutaneous and subcutaneous tumours: A non-randomized phase II clinical trial of a novel palliative therapy

Skin metastases can occur from any tumour entity and often cause distress. Surgery or radiation are options, limitations arise from size or location and prior treatments. Electroporation (EP) creates transient pores in cell membranes by short high-voltage pulses allowing diffusion of molecules into the cells. EP with systemic chemotherapy is a standard therapy for cutaneous tumours, causing cell death of tumour cells and skin tissue recovering. Calcium-ions (Ca) are part of various signalling pathways. Intracellular Ca overload induces celldeath through ATP depletion as demonstrated in vitro and vivo, with healthy cells recovering. Intratumoural Ca-injection before EP-treament of affected areas is a new, promising palliative treatment reducing use of chemotherapy and interactions anti-tumour-therapies. The purpose of this trial was to investigate CaEP as therapy for cutaneous and subcutaneous tumours. Approval of German and Danish Drug Oversight Agencies and local Ethics Committees was obtained prior to the start of the trial. It was supported from the European Fund for Regional Development as part of the “Changing Cancer Care”-project. This multi-centre non-randomized single-arm phase II trial investigates the use of CaEP in cutaneous and subcutaneous tumours of any cancer histology. All included patients receive CaEP. Primary endpoint is clinical response rate at two months. Response rate is defined as number of responding to treated lesions, evaluated by reduction in size, documented with clinical photography. Secondary endpoints include: treatment response at time points month 1, 3, 4, 6 and 12; histological tissue changes in biopsies one year after treatment; response on MRI on a subset of patients; quality-of-life assessments before treatment, after 2 and 12 months using the EORTC QLQ-C15-PAL questionnaire; examining systemic immune response through routine scans; response due to tumour histology; response rate of all tumours treated; response rate per patient; current of applied pulses and qualitative interviews of one subset of patients. The study is conducted at three cancer centres in Germany and Denmark. EudraCT 2019-004314-34; NCT04225767. The authors.

The role of ion-lipid interactions and lipid packing in transient defects caused by phenolic compounds

The transient disruption of membranes for the passive permeation of ions or small molecules is a complex process relevant to understanding physiological processes and biotechnology applications. Phenolic compounds are widely studied for their antioxidant and antimicrobial properties, and some of these activities are based on the interactions of the phenolic compound with membranes. Ions are ubiquitous in cells and are known to alter the structure of phospholipid bilayers. Yet, ion-lipid interactions are usually ignored when studying the membrane-altering properties of phenolic compounds. This study aims to assess the role of Ca2+ ions on the membrane-disrupting activity of two phenolic acids and to highlight the role of local changes in lipid packing in forming transient defects or pores. Results from tethered bilayer lipid membrane electrical impedance spectroscopy experiments showed that Ca2+ significantly reduces membrane disruption by caffeic acid methyl ester and caffeic acid. As phenolic acids are known metal chelators, we used UV-vis and fluorescence spectroscopy to exclude the possibility that Ca2+ interferes with membrane disruption by binding to the phenolic compound and subsequently preventing membrane binding. Molecular dynamics simulations showed that Ca2+ but not caffeic acid methyl ester or caffeic acid increases lipid packing in POPC bilayers. The combined data confirm that Ca2+ reduces the membrane-disrupting activity of the phenolic compounds, and that Ca2+-induced changes to lipid packing govern this effect. We discuss our data in the context of ion-induced pores and transient defects and how lipid packing affects membrane disruption by small molecules.

Laser-enabled delivery of antiretroviral drugs into HIV-1 infected TZM-bl cells.

The use of femtosecond laser to create sub-microscopic transient pores on the cell membrane allowing exogenous material into mammalian cells has become a very efficient optical delivery method over the past decade. This study focuses on laser-enabled delivery of antiretroviral (ARV) drugs into HIV-1 infected TZM-bl cells in vitro. A 1kHz femtosecond laser emitting at a wavelength of 800 nm was used to photoporate cells at 6.5μW. Trypan blue was used for characterisation and its uptake was quantified using Matlab software. Cell membrane damage was assessed using the lactate dehydrogenase (LDH) assay while HIV-1 infection was assessed using luciferase assay. Our results showed successful delivery of ARVs into HIV-1 infected cells without compromising their cell membranes, subsequently reducing the level of infection. The LDH assay showed no significant cell membrane damage of laser-treated cells, and the luciferase assay demonstrated significant reduction in the level of HIV-1 infection.

Antitumor Proliferation and Related Mechanism of Ultrasound Irradiation Combined with Safflower Yellow.

