Microperfusion System Research Articles

Safety and efficacy of a novel transbronchial radiofrequency ablation system for lung tumours: One year follow-up from the first multi-centre large-scale clinical trial (BRONC-RFII).

Radiofrequency ablation (RFA) is an emerging treatment of lung cancer, yet it is accompanied by certain safety concerns and operational limitations. This first multi-centre, large-scale clinical trial aimed to investigate the technical performance, efficacy and safety of an innovative transbronchial RFA system for lung tumours. The study enrolled patients with malignant lung tumours who underwent transbronchial RFA using an automatic saline microperfusion system between January 2021 and December 2021 across 16 medical centres. The primary endpoint was the complete ablation rate. The performance and safety of the technique, along with the 1-year survival rates, were evaluated. This study included 126 patients (age range: 23-85 years) with 130 lung tumours (mean size: 18.77 × 14.15 mm) who had undergone 153 transbronchial RFA sessions, with a technique success rate of 99.35% and an average ablation zone size of 32.47 mm. At the 12-month follow-up, the complete ablation rate and intrapulmonary progression-free survival rates were 90.48% and 88.89%, respectively. The results of patients with ground-glass nodules (GGNs) were superior to those of the patients with solid nodules (12-month complete ablation rates: solid vs. pure GGN vs. mixed GGN: 82.14% vs. 100% vs. 96.08%, p = 0.007). No device defects were reported. Complications such as pneumothorax, haemoptysis, pleural effusion, pulmonary infection and pleural pain were observed in 3.97%, 6.35%, 8.73%, 11.11% and 10.32% of patients, respectively. Two subjects died during the follow-up period. Transbronchial RFA utilizing an automatic saline microperfusion system is a viable, safe and efficacious approach for the treatment for lung tumours, particularly for patients with GGNs.

3D culture of ovarian follicles in granular and nanofibrillar hydrogels

3D culture of ovarian follicles in hydrogel matrices is an important emerging tool for basic scientific studies as well as clinical applications such as fertility preservation. For optimizing and scaling 3D culture of preantral follicles, there is a need for identifying biomaterial matrices that simplifies and improves the current culture procedures. At present, microencapsulation of follicles in alginate beads is the most commonly used approach. However, this technique involves notable manual handling and is best suited for encapsulation of single or several follicles. As a potential alternative, we here explore the suitability of different particle-based hydrogel matrices, where follicles can easily be introduced in tunable 3D environments, in large numbers. Specifically, we study the growth of secondary murine follicles in microgranular alginate and nanofibrillar cellulose matrices, with and without cell-binding cues, and map follicle growth against the viscoelastic properties of the matrices. We cultured follicles within the particle-based hydrogels for 10 days and continuously monitored their size, survival, and tendency to extrude oocytes. Interestingly, we observed that the diameter of the growing follicles increased significantly in the particle-based matrices, as compared to state-of-the-art alginate micro-encapsulation. On the other hand, the follicles displayed an increased tendency for early oocyte extrusion in the granular matrices, leading to a notable reduction in the number of intact follicles. We propose that this may be caused by impaired diffusion of nutrients and oxygen through thicker matrices, attributable to our experimental setup. Still, our findings suggest that viscoelastic, granular hydrogels represent promising matrices for 3D culture of early-stage ovarian follicles. In particular, these materials may easily be implemented in advanced culturing devices such as micro-perfusion systems.

Microperfusion cell culture system for promoted cell growth using non-thermal atmospheric pressure plasma exposure

Promoting cell growth is demanded in various applications, such as drug screening and regenerative medicine. Recently, non-thermal atmospheric pressure plasma (NTAPP), which can generate reactive oxygen and nitrogen species under atmospheric conditions, has been used to promote cell growth. In this study, microperfusion systems using the stimulation of NTAPP exposure were developed to induce cell growth. NTAPP was generated through the dielectric barrier discharge method. When NTAPP exposure was applied to pure water for 4 min, concentrations of nitrate, nitrite, and hydrogen peroxide reached 250, 20, and 0.5 mg l−1, respectively. Further, C2C12 murine myoblast cells were injected into the microperfusion system, stimulated with NTAPP exposure, and cultured for 3 d. Then, the liquid medium was changed to initiate cell differentiation. The formation of tubular structures was observed after incubation for 3 d.

