Microfluidic Transfection System and Temperature Strongly Influence the Efficiency of Transient Transfection.

For the process of transient transfection (TTF), DNA is often transported into the cells using polyplexes. The polyplex uptake and the subsequent transient expression of the gene of interest are of great importance for a successful transfection. In this study, we investigated a 3D-printed microfluidic system designed to facilitate direct TTF for suspension of CHO-K1 cells. The results demonstrate that this system achieves significantly better results than the manual approach. Furthermore, the effect of both post-transfection incubation time (t) and temperature (T) on polyplex uptake was explored in light of the membrane phase transitions. Attention was paid to obtaining the highest possible transfection efficiency (TFE), viability (V), and viable cell concentration (VCC). Our results show that transfection output measured as product of VCC and TFE is optimal for t = 1 h at T = 22 °C. Moreover, post-transfection incubation at T = 22 °C with short periods of increased T at T = 40 °C were observed to further increase the output. Finally, we found that around T = 19 °C, the TFE increases strongly. This is the membrane phase transition T of CHO-K1 cells, and those results therefore suggest a correlation between membrane order and permeability (and in turn, TFE).

Cell Membrane State, Permeability, and Elasticity Assessment for Single Cells and Cell Ensembles.

The phase state and especially phase transitions of synthetic lipid membranes are known to drastically modulate mechanical membrane properties like permeability and bending modulus. Although the main transition of lipid membranes is typically detected employing differential scanning calorimetry (DSC), this technique is not suitable for many biological membranes. Moreover, often single cell data on the membrane state or order is of interest. We here first describe how to use a membrane polarity-sensitive dye, Laurdan, to optically determine the order of cell ensembles over a wide temperature range from T = -40 °C to +95 °C. This allows to quantify the position and width of biological membrane order-disorder transitions. Second, we show that the distribution of membrane order within a cell ensemble allows for correlation analysis of membrane order and permeability. Third, combining the technique with conventional atomic force spectroscopy allows for the quantitative correlation of an overall effective Young's modulus of living cells with the membrane order.

Transport Across Cell Membranes is Modulated by Lipid Order.

This study measures the uptake of various dyes into HeLa cells and determines simultaneously the degree of membrane lipid chain order on a single cell level by spectral analysis of the membrane-embedded dye Laurdan. First, this study finds that the mean generalized polarization (GP) value of single cells varies within a population in a range that is equivalent to a temperature variation of 9 K. This study exploits this natural variety of membrane order to examine the uptake as a function of GP at constant temperature. It is shown that transport across the cell membrane correlates with the membrane phase state. Specifically, higher membrane transport with increasing lipid chain order is observed. As a result, hypothermal-adapted cells with reduced lipid membrane order show less transport. Environmental factors influence transport as well. While increasing temperature reduces lipid order, it is found that locally high cell densities increase lipid order and in turn lead to increased dye uptake. To demonstrate the physiological relevance, membrane state and transport during an in vitro wound healing process are analyzed. While the uptake within a confluent cell layer is high, it decreases toward the center where the membrane lipid chain order is lowest.

Shear stress induced lipid order and permeability changes of giant unilamellar vesicles.

BACKGROUND: The permeability of a lipid bilayer is a function of its phase state and depends non-linearly on thermodynamic variables such as temperature, pressure or pH. We investigated how shear forces influence the phase state of giant unilamellar vesicles and their membrane permeability. METHODS: We determined the permeability of giant unilamellar vesicles composed of different phospholipid species under shear flow in a tube at various temperatures around and far off the melting point by analyzing the release of fluorescently labelled dextran. Furthermore, we quantified phase state changes of these vesicles under shear forces using spectral decomposition of the membrane embedded fluorescent dye Laurdan. RESULTS: We observed that the membrane permeability follows a step function with increasing permeability at the transition from the gel to the fluid phase and vice versa. Second, there was an all-or-nothing permeabilization near the main phase transition temperature and a gradual dye release far off the melting transition. Third, the Laurdan phase state analysis suggests that shear forces induce a reversible melting temperature shift in giant unilamellar vesicle membranes. MAJOR CONCLUSIONS: The observed effects can be explained best in a scenario in which shear forces directly induce membrane pores that possess relatively long pore lifetimes in proximity to the phase transition. GENERAL SIGNIFICANCE: Our study elucidates the release mechanism of thermo-responsive drug carriers as we found that liposome permeabilization is not continuous but quantized. Furthermore, the shear force induced melting temperature shift must be taken into consideration when thermo-responsive liposomes are designed.

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy.

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from -40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.