Ultrasound irradiation is now the best method for evaluating benign and malignant tumor nodules. Chemotherapy has always played an important role in the treatment of malignant tumors. With the large-scale application of chemotherapy drugs, the problem of multidrug resistance of tumors has become more and more prominent, which has become one of the difficulties in tumor chemotherapy. This study mainly explores the antitumor proliferation and related mechanisms of ultrasound irradiation combined with safflower yellow. The breast cancer cell line 4T1 derived from BALB/c mice was selected. BALB/c is an albino laboratory mouse, which, like many commonly used sublines, originated from Mus musculus. BALB/c mice have been bred for more than 200 generations in research institutions around the world and are widely used in animal experiments in immunology and physiology. When the cell proliferation reached 80%–90% of the bottom area of the culture flask, it was resuspended, passaged, frozen, and resuscitated according to experimental needs. The 4T1 breast cancer cell line was cultured by conventional methods. 4T1 breast cancer cells in the logarithmic proliferation phase were collected. After 0.25% was digested with pancreatin, it was washed twice with PBS to adjust the concentration to 1 × 107/mL. A 0.1 mL tumor cell suspension was subcutaneously inoculated on the edge of the mouse chest, thereby establishing a breast cancer model of BALB/c mice. After 6-15 days, the tumor volume grew rapidly and became larger. When the length of the tumor is 2.5 × 2.5, the modeling is successful. Ultrasound-targeted microbubble destruction technology, as a novel drug delivery method with high efficiency and low toxicity, can form transient pores (sonoporation effect) on the cell surface, widen the intercellular space, and increase the membrane permeability, and thus effectively. The transport of drugs, genes, proteins, etc., is promoted to target organs and tissues. Tumor-forming mice were randomly divided into the following four groups: control group, safflower yellow group, ultrasound irradiation group, and ultrasound irradiation combined with safflower yellow group. From the second day of inoculation to the end of the experiment, the body weight of the mice successfully inoculated with 4T1 cells was measured every day; from the 5th day, tumors in each group were calculated body volume and tumor inhibition rate (TIR) of each group. The combined treatment group has a higher tumor inhibition rate than the ultrasound irradiation group, and the difference is statistically significant (P < 0.05). Ultrasound irradiation combined with safflower yellow pigment can effectively inhibit tumor proliferation, maintain, or even improve the efficacy of chemotherapy, thereby improving the patient's tolerance to chemotherapy.

Calcium Electroporation Reduces Viability and Proliferation Capacity of Four Uveal Melanoma Cell Lines in 2D and 3D Cultures.

Simple SummaryCalcium electroporation (CaEP) is an innovative anti-tumor treatment modality that induces cell death by introducing supraphysiological concentrations of calcium into cells with a limited effect on normal cells. The objective of the present study is to assess the effect of CaEP in uveal melanoma (UM) cell lines in comparison to electrochemotherapy (ECT) with bleomycin using 2D monolayer cell cultures as well as 3D tumor spheroid models in four different UM cell lines. The morphological changes of the spheroids, the cell viability, growth rate as well as the cytotoxic effect of electroporation (EP) with calcium chloride and bleomycin were evaluated with various drug concentrations. The results of CaEP and ECT both suggest a comparable dose-dependent reduction in cell viability and proliferation rate in all tested 2D cell lines and 3D tumor spheroids. These data point out that CaEP is an established anticancer treatment causing cell death by ATP depletion in in vitro and in vivo, representing an efficient alternative therapy with a lower cytotoxic potency for the local UM tumor control.Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non-cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye.

Characterization of spatio-temporal distributions of wave-induced pore pressure in a non-cohesive seabed: Amplitude-attenuation and phase-lag

While ocean waves propagating over a porous seabed, the amplitude of excess pore pressure would attenuate into the seabed (termed as “amplitude-attenuation”), which is always accompanied by “phase-lag”. In this study, the amplitude-attenuation and the phase-lag phenomena in a fine-sand bed and a medium-sand bed were physically modeled in a large wave flume. A relative rigidity of soil-skeleton to pore-fluid is introduced to evaluate the seabed compressibility. Explicit expressions of the amplitude-attenuation and the phase-lag are derived and then validated with the experimental results. Parametric analyses indicate that, for the relative rigidity of the seabed much larger than 1.0, the amplitude-attenuation and the phase-lag along the non-dimensional soil depth would become more significant for larger wave period, but for smaller values of the combined rigidity-permeability parameter of the soil. A non-dimensional parameter (Ic) is further derived to characterize the combined effects of wave parameters and soil properties. Simplified spatio-temporal correlation of transient pore pressure is finally established for Ic ≫ 1.0, indicating the more rapidly the pore pressure amplitude attenuates within the seabed, the more significant the phase-lag becomes, correspondingly.