Development of an integrated microperfusion-EEG electrode for unbiased multimodal sampling of brain interstitial fluid and concurrent neural activity

Objective. To modify off-the-shelf components to build a device for collecting electroencephalography (EEG) from macroelectrodes surrounded by large fluid access ports sampled by an integrated microperfusion system in order to establish a method for sampling brain interstitial fluid (ISF) at the site of stimulation or seizure activity with no bias for molecular size. Approach. Twenty-four 560 µm diameter holes were ablated through the sheath surrounding one platinum–iridium macroelectrode of a standard Spencer depth electrode using a femtosecond UV laser. A syringe pump was converted to push–pull configuration and connected to the fluidics catheter of a commercially available microdialysis system. The fluidics were inserted into the lumen of the modified Spencer electrode with the microdialysis membrane removed, converting the system to open flow microperfusion. Electrical performance and analyte recovery were measured and parameters were systematically altered to improve performance. An optimized device was tested in the pig brain and unbiased quantitative mass spectrometry was used to characterize the perfusate collected from the peri-electrode brain in response to stimulation. Main results. Optimized parameters resulted in >70% recovery of 70 kDa dextran from a tissue analog. The optimized device was implanted in the cortex of a pig and perfusate was collected during four 60 min epochs. Following a baseline epoch, the macroelectrode surrounded by microperfusion ports was stimulated at 2 Hz (0.7 mA, 200 µs pulse width). Following a post-stimulation epoch, the cortex near the electrode was stimulated with benzylpenicillin to induce epileptiform activity. Proteomic analysis of the perfusates revealed a unique inflammatory signature induced by electrical stimulation. This signature was not detected in bulk tissue ISF. Significance. A modified dual-sensing electrode that permits coincident detection of EEG and ISF at the site of epileptiform neural activity may reveal novel pathogenic mechanisms and therapeutic targets that are otherwise undetectable at the bulk tissue level.

Immobilization of Recombinant Fluorescent Biosensors Permits Imaging of Extracellular Ion Signals.

Given the importance of ion gradients and fluxes in biology, monitoring ions locally at the exterior of the plasma membrane of intact cells in a noninvasive manner is highly desirable but challenging. Classical targeting of genetically encoded biosensors at the exterior of cell surfaces would be a suitable approach; however, it often leads to intracellular accumulation of the tools in vesicular structures and adverse modifications, possibly impairing sensor functionality. To tackle these issues, we generated recombinant fluorescent ion biosensors fused to traptavidin (TAv) specifically coupled to a biotinylated AviTag expressed on the outer cell surface of cells. We show that purified chimeras of TAv and pH-Lemon or GEPII 1.0, Förster resonance energy transfer-based pH and K+ biosensors, can be immobilized directly and specifically on biotinylated surfaces including glass platelets and intact cells, thereby remaining fully functional for imaging of ion dynamics. The immobilization of recombinant TAv–GEPII 1.0 on the extracellular cell surface of primary cortical rat neurons allowed imaging of excitotoxic glutamate-induced K+ efflux in vitro. We also performed micropatterning of purified TAv biosensors using a microperfusion system to generate spatially separated TAv–pH-Lemon and TAv–GEPII 1.0 spots for simultaneous pH and K+ measurements on cell surfaces. Our results suggest that the approach can be greatly expanded by immobilizing various biosensors on extracellular surfaces to quantitatively visualize microenvironmental transport and signaling processes in different cell culture models and other experimental settings.

Efficacy of Sub-Tenon Micro-Perfusion of Cyclophosphamide in Rabbits with Severe Ocular Inflammation.

