mrna transfection, hek293

Bookshelf For cultured cells LNP size is expected to play a lesser role in enabling delivery of the cargo due to the absence of circulatory and lymphatic systems which minimize tissue penetration by rapidly removing smaller particles. & DeBerardinis, R. J. Mechanisms and Implications of Metabolic Heterogeneity in Cancer. Untreated HEK 293T cells were used as an autoluminescent control. Alexander Shekhtman. 2500ng DNA 5ul of P3000/Lipo and. 2a and c). Methods 1, 171175. HEK293 cells were transiently transfected and the activity of each sgRNA-pair was assessed 72 h after transfection by RT-qPCR for FAAH and FAAH-OUT mRNA expression. Lactate levels reached an isotopic steady state47 that persisted until the labeled glucose was chased. Overlay panels indicate that ROS originate in mitochondria. & Reichl, U. Glycolysis is governed by growth regime and simple enzyme regulation in adherent MDCK cells. and JavaScript. 4) for which the isotopic steady state plateau observed in control cells was replaced by a monotonic decrease in signal strength followed by a rapid exponential decline when the labeled glucose pulse was removed. Small 16, e1903857. Lastly, all wells were stained with 4M MitoSOX in modified PBS for 30min in the growth chamber. Flow was controlled by a peristaltic pump to deliver 100200uL/min. Transcriptional and post-transcriptional limitations of high-yielding, PEI-mediated transient transfection with CHO and HEK-293E cells. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. While transfected DNA is translocated into the nucleus for transcription, transfected RNA remains in the cytosol, where it is expressed within minutes after transfection (mRNA) or bound to mRNA to silence the expression of a target gene (siRNA and miRNA) (see Guidelines for RNA Transfection ). Drug Deliver. https://doi.org/10.1038/mt.2008.200 (2008). Danaei, M. et al. The non-ideality of the leading and trailing edges of the glucose pulse are described by the rise and fall times, tRG and tFG, respectively, which were resolved by fitting the pulse-chase profile to a single phase exponential association/decay (Supplementary Figure4, and Supplementary Table 4). https://doi.org/10.1038/nprot.2011.335 (2011). Kim, J. Article Breindel, L., Burz, D. S. & Shekhtman, A. This was also confirmed by fluorescence microscopic analyses. Thromb. The data suggest that even modified mRNAs are highly unstable, and that utilizing transfection reagents to protect mRNA from degradation is critical for sustained protein expression. Samples treated continuously or for 8 or 12h produced comparable amounts of Luciferase-dependent activity over the same time course (Fig. 3, 13281340. 5, 21082116. Importantly, the change in the concentration of bound metabolite over time can also be described by differentiating (19) and (20) and presenting the result as. Epub 2018 Jan 2. The sample solution was reapplied four times. The times of lactate and alanine production, tP, were significantly slower in LF-mRNA treated HEK 293T cells as compared to control (Fig. One dimensional 13C-edited proton NMR experiments43 were used to monitor carbon that was metabolized from the isotopically labeled glucose43. Numerical solutions for Eqs. Federal government websites often end in .gov or .mil. The Gaussian curve was drawn to ease the comparative visualization of the distribution. Chem. Sciolino, N., Reverdatto, S., Premo, A. et al. where K=1/tC, the reciprocal time constant for metabolite clearance. Transfection of HEK293-EBNA1 Cells in Suspension with - PubMed where yo1, AoR and A1 are constants determined to fit the kinetic flux profiling at the leading edge (Rise) of 13C-glucose pulse. Rasoulianboroujeni, M. et al. No attempt was made to vary the size distribution of the resulting particles. Ionization and structural properties of mRNA lipid - Nature 13th May, 2015. N-1-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. (b) 13C edited heteronuclear single quantum coherence, 1H-13C HSQC, NMR spectrum showing metabolites present in-cell. https://doi.org/10.1016/j.cell.2017.12.006 (2018). Therefore, extracellular metabolites are quickly evacuated from the active volume and do not contribute to the observed NMR signal. The profile for glutamate reached a maximum about the time the labeled glucose pulse was chased from the system. and CMV promoters at 14 days after transfection. The increase is indicative of a change in alanine metabolism in cells treated with LF-mRNA, which could entail an increased time of utilization of the alanine free pool resulting in a higher bound fraction and/or a decrease in alanine production. Liu, A. et al. 17, 944953. 