Deformation and Shape Transition Studies of Single Mammalian Red Blood Cells in View of the Effects of Diseases or Chemical Modifications by Means of Microfluidic Devices

The main focus of the thesis was to study and understand why human RBC change their shape upon increasing shear stress in the microvasculatur. Therefore, human RBCs were observed while flowing in PDMS micro-channels of different structures (even structure, corner structure, spiral structure and zig-zag structure) and tapered glass capillaries. This allowed for the study of RBC morphology upon various levels of flow conditions or shear stress, respectively. Besides the study of the flow behaviour under various flow conditions various types of RBCs were investigated. The following mammalian red blood cells (RBCs) were investigated: healthy human RBCs, chemically modified RBCs as a model system for blood diseases, RBCs suffering from b-Thalassemia and Spherocytosis as well as RBCs from Alpaca (vicugna pacos). The chemical modification was performed by incubating healthy RBCs in suspensions of phosphate buffered saline (PBS) with a small concentration of either formaline, diamide or cholesterol. The chemicals formaline and diamide hardened the cell membrane and cholesterol changed the strain inside the RBC membrane. To characterize the RBC mechanics quantitatively the following physical parameters were used: bending modulus, shearing modulus, area expansion modulus and the transition velocity. First, cell morphology in flow was investigated regarding length and curvature measurements of the two-dimensional cell projection (for example length to width ratio or front and rear curvature). For each cell type the discocyte, slipper and parachute shape was measured regarding width to length ratios and protuberances-length ratios as well as front and rear curvatures. A detailed study of the healthy, chemically modified and diseased shape morphology showed that chemically modified and diseased cells had a different shape and size compared to healthy RBCs. The variations of the measured two-dimensional projection ratios and curvatures showed a correlation to alterations in the cell membrane. Second, in flow systems the mechanical properties were investigated dynamically. While the cells were exposed to a constant hydrodynamic flow field their deformation was measured. The Taylor deformation parameter varied proportionally to changes in mechanical properties of the RBCs. Chemically modified RBCs showed a smoother increase of the Taylor deformation with increasing cell velocity and saturated at lower Taylor deformation levels. Modifications of the RBCs with diamide and formaline incubations decreased the Taylor deformation parameter for the measured flow regimes dramatically. This supported the results from the static measurements. However, the Taylor deformation parameter did not indicate shape changes of RBCs (such as discocyte to slipper or parachute) which were observed upon increasing shear stress. Third, using PDMS micro channels the shape transition from the discocytic rest shape to a slipper or parachute shape of RBCs was characterized further. In a 10 x 10µm (width and height) channel the transition velocities v_c of hundreds of RBCs for different volume flows were measured. It was found that the transition velocity solely depends on mechanical properties of the RBC membrane. Formaline and diamide modifications increased the transition velocity by a factor of about two to three. Low cholesterol content in the membrane as well as high tonicity of the suspension medium increased the transition velocity weakly. In contrast, high cholesterol content and low medium tonicity decreased the transition velocity. Cells from donors with the blood disease b-Thalassemia showed an increase of the transition velocity of around 25%. In the case of Spherocytosis disease a dramatic increase of around 470% was observed. Fourth, using tapered glass capillaries, the shape transition was further investigated to see the effect of different channel heights as well. In contrast to PDMS microchannels glass capillaries had different round shaped cross-sections if they were fabricated with a small taper angle. This experiment enabled for each RBC alteration the drawing of a phase diagram showing the shape morphology in dependency of the cell velocity and squared capillary radius. Alterations in the membrane by chemical modification or blood diseases were visible by different phase boundaries. As a proof of principle transition velocities of the 10 x 10µm PDMS channel experiments were validated for the same flow environments. In addition, phases with very high slipper probabilities were identified at small capillary radii. This was mainly due to the wall interactions of the cells for small capillary radii. The fifth research focus investigated the transition into a slipper and parachute shape in more detail. Therefore, the slipper was observed in a weakly tapered glass capillary and a slip velocity was introduced. The slip velocity is the difference of the experimentally measured cell velocity and the theoretical extrapolated velocity in undisturbed flow at the corresponding capillary radius. In the experiments, the slip velocity increased during the shape transition. As a conclusion the main effect of the shape transition was to lower the flow resistance. These experiments brought new insight to the shape change of human RBCs. However, RBC from alpaca showed a different shape behaviour. In both microfluidic flow systems (glass capillary and PDMS microchannel), the flow motion of Alpaca RBC was investigated as well. At rest these cells were characterized by an elliptical extended shape. Exposed to shear stress, these cells behaved differently from discocytic mammalian RBCs because they deformed much less. However, as a new characteristic, they showed a combination of flow motions of healthy human RBCs: tumbling and swinging. The tumbling motion of RBCs was characterized by an incidental inclination angle with respect to the flow direction whereas the swinging motion showed a periodical change of the inclination angle with respect to the flow direction. Furthermore, the relaxation time after an induced bending of Alpaca RBC was measured by squeezing them through extremely narrow capillaries (2R_(cap)