PurposeTo explore the feasibility of cyclophosphamide (CP) via a sub-Tenon micro-perfusion system (SMS) in rabbits, and assess its therapeutic efficacy in severe ocular inflammation.Materials and MethodsDistribution and pharmacokinetics of CP were evaluated in vivo, and the concentrations of CP in plasma, vitreous humor, and retina/choroid were quantitated by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) at different time points. After induction of severe experimental uveitis, rabbits were divided into three groups (n=8 in each): the SMS group, subconjunctival injection (SI) group, and control group. Clinical inflammatory score was assessed in rabbits. Electroretinography and histopathology were performed on post-treatment day 8. Statistical analyses were performed using Mann–Whitney and Kruskal–Wallis tests. P-value less than 0.05 was considered significant.ResultsThe concentrations of CP in vitreous humor and retina/choroid in the SMS group were significantly higher than that of the SI group at 3, 6, 10, and 24 hours (P<0.01), while plasmatic CP concentrations were comparable at all time points in the SMS group and SI group (P>0.05). The SMS group showed significantly less inflammation compared to the control group and SI group. Furthermore, the restoration of retinal structure and function were more obvious in the SMS group compared with conventional SI application.ConclusionSub-Tenon micro-perfusion of CP exhibited satisfied therapeutic efficacy in rabbits with severe ocular inflammation and may provide a promising alternative for controlling ocular inflammatory disease and immune-mediated ocular diseases.

Dose- and time-dependent effects of actomyosin inhibition on live mouse outflow resistance and aqueous drainage tissues.

Actomyosin contractility modulates outflow resistance of the aqueous drainage tissues and intraocular pressure, a key pathogenic factor of glaucoma. We established methodology to reliably analyze the effect of latrunculin-B (Lat-B)-induced actin depolymerization on outflow physiology in live mice. A voltage-controlled microperfusion system for delivering drugs and simultaneously analyzing outflow resistance was tested in live C57BL/6 mice. Flow rate and perfusion pressure were reproducible within a coefficient of variation of 2%. Outflow facility for phosphate-buffered saline (0.0027 ± 0.00036 μL/min/mmHg; mean ± SD) and 0.02% ethanol perfusions (Lat-B vehicle; 0.0023 ± 0.0005 μL/min/mmHg) were similar and stable over 2 hours (p > 0.1 for change), indicating absence of a ‘washout’ artifact seen in larger mammals. Outflow resistance changed in graded fashion, decreasing dose- and time-dependently over 2 hours for Lat-B doses of 2.5 μM (p = 0.29), 5 μM (p = 0.039) and 10 μM (p = 0.001). Resulting outflow resistance was about 10 times lower with 10 μM Lat-B than vehicle control. The filamentous actin network was decreased and structurally altered in the ciliary muscle (46 ± 5.6%) and trabecular meshwork (37 ± 8.3%) of treated eyes relative to vehicle controls (p < 0.005; 5 μM Lat-B). Mouse actomyosin contractile mechanisms are important to modulating aqueous outflow resistance, mirroring mechanisms in primates. We describe approaches to reliably probe these mechanisms in vivo.

Effect of iron oxide nanoparticles on the permeability properties of Sf21 cells

It was recently reported that nanoparticles could significantly modulate the thermal properties of solutions at subzero temperatures, and as a result, nanoparticles have been widely used in both cryopreservation and cryosurgery. In cryopreservation, the water permeability coefficient of cell membrane is an essential parameter for quantitative investigation of cell dehydration and intracellular ice formation. However, few studies were focused on the effects of nanoparticles on the permeability properties of cell membrane. In order to optimize the processes of cryopreservation with nanoparticles, we measured the permeability properties of Sf21 cells in the presence of iron oxide nanoparticles in this study. The responses of Sf21 cells with iron oxide nanoparticles were obtained by the microperfusion system at −2, 5, 15 and 25 °C, respectively. The osmotically inactive cell volume (Vb), the cell membrane hydraulic conductivity (Lp) and it's activation energy (ELp), and the reference value of Lp at the reference temperature (Lpg) with 0.02%, 0.1% and 0.5% (w/w) iron oxide nanoparticles were determined by 2-parameter (2-p) model at −2, 5, 15 and 25 °C. We analyzed the effects of iron oxide nanoparticles on the permeability properties of the Sf21 cells. The results indicated that iron oxide nanoparticles have a significant influence on membrane permeability properties (Lpg and ELp) of Sf21 cells. The introduction of iron oxide nanoparticles tends to increase the values of Vb and Lpg, while decrease the value of ELp. These findings may provide a new route to optimize the biomaterial cryopreservation.

