Use of inert gas jets to measure the forces required for mechanical gene transfection

ABSTRACT: BACKGROUND: Transferring genes and drugs into cells is central to how we now study, identify and treat diseases. Several non-viral gene therapy methods that rely on the mechanical disruption of the plasma membrane have been proposed, but the success of these methods has been limited due to a lack of understanding of the mechanical parameters that lead to cell membrane permeability. METHODS: We use a simple jet of inert gas to induce local transfection of plasmid DNA both in vitro (HeLa cells) and in vivo (chicken chorioallantoic membrane). Five different capillary tube inner diameters and three different gases were used to treat the cells to understand the dependency of transfection efficiency on the dynamic parameters. RESULTS: The simple setup has the advantage of allowing us to calculate the forces acting on cells during transfection. We found permeabilization efficiency was related to the dynamic pressure of the jet. The range of dynamic pressures that led to transfection in HeLa cells was small (200 +/- 20 Pa) above which cell stripping occurred. We determined that the temporary pores allow the passage of dextran up to 40 kDa and reclose in less than 5 seconds after treatment. The optimized parameters were also successfully tested in vivo using the chorioallantoic membrane of the chick embryo. CONCLUSIONS: The results show that the number of cells transfected with the plasmid scales with the dynamic pressure of the jet. Our results show that mechanical methods have a very small window in which cells are permeabilized without injury (200 to 290 Pa). This simple apparatus helps define the forces needed for physical cell transfection methods.     

Stroke frequency in front crawl: its mechanical link to the fluid forces required in non-propulsive directions

Two hypotheses were tested: (a) stroke frequency is predictable from the amplitudes of bodyroll and the turning effect around the body's long-axis generated by the non-propulsive fluid forces (that is, the torque driving bodyroll), and (b) swimmers exhibit at least one alteration in the factors influencing the bodyroll cycle as they increase the stroke frequency for faster swimming, so that they can reduce the fluid forces "wasted" in non-propulsive directions. The mechanical formula that links stroke frequency and the kinetics of bodyroll was derived on the basis of Euler's equation of motion. Experimental data were collected from competitive swimmers to validate the derived mechanical relations and to examine the strategy that skilled swimmers would use to change the stroke frequency as they swam faster. A strong correlation (slow: r = 0.70 and fast: r = 0.85) and a non-significant difference between the observed stroke frequency and the formula-based estimates supported the first hypothesis. As the subjects increased stroke frequency (38%) for faster swimming, bodyroll decreased (19%) and the trunk twist increased (40%). The combined alterations resulted in a small reduction in the shoulder roll (12%), enabling the swimmers to gain the benefits associated with a large rolling action of the upper trunk, while limiting the amount of increase in the turning effect of fluid forces in non-propulsive directions (40%). The second hypothesis was, therefore, supported. The derived mechanical formula provides a theoretical basis to explore mechanisms underlying the interrelations among stroke frequency, stroke length and swimming speed, and sheds light on a possible reason that swimmers generally adopt six-beat kicks.     

Correction of inert gas washin or washout for gas solubility in blood

We show that when an inert gas is washed into the lungs its retention in the blood during any one breath is approximately proportional to its solubility. This relationship makes possible the correction of washin or washout data for blood uptake or release, provided that two gases of different solubility are used simultaneously. The method automatically allows for the characteristics of an individual washin or washout and for the occurrence of recirculation within a fairly short washin or washout period. It has been tested in models with nonuniform ventilation and perfusion and closely approximates the behavior of a truly insoluble gas. In the derived ventilation distribution, gas solubility appears as ventilation to units of low turnover. In the case of N2 this effect is small but causes appreciable overestimation of lung volume. The recovered dead space and main alveolar distribution are insignificantly affected.     

Reproducibility of the multiple inert gas elimination technique

Although measurement errors in the multiple inert gas elimination technique have a coefficient of variation of approximately 3%, small biological fluctuations in ventilation, blood flow, or other variables must contribute additional variance to this method of assessing ventilation-perfusion (VA/Q) mismatch. To determine overall variance of computed indices of VA/Q mismatch, an analysis of variance was carried out using a total of 400 duplicate pairs of inert gas samples obtained from canine (N = 118) and human (N = 282) studies in the past 2 years. In both sets VA/Q mismatch ranged from minimal (2nd moment of ventilation and blood flow distributions, log SDV and log SDQ, respectively approximately equal to 0.3 each) to severe (log SDV and log SDQ approximately equal to 2.0). Differences between duplicate log SD values were computed and found to be a constant fraction of the mean log SD of each duplicate pair, averaging 13% for both canine and human ventilation and blood flow data. The resultant coefficient of variation for a single measurement of log SD about its mean averaged 8.6% for all data combined. This analysis demonstrates excellent reproducibility of these dispersion indices over a wide range of conditions, and if the mean of duplicate values is used, thus reducing variability by square root 2 to 6.1%, log SD can be estimated with an approximately 95% confidence limit of +/- 12%.     