Porous Liquids: Computational Design for Targeted Gas Adsorption.

In this Perspective, we present the unique gas adsorption capabilities of porous liquids (PLs) and the value of complex computational methods in the design of PL compositions. Traditionally, liquids only contain transient pore space between molecules that limit long-term gas capture. However, PLs are stable fluids that that contain permanent porosity due to the combination of a rigid porous host structure and a solvent. PLs exhibit remarkable adsorption and separation properties, including increased solubility and selectivity. The unique gas adsorption properties of PLs are based on their structure, which exhibits multiple gas binding sites in the pore and on the cage surface, varying binding mechanisms including hydrogen-bonding and π-π interactions, and selective diffusion in the solvent. Tunable PL compositions will require fundamental investigations of competitive gas binding mechanisms, thermal effects on binding site stability, and the role of nanoconfinement on gas and solvent diffusion that can be accelerated through molecular modeling. With these new insights PLs promise to be an exceptional material class with tunable properties for targeted gas adsorption.

Electrochemotherapy in the Treatment of Kaposi’s Sarcoma: A Single Centre Cohort Study and a Comparison with the Literature Data

Background: Kaposi's sarcoma (KS) is a tumor of endothelial derivation, which primarily affects the skin and is mainly related to the type 8 Human Herpesvirus (HHV8). Its onset is favored by immunosuppression, although the most common form is the classic or spo-radic KS mainly developing in elderly men of Mediterranean and Eastern European origin. Dif-ferent therapeutic options are available, depending on the clinical variant, progression pattern, and comorbidities. The treatment of localized forms includes surgical excision, laser treatment, cryosurgery, radiotherapy, imiquimod 5%, and intra-lesion injection of cytotoxic drugs; on the other hand, the treatment of widespread disease encompasses radiotherapy and chemotherapy. Methods: In this scenario, Electrochemotherapy (ECT), has shown to be an effective alternative to traditional treatment for disseminated KS skin lesions. The rationale of ECT relies on the lo-cal application of short, high-voltage electric pulses, able to open transient pores in the cell membrane (reversible electroporation that increases the delivery of some poorly permeant cyto-toxic agents into the cytosol. Results: Herein, we performed a retrospective analysis on 9 KS patients treated with ECT at our center between June 2016 and January 2020. The rate of Complete Response (CR) was 77.8% after the first cycle of treatment and 88.9% after the second course, with an overall response (OR) of 100%. Conclusion: Sustained local control of treated lesions was present in 77.8% of patients 6 months after the treatment and all of them reported only mild local toxicity, together with an excellent functional and cosmetic outcome, in agreement with data obtained from the compari-son with the recent literature.

A Novel Radial Basis Function Approach for Infiltration-Induced Landslides in Unsaturated Soils

In this article, the modeling of infiltration--induced landslides, in unsaturated soils using the radial basis function (RBF) method, is presented. A novel approach based on the RBF method is proposed to deal with the nonlinear hydrological process in the unsaturated zone. The RBF is first adopted for curve fitting to build the representation of the soil water characteristic curve (SWCC) that corresponds to the best estimate of the relationship between volumetric water content and matric suction. The meshless method with the RBF is then applied to solve the nonlinear Richards equation with the infiltration boundary conditions. Additionally, the fictitious time integration method is adopted in the meshless method with the RBF for tackling the nonlinearity. To model the stability of the landslide, the stability analysis of infinite slope coupled with the nonlinear Richards equation considering the fluctuation of transient pore water pressure is developed. The validation of the proposed approach is accomplished by comparing with exact solutions. The comparative analysis of the factor of safety using the Gardner model, the van Genuchten model and the proposed RBF model is provided. Results illustrate that the RBF is advantageous for reconstructing the SWCC with better estimation of the relationship than conventional parametric Gardner and van Genuchten models. We also found that the computed safety factors significantly depend on the representation of the SWCC. Finally, the stability of landslides is highly affected by matric potential in unsaturated soils during the infiltration process.

Influence of Electroporation Medium on Delivery of Cell-Impermeable Small Molecules by Electrical Short-Circuiting via an Aqueous Droplet in Dielectric Oil: A Comparison of Different Fluorescent Tracers.

Membrane permeabilization stimulated by high-voltage electric pulses has been used to deliver cell-impermeable exogenous molecules. The electric field effect on the cells depends on various experimental parameters, such as electric field strength, the number of electric pulses, and the electroporation medium. In this study, we show the influence of the electroporation medium on membrane permeabilization stimulated by electrical short-circuiting via an aqueous droplet in dielectric oil, a novel methodology developed by our previous investigations. We investigated the membrane permeabilization by three methods, influx of calcium ions, uptake of nucleic acid-binding fluorophores (YO-PRO-1), and calcein leakage. We demonstrated that the external medium conductivity had a significant impact on the cells in all described experiments. The short-circuiting using a low-conductivity electroporation medium enhanced the formation of both transient and irreversible membrane pores. We also found that clathrin-mediated endocytosis contributed to YO-PRO-1 uptake when a cell culture medium was used as an electroporation medium.