4). As a result, mostly intracellular labeled lactate is observed. https://doi.org/10.1002/smll.201903857 (2020). To ascertain the role of mRNA in the interaction with LF, LNPs were assembled with and without mRNA, and negatively stained with uranyl acetate for electron microscopic imaging (Fig. Assuming that the labeled metabolite undergoes chemical exchange between free and bound states in the presence of the unlabeled counterpart and that the concentration of bound metabolites are much smaller than either the total concentration of metabolites or the concentration of binding sites, the f1(A*, t) term becomes68 (see also Eqs. Mammalian cells such as the human embryonic kidney 293 (HEK-293) and the Chinese hamster ovary (CHO) cells are widely used as hosts to express recombinant proteins to study their structural, biophysical, and pharmacological properties ( Baldi et al., 2007; Dalton and Barton, 2014 ). Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. 34, 652664. High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices. https://doi.org/10.1038/s41578-021-00358-0 (2021). The inclusion of mRNA cargo in LF LNPs resulted in a larger, less polydisperse population of particle sizes with the mean of 13512nm (Fig. To introduce the Cap1 structure on the 5 end, mRNA was denatured at 65C for 10min and the ScriptCap Cap1 Capping System (Cellscript) was used according to the manufacturers guidelines. LF-mediated transfection of the same mRNA resulted in sustained protein expression. & Karatasos, K. Complexation of single stranded RNA with an ionizable lipid: An all-atom molecular dynamics simulation study. 21, S59S59 (2013). Cavanagh, J., Fairbrother, W. J., Palmer, A. G., Rance, M. & Skelton, N. J. https://doi.org/10.1038/Nchembio.2077 (2016). 3a. The model predicts the competition between labeled and unlabeled metabolites for binding sites will shorten tP and lengthen tC (Eqs. Plasmid pTK305 was amplified and linearized with restriction enzyme AscI (New England Biolabs), extracted with phenol/chloroform and precipitated with ethanol/ammonium acetate. Messenger RNAs alone are too unstable to be used as transfecting agents even those containing modified bases. Control Release 217, 337344. 75, 191197. HEK 293T cells treated with LNPs containing mRNA were able to access nutrients and produce protein for more than 24h implying that the inevitable release of pseudouridine upon mRNA degradation did not inhibit transcription of the mRNA product. Chem.-Int Edition 59, 23042308. Pharmaceutics 10, 57. https://doi.org/10.3390/pharmaceutics10020057 (2018). Transient transfection of serum-free suspension HEK 293 cell culture Beckonert, O. et al. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. They observed that an N- terminal fusion led to a 4-5 fold reduction in expression levels which was not observed in this study. A feasibility study of different commercially available serum-free mediums to enhance lentivirus and adeno-associated virus production in HEK 293 suspension cells. The purified transcript included a~150-nucleotide 3 poly(A) tail and 5 Cap124 to further facilitate protein translation (Supplementary Figure1). Messenger RNA in lipid nanoparticles rescues HEK 293 cells - Nature Note that tP=1/(J/A+K) is the characteristic timescale of the rise in the NMR observed metabolite signal for the leading edge of the 13C-glucose pulse and numerically corresponds to the time needed to go from the initial condition to 1/e~0.63 of the saturation level. The cells were re-suspended in 80L of modified PBS and 10L were aliquoted into each of four wells of a black 384w plate (# 784,086, Greiner Bio-One). Improved translation efficiency of