Influence of blood flow on cutaneous permeability to inert gas

A thermally regulated Plexiglas chamber was designed for investigation of transcutaneous diffusion of N2 and helium (He) in the human hand. Influence of cutaneous blood flow in this process was studied simultaneously with gas diffusion measurements. Changes in cutaneous blood flow (Q, in ml X min-1 X 100 ml tissue-1) were effected by altering ambient temperature (T) from 20 to 40 degrees C (Q = 0.08 X 100.07T). We found that the rate of inert gas diffusion through human skin, expressed as conductance (G, in ml STPD X h-1 X m-2 X atm-1), increases exponentially as a function of blood flow, and was indistinguishable between He and N2 (G = 21.19 X 100.0124Q). The permeability, diffusion coefficient per unit diffusion distance (D/h, in cm/h), also rose exponentially as a function of blood flow. But permeability for He (D/h = 0.1748 X 100.0203Q) was greater than that for N2 (D/h = 0.1678 X 100.0114Q). As cutaneous blood flow rises, because of increased temperature, the apparent diffusion distance falls linearly for both N2 and He. The change is more prominent for He than for N2 diffusion. Estimated replacement time for the body stores of N2 by transcutaneous diffusion alone was shortened from 26.8 h at 31 degrees C to 15.1 h at 37 degrees C. It is suggested from this study that beneficial results may be derived during decompression procedure 1) by maintaining an appropriate transcutaneous pressure gradient of inert gases, and 2) by elevating ambient temperature.     

Adaptation of the inert gas FRC technique for use in heavy exercise

We automated the inert gas rebreathe technique for measurement of end-expiratory lung volume (EELV) during heavy exercise. We also assessed the use of two gas tracers (He and N2) vs. a single gas tracer (He) for measurement of this lung volume and compared the two-tracer EELV to changes in the inspiratory capacity (defined with transpulmonary pressure) and shifts in the end-expiratory pressure from rest through heavy exercise. A computer program switched a pneumatic valve when flow crossed zero at end expiration and defined points in the He and N2 traces for calculation of EELV. An inherent delay of the rebreathing valve (50 ms) caused virtually no error at rest and during light exercise and an error of 74 +/- 9 ml in the EELV at peak inspiratory flow rates of 4 l/s. The measurement of EELV by the two gas tracers was closely correlated to the single-gas tracer measurement (r = 0.97) but was consistently higher (120 +/- 10 ml) than when He was used alone. This difference was accentuated with increased work rates (2-5% error in the EELV, rest to heavy exercise) and as rebreathe time increased (2-7% error in the EELV with rebreathe times of 5-20 s for all work loads combined). The double-gas tracer measurement of EELV agreed quite well with the thoracic gas volume at rest (P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of flow cytometry to rapidly optimize the transfection of animal cells

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.     

A tidal breathing model for the multiple inert gas elimination technique.

The tidal breathing lung model described for the sine-wave technique (D. J. Gavaghan and C. E. W. Hahn. Respir. Physiol. 106: 209-221, 1996) is generalized to continuous ventilation-perfusion and ventilation-volume distributions. This tidal breathing model is then applied to the multiple inert gas elimination technique (P. D. Wagner, H. A. Saltzman, and J. B. West. J. Appl. Physiol. 36: 588-599, 1974). The conservation of mass equations are solved, and it is shown that 1) retentions vary considerably over the course of a breath, 2) the retentions are dependent on alveolar volume, and 3) the retentions depend only weakly on the width of the ventilation-volume distribution. Simulated experimental data with a unimodal ventilation-perfusion distribution are inserted into the parameter recovery model for a lung with 1 or 2 alveolar compartments and for a lung with 50 compartments. The parameters recovered using both models are dependent on the time interval over which the blood sample is taken. For best results, the blood sample should be drawn over several breath cycles.     