Physical Modeling for Large-Scale Landslide with Chair-Shaped Bedrock Surfaces under Precipitation and Reservoir Water Fluctuation Conditions

The deformation and failure mechanisms of historical landslides, characterized with different types of bedrock surface shapes which are known to have been induced by rainfall and reservoir water fluctuations, is an important issue currently being addressed by many researchers. The Zhaoshuling Landslide of the Three Gorges Reservoir Region, which was characterized with a chair-shaped bedrock surface under rainfall and reservoir water fluctuation conditions, was selected as an example in this study’s physical modeling process. The results of different parameters, including the displacements, pore water pressure, and total soil pressure during the landslide event, revealed that the Zhaoshuling Landslide with a chair-shaped bedrock surface had been extremely sensitive to heavy rainfall coupled with the rapid lowering of the water levels. Then, based on the data analysis results of the monitoring of the rainfall and groundwater levels, as well as the reservoir water levels, a conceptual model was put forward to explain the failure mechanisms. It was believed that the chair-shaped bedrock at the toe of the slope had been subjected to a localized zone of high transient pore water pressure, which had significantly adverse effects on the mechanisms of the slope stability.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1–like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Mechanoporation enables rapid and efficient radiolabeling of stem cells for PET imaging

Regenerative medicine uses the patient own stem cells to regenerate damaged tissues. Molecular imaging techniques are commonly used to image the transplanted cells, either right after surgery or at a later time. However, few techniques are fast or straightforward enough to label cells intraoperatively. Adipose tissue-derived stem cells (ADSCs) were harvested from knee joints of minipigs. The cells were labeled with PET contrast agent by flowing mechanoporation using a microfluidic device. While flowing through a series of microchannels, cells are compressed repeatedly by micro-ridges, which open transient pores in their membranes and induce convective transport, intended to facilitate the transport of 68Ga-labeled and lipid-coated mesoporous nanoparticles (MSNs) into the cells. This process enables cells to be labeled in a matter of seconds. Cells labeled with this approach were then implanted into cartilage defects, and the implant was imaged using positron emission tomography (PET) post-surgery. The microfluidic device can efficiently label millions of cells with 68Ga-labeled MSNs in as little as 15 min. The method achieved labeling efficiency greater than 5 Bq/cell on average, comparable to 30 min-long passive co-incubation with 68Ga-MSNs, but with improved biocompatibility due to the reduced exposure to ionizing radiation. Labeling time could also be accelerated by increasing throughput through more parallel channels. Finally, as a proof of concept, ADSCs were labeled with 68Ga-MSNs and quantitatively assessed using clinical PET/MR in a mock transplant operation in pig knee joints. MSN-assisted mechanoporation is a rapid, effective and straightforward approach to label cells with 68Ga. Given its high efficiency, this labeling method can be used to track small cells populations without significant effects on viability. The system is applicable to a variety of cell tracking studies for cancer therapy, regenerative therapy, and immunotherapy.

A phenomenological description of the transient single-phase pore velocity period using the resistance force-velocity relationship

A new approach to determine the transient period towards steady state pore flow velocity for fluids propagating through porous media under constant pressure condition is presented. The transient expression relates to the mean pore velocity rather than the fluid pressure conventional considered when characterizing transient behavior in porous media. It is based on the general, resistance force-velocity relationship, and is therefore analogous to the approach used when calculating transient periods for objects falling through resisting air and for the increase in electric currents towards respective steady state values. The transient is caused by inertia forces and characterized by a relaxation time comprising fluid density and viscosity together with porous medium properties as porosity and absolute permeability. Results show that the transient period increases with decreasing medium porosity and fluid viscosity and with increasing fluid density and absolute permeability of the medium. The transient period is negligibly small for typical fluid/medium property values characterizing typical subterrain sandstone reservoirs. Significant transient periods, occasionally observed during laboratory fluid injection tests, are therefore caused by other time-dependent processes not captured by the transient expression presented herein, e.g., fines migration or electrokinetic phenomena. Cited as: Standnes, D. C. A phenomenological description of the transient single-phase pore velocity period using the resistance force-velocity relationship. Advances in Geo-Energy Research, 2022, 6(2): 104-110. https://doi.org/10.46690/ager.2022.02.03