therapeutic mRNA - PubMed For effective pulmonary drug delivery, LNP-mRNA formulations must be stably aerosolized. https://doi.org/10.1161/Circresaha.117.311311 (2018). The site is secure. Commun. The plate was read on a Synergy H1 plate reader (BioTek) with excitation at 510nm and emission at 580nm. https://doi.org/10.1016/j.cell.2018.03.055 (2018). Up to date, the only transfection . The plasmid pTK305 used for in vitro transcription of firefly Luciferase mRNA was purchased from Addgene (plasmid # 66,812; http://n2t.net/addgene:66812; RRID:Addgene_66812). To add a 3 poly(A) tail the mRNA was again denatured at 65C for 10min and the A-Plus Poly(A) Polymerase Tailing Kit (Cellscript) was used according to manufacturers guidelines. 4a). Using various chemical or physical methods, this gene transfer technology enables the study of gene function and protein expression in a cellular environment. Importantly, the change in the concentration of bound metabolite over time can be described by differentiating (14) and (15), and presenting the result as. Biochem. Stars indicate significance: p<.05 (*), p<.01 (**), and p<.001 (***). 10, e1003885. During all the stages the individual metabolites are monitored as they progress through metabolic pathways. ~3107 cells were suspended in cell flow buffer, 0.4% BSA, 0.04% EDTA, 20% Percoll and 5 L of Tween-20 to which modified firefly Luciferase mRNA in storage buffer was added to a final concentration of 300M and final volume of 3mL. It is also possible that alanine levels may be influenced by rewiring of glutamine metabolism in defective mitochondria49, and, if mitochondria are compromised by the generation of reactive oxygen species, ROS, then production of glutamate may also be compromised. To further investigate the perturbations associated with alanine metabolism a step function of labeled glucose was applied to cells treated with LF for 10h (Fig. Product Lipofectamine 2000 Reagent is a proprietary formulation Descriptionfor transfecting nucleic acids into a wide range of eukaryotic cells. The reaction was terminated by adding one half volume of LiCl/EDTA solution and redissolved in RNAse-free water. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. 6b)51. This was plotted in GraphPad Prism to create an x,y visualization of the 13C-supplemented medium pulse/peak for each metabolite over the course of each experiment. The CaCl2 solution was decanted and replaced with 25mL of KH salts supplemented with 5mM glucose, 0.4mM palmitate, 0.4mM BSA, and 70mU/L of insulin. (d) Size distribution of the LNPs containing Luciferase mRNA. 189, 101787. https://doi.org/10.1016/j.pneurobio.2020.101787 (2020). official website and that any information you provide is encrypted 2.5 L of 1mg/mL of linear polyethylenimine, PEI, was combined with 0.5g of Luciferase mRNA in 47L of Opti-MEM and incubated for 20min at room temperature. 1a, Supplementary Table 1). To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The growth medium was changed to Opti-MEM/5% FBS and 0.5g of luciferase mRNA was combined with either 1.5L of FuGENE 6, 1.5L of FuGENE HD, 2L of ViaFect or 1L of LF in Opti-MEM and incubated at room temperature according the manufacturers recommendations. Through transfection using the polycationic polymer polyethylenimine (PEI) at high . Correspondence to At the trailing edge of the 13C-glucose pulse, changes in the concentration of a free 13C-labeled metabolite, A*, can also be described by the KFP equation by taking into account the non-ideality of the 13C-glucose pulse and adding the phenomenological term f2(A*, t)=K A* exp(t), Assuming that the same condition, t<<1, holds true, the solution of Eq. 65, 1420. Schematic drawing of the two bicistronic plasmids used in the experiments. This result confirms that intracellular metabolite binding modulates KFP in in-cell NMR based metabolomics. The control contained no mRNA and no transfection reagent to assess baseline luminescence. Prior to each trial a proton-decoupled 31P spectrum was collected with 5k scans and 1s recycle delay to allow the metabolic state of the cell to be assessed. Live cells maintain homeostatic levels of ATP that can be