Electrochemical label-free degranulation monitoring for in-situ evaluation of cellular function.

We fabricated a degranulation monitoring device, combining ion-sensitive field-effect transistor (ISFET) and microperfusion system. The electrical properties of ISFET were maintained even after immobilization of RBL-2H3 mast cells on the sensor. We successfully demonstrated in-situ monitoring of degranulation from stimulated RBL-2H3 cells by ionomycin. Potential change was induced by the release of acid-granule contents, which result in local pH decrease on the sensor under physiological conditions. This microdevice is expected to contribute as a platform technology for evaluating induced immune responses by chemical compounds.

In vivo Temperature Sensitivity of the Calcium Affinity of fluo-5F and mag-fluo4

The role of calcium ion (Ca2+) in diverse cellular signaling pathways has been identified and investigated principally by using fluorescent chelators as indicators. Using the biophysical characteristics of a fluorescent Ca2+ indicator allows investigators to convert measured fluorescence intensity into values of free Ca2+ concentration ([Ca2+]). Troublingly, the characteristics of Ca2+-indicators are dependent on numerous environmental factors. Determining the behavior of each indicator in vivo under comparable experimental conditions is necessary for quantitative measurements. Temperature is one environmental factor that strongly affects Ca2+ indicator behavior. A change in temperature can change the intrinsic physical characteristics of the indicator (e.g., Ca2+ affinity, quantum efficiency, and fluorescence lifetime), as well as change the cellular processes that affect indicator performance (e.g., extrusion by transporters, cellular pH). Therefore, experiments were designed to estimate the disassociation constants (KD) of the Ca2+ indicators fluo-5F and mag-fluo4 (loaded into the cytosol and sarcoplasmic reticulum, respectively) at room and physiological temperatures in murine ventricular myocytes. Ca2+ access to cytosolic compartment was established using a Ca2+ ionophore, while sarcoplasmic reticulum (SR) access was established by sarcolemma permeabilization with saponin in the presence of caffeine. Once access was established, buffers with known [Ca2+] were rapidly applied using a micro-perfusion system with temperature feedback control. We observed that, increasing from room temperature to 36°C caused a modest decrease in the KD of both fluo-5F and mag-fluo4. Both indicators also displayed a decrease in dynamic range when heated. Additionally, temperature-dependent extrusion was observed for indicators loaded into the cytosol, but not those loaded into the SR. These findings are important in enabling quantitative interpretation of measurements in living cells under physiological conditions.

C-2020: Determination of membrane permeability of porcine adipose derived-stem cell