Stabilizing to disruptive transition of focal adhesion response to mechanical forces

Strong mechanical forces can, obviously, disrupt cell–cell and cell–matrix adhesions, e.g., cyclic uniaxial stretch induces instability of cell adhesion, which then causes the reorientation of cells away from the stretching direction. However, recent experiments also demonstrated the existence of force dependent adhesion growth (rather than dissociation). To provide a quantitative explanation for the two seemingly contradictory phenomena, a microscopic model that includes both integrin–integrin interaction and integrin–ligand interaction is developed at molecular level by treating the focal adhesion as an adhesion cluster. The integrin clustering dynamics and integrin–ligand binding dynamics are then simulated within one unified theoretical frame with Monte Carlo simulation. We find that the focal adhesion will grow when the traction force is higher than a relative small threshold value, and the growth is dominated by the reduction of local chemical potential energy by the traction force. In contrast, the focal adhesion will rupture when the traction force exceeds a second threshold value, and the rupture is dominated by the breaking of integrin–ligand bonds. Consistent with the experiments, these results suggest a force map for various responses of cell adhesion to different scales of mechanical force.     

Modelling inert gas exchange in tissue and mixed-venous blood return to the lungs

Inert gas exchange in tissue has been almost exclusively modelled by using an ordinary differential equation. The mathematical model that is used to derive this ordinary differential equation assumes that the partial pressure of an inert gas (which is proportional to the content of that gas) is a function only of time. This mathematical model does not allow for spatial variations in inert gas partial pressure. This model is also dependent only on the ratio of blood flow to tissue volume, and so does not take account of the shape of the body compartment or of the density of the capillaries that supply blood to this tissue. The partial pressure of a given inert gas in mixed-venous blood flowing back to the lungs is calculated from this ordinary differential equation. In this study, we write down the partial differential equations that allow for spatial as well as temporal variations in inert gas partial pressure in tissue. We then solve these partial differential equations and compare them to the solution of the ordinary differential equations described above. It is found that the solution of the ordinary differential equation is very different from the solution of the partial differential equation, and so the ordinary differential equation should not be used if an accurate calculation of inert gas transport to tissue is required. Further, the solution of the PDE is dependent on the shape of the body compartment and on the density of the capillaries that supply blood to this tissue. As a result, techniques that are based on the ordinary differential equation to calculate the mixed-venous blood partial pressure may be in error.     

The secretion of inert gas into the swim-bladder of fish

The composition of the gas mixture secreted into the swim-bladders of several species of fish has been determined in the mass spectrometer. The secreted gas differed greatly from the gas mixture breathed by the fish in the relative proportions of the chemically inert gases, argon, neon, helium, and nitrogen. Relative to nitrogen the proportion of the very soluble argon was increased and the proportions of the much less soluble neon and helium decreased. The composition of the secreted gas approaches the composition of the gas mixture dissolved in the tissue fluid. A theory of inert gas secretion is proposed. It is suggested that oxygen gas is actively secreted and evolved in the form of minute bubbles, that inert gases diffuse into these bubbles, and that the bubbles are passed into the swim-bladder carrying with them inert gases. Coupled to a preferential reabsorption of oxygen from the swim-bladder this mechanism can achieve high tensions of inert gas in the swim-bladder. The accumulation of nearly pure nitrogen in the swim-bladder of goldfish (Carassius auratus) is accomplished by the secretion of an oxygen-rich gas mixture followed by the reabsorption of oxygen.     

Use of pressure insoles to calculate the complete ground reaction forces

A method to calculate the complete ground reaction force (GRF) components from the vertical GRF measured with pressure insoles is presented and validated. With this approach it is possible to measure several consecutive steps without any constraint on foot placement and compute a standard inverse dynamics analysis with the estimated GRF.     

Calculating the distribution of ventilation-perfusion ratios from inert gas elimination data

It is well known that the major cause of hypoxemia in lung disease is ventilation-perfusion (VA/Q) inequality, but it has been extremely difficult to measure the distribution of ventilation-perfusion ratios except in terms of unrealistically simple (albeit useful) models. The multiple inert gas elimination technique provides considerable information concerning the shape, position, and dispersion of the VA/Q distribution, although it cannot precisely define all features of the distribution. Although there are many techniques for obtaining information about the distribution from inert gas elimination data, we have found the most flexible and useful approach to be a multicomponent analysis with enforced smoothing, sometimes known as ridge regression. This presentation describes in some detail the physiological and mathematical principles principles involved in the transformation of inert gas elimination data into a representative distribution of ventilation-perfusion ratios by enforced smoothing techniques. It is important to realize that with this approach and any other approach aimed at estimating the distribution of ventilation-perfusion ratios, the results must be properly interpreted.