easily discerned in a 1D spectrum. One g of mRNA in 25L of Opti-MEM was added and incubation continued for 20min. . Accumulation of sub-100nm polymeric micelles in poorly permeable tumours depends on size. Among the human cell lines, HEK293 is widely utilized due to its high transfectivity, rapid growth rate, and ability to grow in a serum-free, suspension culture. Results showed no reduction in ATP levels over the course of each experiment (Supplementary Figure3). The CAG promoter maintains highlevel transgene expression in HEK293 6, 815823. Hertig, D. et al. Encapsulating mRNA increased the average size of the LNPs from 29 to 135nm and resulted a more symmetric dispersity, parameters critical for stability and efficacy. Please enable it to take advantage of the complete set of features! Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins . J. Pharmacol. Luminescence of HEK 293T cells treated with LF-mRNA for different times. 2008 Jun;50(Pt 2):121-32. doi: 10.1042/BA20070081. Cell Syst. However, the expression for IF is simplified when the metabolite binding is non-specific and t<<1. Formation of LNPs containing nucleic acid cargo arises through electrostatic interactions between the phosphate backbone of the nucleic acid polymer and the positively-charged lipids59. J. Biomol. Optimizing the transient transfection process of HEK-293 - PubMed Shekhtman, A., Breindel, L., Sciolino, N., Burz, D. & Sulchek, T. In-cell NMR based technology to study protein interactions. Anal. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures. 5a). One of the wells, containing non-transfected HEK 293T cells was treated with 1M of Antimycin-A. . If the total concentrations of labeled and unlabeled metabolites and binding sites are [A]tot, [A*]tot, and N, respectively, then [A*]=[A*]tot[A*R], [A]=[A]tot [AR] and [R]=N[A*R][AR]. The resulting polyadenylated, capped N1-methylpseudouridine mRNA was dissolved in RNAse-free water at concentration of 1mg/mL, aliquoted and stored at 70C. 2, 125127. 45, 3038. After a 20min incubation, transfection mixtures were added to the plates, and transfections allowed to proceed in the growth chamber at 37C, 5% CO2, for 1011h. Three 15cm culture dishes (Corning) containing culture medium were seeded with 4106 HEK 293T cells and incubated in 5% CO2 at 37C for 4872h until~80% confluence (~1.2107 cells/plate). LNPs form polydisperse liposomal structures with aqueous interiors stabilized by a surfactant bilayer; the aqueous core provides a stable environment for mRNA, reducing premature degradation relative to naked mRNA30,31. The kinetic profiles generated for each was analyzed by using first order kinetics to yield time constants, tP and tC, for the production and clearance of labeled metabolites from cells respectively. Arterioscler. The bioreactor maintains high levels of phosphate-containing metabolites for>24h, consistent with a high energy charge and metabolically active cells33. Celebi Torabfam G, Yetisgin AA, Erdem C, Cayli A, Kutlu O, Cetinel S. Cytotechnology. 6, 10601083. A two-channel polydimethylsiloxane, PDMS, Volume Exchange for Convective Transfer, VECT, device with rigid, 9.6m microchannels was prepared as previously described61,62. Mol. The .gov means its official. Let. Commun. Frontiers | An Overview of Methods and Tools for Transfection of Cells were harvested as described above60, passed through a 40m filter to reduce clumping and aggregation, and counted. Protoc. Once the beads were cast and transferred to the NMR tube, the cells were equilibrated with 12C glucose in KH medium to allow the beads to settle into their final position within the NMR tube, and to establish a metabolic steady-state prior to the introduction of 13C-glucose medium. Although HEK293 cells have some merits in terms of the production of recombinant therapeutic proteins, low protein yield is an issue that needs to be resolved. Kariko, K. et al. These equations can always be fit using an exponential function like Eq. 2, 13691372. Note that the characteristic time of the clearance in the NMR observed metabolite signal, tC=1/(J/A-K), is larger than tP. HEK 293T cells were grown and treated like the ROS plate assays with the following modifications. Viruses. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. Cells were transfected with modified Luciferase mRNA for 24h using five different reagents and the resulting Luciferase luminescence activity was quantified. Protein NMR spectroscopy (Academic Press, 2007). Chem. https://doi.org/10.1021/acs.molpharmaceut.9b01182 (2020). (18) with an R2 factor better than 0.99 indicating that an exponential is a good approximation even in the general case. Expressing EGFP by diretly transfecting mRNA? | ResearchGate Both genes, Transfection efficiency using different DNAPEI, Transfection efficiency using different DNAPEI ratios. 13C-labeled metabolites are observed against an unlabeled background. As LNP-mediated mRNA vaccines become more widely available as a therapeutic treatment, understanding the effect of LNPs on cells and tissues becomes more important5. 84, 916. To establish an analysis window for metabolic studies, the extent of protein expression was examined by incubating HEK 293T cells with LF-mRNA LNPs for different times. https://doi.org/10.1016/j.mib.2018.02.004 (2018). Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. Vector pCMV-EGFP-EPO carried the EGFP gene in the first position and the human EPO gene in the second (, Transfection efficiency using different DNAPEI ratios. Haas, E. J., Angulo, F. J. The. Mater. Koczula, K. M. et al. Question. Will the lipids that constitute LNPs damage plasma and internal cell membranes? 27, 441464. To estimate kinetic parameters for the clearance of labeled metabolites from the free pool, the trailing edges of the profiles were also fit to a two-phase exponential: where tC is the clearance time for a 63% reduction in the metabolite signals, yCo is the relative concentration when the signal begins to decrease, and AFo and AC are constants. The cells were washed with PBS and incubated for 10min with 1g/mL Hoechst 33,258 dye in PBS at room temperature. Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics. The dynamic response of cells to drug treatments can be assessed by metabolic profiling, which is generally accomplished by using mass spectroscopy or NMR spectroscopy12,13. mRNA Transfection into CHO-Cells Reveals Production Bottlenecks The performance of the device was optimized to introduce a pulse of labeled glucose with a rise time,~10min, that is faster than the characteristic time of glycolysis, 1020min36. Unauthorized use of these marks is strictly prohibited. Note that LF reagent used in these experiments is composed of a mixture of polycationic and phospholipids. Today 27, 17631773 (2022). Chem. Cells were washed with PBS and lysed by adding of 50L of 1X Luciferase Cell Culture Lysis Reagent (Promega). (14) and (15) are linear and can be solved exactly68. Hou, X. C., Zaks, T., Langer, R. & Dong, Y. 3b insets)44. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions. https://doi.org/10.1021/acs.biochem.5b00036 (2015). Cells were harvested from the wells with trypsinEDTA as described above and transferred to 1.5mL Eppendorf tubes, washed twice with warm modified PBS, PBS containing 1mM MgCl2, 1mM CaCl2 and 5.5mM glucose. More recent real time NMR-based metabolomic studies use a bioreactor to maintain cells in a physiologically active state and can detect free intracellular metabolites, but lack the ability to monitor key reactions such as glycolysis that take place over the course of minutes16,17,18. 2022 Dec;74(6):635-655. doi: 10.1007/s10616-022-00551-1. Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). HEK 293T cells were previously utilized to monitor the uptake and expression of Luciferase mRNA21,22. Control Release 217, 345351. (14) and (15) in the general case when [AR] or [A*R] are comparable to [A]tot or [A*]tot is presented in Supplementary Figure5. Transfection mixtures were scaled up 100-fold, i.e. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Dots represent data points obtained using biological replicate samples. 