Adipose-derived stem cells (ADSCs), as an abundant and accessible source of adult stem cells, self-renew and differentiate into various cell lines and have promising potential in tissue engineering and as an alternative to the use of regenerative medicine strategies. In order to meet clinically urgent demand of ADSCs, the cell cryopreservation was applied to the ADSCs long-term preservation. To optimize the cryoprotective agents (CPAs) addition, removal and the freezing as well as thawing processes, we measured the transport properties of porcine adipose-derived stem cells (pADSCs). In this study, the microperfusion system was used to measure transport properties of porcine adipose-derived stem cells (pADSCs) membrane. The membrane permeability coefficient to water (Lp), the membrane permeability coefficient to Me2SO, EG and glycerol (Ps), and the activation energies and reference membrane coefficient have been investigated at 28, 18, 8 and −2 °C, respectively. We determined the inactive cell (Vb), the hydraulic conductivity (Lp) and the CPAs permeability (Ps) at various temperature in 2-paramerter (2 − p) model. No significant difference in the Me2SO and EG permeability was obtained at experimental temperature and the effect of the temperature on the transport properties of the pADSC membrane was analyzed. However, the glycerol permeability of pADSCs was too small to be precisely measured. We obtained the parameter of pADSCs with 1.5 M glycerol at 32 °C and determined the accurate activation energies of glycerol permeability and the referent of glycerol permeability. Membrane permeability of porcine Adipose Derived-Stem cell quantitatively measured was used to minimize the excessive volume shrinkage and the toxicity with CPAs addition.

Measurement of the biophysical properties of porcine adipose-derived stem cells by a microperfusion system

Adipose-derived stem cells (ADSCs), which are an accessible source of adult stem cells with capacities for self-renewal and differentiation into various cell types, have a promising potential in tissue engineering and regenerative medicine strategies. To meet the clinical demand for ADSCs, cryopreservation has been applied for long-term ADSC preservation. To optimize the addition, removal, freezing, and thawing of cryoprotective agents (CPAs) applied to ADSCs, we measured the transport properties of porcine ADSCs (pADSCs). The cell responses of pADSCs to hypertonic phosphate-buffered saline and common CPAs, dimethyl sulfoxide, ethylene glycol, and glycerol were measured by a microperfusion system at temperatures of 28, 18, 8, and −2°C. We determined the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), and CPA permeability (Ps) at various temperatures in a two-parameter model. Then, we quantitatively analyzed the effect of temperature on the transport properties of the pADSC membrane. Biophysical parameters were used to optimize CPA addition, removal, and freezing processes to minimize excessive shrinkage of pADSCs during cryopreservation. The biophysical properties of pADSCs have a great potential for effective optimization of cryopreservation procedures.

Note: A micro-perfusion system for use during real-time physiological studies under high pressure

We construct a micro-perfusion system using piston screw pump generators for use during real-time, high-pressure physiological studies. Perfusion is achieved using two generators, with one generator being compressed while the other is retracted, thus maintaining pressurization while producing fluid flow. We demonstrate control over perfusion rates in the 10-μl/s range and the ability to change between fluid reservoirs at up to 50 MPa. We validate the screw-pump approach by monitoring the cyanide-induced response of UV-excited autofluorescence from Saccharomyces cerevisiae under pressurization.

Development of electrolysis pump for subepidermal microperfusion system

Development of electrolysis pump for subepidermal microperfusion system

Mechanistic insight into CM18-Tat11 peptide membrane-perturbing action by whole-cell patch-clamp recording.

The membrane-destabilization properties of the recently-introduced endosomolytic CM18-Tat11 hybrid peptide (KWKLFKKIGAVLKVLTTG-YGRKKRRQRRR, residues 1–7 of cecropin-A, 2–12 of melittin, and 47–57 of HIV-1 Tat protein) are investigated in CHO-K1 cells by using the whole-cell configuration of the patch-clamp technique. CM18-Tat11, CM18, and Tat11 peptides are administered to the cell membrane with a computer-controlled micro-perfusion system. CM18-Tat11 induces irreversible cell-membrane permeabilization at concentrations (≥4 µM) at which CM18 triggers transient pore formation, and Tat11 does not affect membrane integrity. We argue that the addition of the Tat11 module to CM18 is able to trigger a shift in the mechanism of membrane destabilization from “toroidal” to “carpet”, promoting a detergent-like membrane disruption. Collectively, these results rationalize previous observations on CM18-Tat11 delivery properties that we believe can guide the engineering of new modular peptides tailored to specific cargo-delivery applications.

A Micro-Perfusion System for the Fluorescence-Based Monitoring of Physiological Responses to High Hydrostatic Pressures

Hydrostatic pressures of 102 to 103 atm affect a range of cellular processes, including motility, cell division, nutrient uptake, fermentation, translation and transcription, protein synthesis, and ultimately viability. In order to perform the real time monitoring of pressure effects, we present a micro-perfusion system designed for spectroscopic measurements on cellular systems under high pressure. The system consists of an optically-compatible pressure chamber and interchangeable fluid reservoirs. Perfusion is achieved using a dual pressure-generator configuration, where one positive-displacement generator is compressed while the other is retracted, thus maintaining a pressurized volume while achieving fluid flow. Control over perfusion rates (typically in the 10 μl/s range) and the ability to change between fluid reservoirs while under pressure (up to 600 atm) are demonstrated. Next, the system is used for the time-gated, spectral monitoring of endogenous NADH fluorescence under pressure. Spectrofluorimetric measurements utilize a nitrogen-discharge laser for sub-nanosecond pulse width, 337-nm wavelength excitation, and an intensified CCD coupled to a spectrograph for nanosecond-gated spectral detection. Because emission from NADH is routinely used at ambient pressure for monitoring mitochondrial function, we validate the system by observing the pressurized response of Saccharomyces cerevisiae (baker's yeast) to mitochondrial functional modifiers (e.g., cyanide). Because the system is compatible with both spectroscopy and sub-cellular resolution microscopy imaging, the system represents a robust tool for investigating the biophysical effects of pressure on cellular systems.

Ex vivo microperfusion system of the adipose organ: a new approach to studying the mobilization of adipose cell populations

Adipose tissue (AT) is a dynamic organ that expands and contracts rapidly. It is composed of adipocytes and of cell populations among which immune cells and mesenchymal progenitors known as adipose stromal cells (ASCs). The AT cell turnover has been extensively studied. Surprisingly it has only been viewed as the result of both cell proliferation/death and cell infiltration. Nevertheless, both immune cells and ASCs exhibit migration abilities; therefore their egress from AT in response to physiological/pathophysiological stimuli has to be considered. To do so, the aim of the present work was to develop a model allowing the study of cell release from the adipose organ. Mesenteric (Mes) ATs were isolated from 9-week-old C57BL/6 male mice and were catheterized via the superior mesenteric artery and were perfused with a saline solution. After an equilibration period, the mesenteric fat pad was perfused with CXCL12 (stromal-derived factor-1, SDF-1) or sphingosine 1-phosphate (S1P) to trigger cell mobilization and perfusates were collected every 30 min for subsequent flow cytometry analyses. We report here that CXCL12 induces the specific release of ASCs from MesAT thus demonstrating that ASCs are specifically mobilized from fat depots by a CXCL12-dependent pathway. Moreover, we showed that leukocyte mobilization can be triggered via a S1P-dependent pathway. We have developed a microperfusion model of an intact fat depot allowing the study of AT cell release in response to various molecules. The perfusion system described here demonstrates that ASCs and leukocytes can be pharmacologically mobilized from AT. Therefore, AT microperfusion might constitute an appropriate and reliable approach for evaluating the mobilization of different cell populations from AT in various physiological and pathophysiological contexts. Such a model might help in identifying factors and drugs controlling AT cell release, impacting the medical fields of regenerative medicine and of obesity or its associated comorbidities.

Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system

Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.