293s cells cultured in serum-free DMEM-FS medium, Growth and productivity of transiently transfected 293s cells growing in serum-free DMEM-FS medium, Progress of 293s cell cultivation from an early ( filled symbols ) and, Progress of 293s cells from an early ( filled symbols ) and a, 293s cells from an early ( filled symbols ) and a late (, 293 s cells from an early ( filled symbols ) and a late, Progress of 293s cells from the early passage (see Fig. Google Scholar. Antib Ther. & Mahan, L. C. The kinetics of competitive radioligand binding predicted by the law of mass-action. Angew. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. CAS Excess solution was removed by blotting with Whatman paper between each step63. A map of protein-metabolite interactions reveals principles of chemical communication. Electron leakage from the electron transport system on the inner membrane leads to partial reduction of oxygen to form superoxide, an ROS, that can ultimately result in mitophagy. mRNA synthesis was performed according to the manufacturers protocol. Frontiers | HEK293 Cell Line as a Platform to Produce Recombinant Protein Sci. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. Rehberg, M., Ritter, J. Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. 12, 757758 (2021). (A) The mRNA expressions of report gene (eGFP) and internal reference gene (GAPDH) were measured by qRT-PCR. Aranibar, N. et al. (a). Trypsin was neutralized by using a fivefold dilution with complete medium, cells were counted and pelleted by centrifugation at 200g for 10min. PMC Error bars represent mean deviation. Before the end of the 4h incubation, Antimycin A, which inhibits the electron transport complex, was added to another well at concentration of 1.5M and incubated for 1h. The well was washed once with warm OptiMEM/5% FBS and replenished with 2mL of the same medium. Analytical tools to study cell physiology are critical for optimizing drug-host interactions. 12, 482-+. Following injection, 500 spectra were collected consecutively over a 5.5h period allowing the disposition of the 13C-glucose to be monitored as it progressed through different metabolic pathways. Transient plasmid transfection falls short of the specific productivities of stable producer cells, making it unsuitable for the elucidation of high specific productivity bottlenecks. PMCID: PMC3973801 DOI: 10.1007/s10616-013-9601-3 Abstract Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. (1) and (2) takes into account the non-ideality of the 13C-glucose pulses due to diffusion (Fig. White, C. R., Phillips, N. F. & Seymour, R. S. The scaling and temperature dependence of vertebrate metabolism. Piazza, I. et al. To assay for luciferase activity 20L of lysate was mixed with 100L of Luciferase Assay Reagent (Promega) in 96-well white plate (Greiner Bio-One) and immediately read on a Synergy H1 Hybrid Multi-Mode plate reader (BioTek Instruments). (c) Normalized 13C-glucose pulses chased with unlabeled glucose in non-transfected HEK 293T cells: (I) Prior to the pulse cells are fed unlabeled glucose in modified KH buffer; (II) During the incorporation stage a 15mL pulse of 13C-glucose is introduced through the injectable loop; (III) the 13C-glucose reaches a plateau concentration; (IV) During the clearance stage the 13C-glucose is eliminated from the system. CAS Safety of the BNT162b2 mRNA Covid-19 vaccine in a nationwide setting. 293T cells are easily transfected with IVT mRNA using many commercially available lipofection methods (e.g. Rajendra Y, Kiseljak D, Baldi L, Wurm FM, Hacker DL. and CMV promoters at 14 days after transfection. Glutamate profiles exhibited consistently higher experimental error in cells treated with LF or LF-mRNA. ROS production in HEK 293T cells in response to LNP exposure with and without mRNA. 0.79mm) with a blunt 21-gauge needle. This may explain how empty LNPs elicit greater ROS production than those that are cargo-laden and, in combination with the requirement for proper mitochondrial export of glutamate in the primary biosynthesis of alanine, why only the alanine profiles differed in the absence and presence of mRNA. Rosenblum, D. et al. The atomizer consisted of a vertically oriented pipette with an airflow of 5.5L/min. The two-phase decay model was used to fit the trailing edge.
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