129 Effect of different nanoparticles on the SF21 cell membrane transport properties to water and glycerol

Many researchers focus on the cell toxicity of nanoparticles and drug transportation with the nanoparticles. Recently cryobiologists found that the efficiency of cryopreservation of many cells and tissues is good when nanoparticles added to the cryoprotectant solution. However, the mechanism is not understood. Previous study demonstrated that the ice growth is accelerated and devitrification temperatures of cryoprotectant solutions are significantly lowered in these nanoparticles cryoprotectant solutions, and the nanoparticles could change the structure and property of cell membrane. To obtain the optimal protocols for freezing, thawing, and addition/removal of cryoprotectant agent (CPA), the membrane transport properties need to be determined. In our study, we applied the microperfusion system to the measurement of the SF21 cell membrane transport properties to water and glycerol with hydroxyapatite TiO 2 SiO 2 and AlO 3 at 20 °C. We found that the SF21 cell membrane transport properties to water and glycerol is significantly different with nanoparticles. The finding can be used to improve the efficiency of SF21 cell conventional and vitrified cryopreservation, also we should pay attention to the application of the nanoparticles in cryogenics. Source of funding: National Natural Science Foundation of China (No. 51076149). Conflict of interest: None declared. jywong@mail.ustc.edu.cn

127 Measuring of Sf21 cell membrane biophysical parameters using a new nondilute solute transport equation

In cryobiology, Kedem-Katchalsky model (KK) and two-parameter model (2P) has been used to predict cell volume changes. However, the KK model might lead to conceptual errors when used for cell membranes and the 2P model is based on ideal and dilute solution. Recently, Heidi Y. Elmoazzen et al. (Heidi Y. Elmoazzen, Janet A. W. Elliott, and Locksley E. McGann, “Osmotic Transport across Cell Membranes in Nondilute Solutions: A New Nondilute Solute Transport Equation,” Biophysical Journal, vol. 96, pp. 2559–571, 2009) had proposed a new nondilute solute transport equation which could fit or predict cell osmotic volume changes more appropriate. The purpose of the article is to verify the validity of the new equation. We used SF21 cell volume changes in different concentrations of Glycerol and different temperatures to fit the new model to water permeability coefficient ( L ) and solute permeability coefficient ( P ) of cell membrane. The experiment was carried out by using microperfusion system designed by our lab. What’s more, the extracellular concentration of CPA was simulated by using a universal self-adaptive time-varying function, which is derived by Gang Zhao (Gang Zhao, “A Universal Self-Adaptive Time-Varying Function for Extracellular Concentration during Osmotic Shift for Curve-Fitting Permeability Coefficients of Cell Membrane,” Cryoletters, vol. 31, pp. 279–90, 2010). Mean (means ± SD) L values in the presence of 2.0 M Glycerol at 25, 20, 15, and 10 °C were determined to be 0.68 ± 0.29, 0.78 ± 0.33, 0.77 ± 0.20, and 0.67 ± 0.16 10-28mol2/ (minatm μ m5). The P values were 4.6 ± 0.1.69, 1.11 ± 0.99, 3.84 ± 0.65, and 2.96 ± 2.81 10–32 ml 2 / (minatm μ m 5 ). L values in the presence of 1.5 M Glycerol at 25, 20, 15, and 10 °C were determined to be 0.97 ± 0.61, 1.27 ± 0.64, 0.818 ± 0.29, and 0.533 ± 0.17 10–28mol 2 / (minatm μ m 5 ). The P values were 4.18 ± 2.51, 3.00 ± 1.62, 1.14 ± 0.58, and 2.3 ± 1.53 10–32 mol 2 / (minatm μ m 5 ). L values in the presence of 1.0 M Glycerol at 25, 20, 15, and 10 °C were determined to be 1.78 ± 0.65, 1.24 ± 0.66, 1.02 ± 0.26, and 1.15 ± 0.39 10–28 mol 2 / (minatm μ m 5 ). The P values were 2.67 ± 0.94, 2.70 ± 1.53, 1.45 ± 0.96, and 0.79 ± 0.40 10–32 mol 2 / (minatm μ m 5 ). From fitting lines of the experiment data, we could find out the new nondilute equation is very similar to 2 P model, which demonstrates, in our opinion, the new nondilute equation can be used to fit and predict cell volume changes. Therefore, more experiments should be done to prove which one might lead to a better result. Source of funding: This work was supported by the National Natural Science Foundation of China (Nos. 51076149, 51276179) Conflict of interest: None declared. zhaog@ustc.edu.cn