**3. Visualization of CVs**

Isolation of CVs from blood first requires removal of blood cells, in particular erythrocytes. Namely, to their prevailing abundance in blood of healthy humans, they present an obstacle in observation of the effects of other blood components. Usually, this is performed by centrifugation at a relatively low speed. Upon centrifugal force particles in the blood are inclined to move toward the bottom of the tube. The motion is roughly determined by the centrifugal force, buoyancy, and resistance force approximated by the Stokes law. It can be seen by equilibrating the forces that the speed of particles is proportional to the square of the particle radius and proportional to its density which means that larger particles will move faster. Besides being the most abundant, erythrocytes are also relatively large. If approaching each other close enough they form rolleaux which effectively speeds up their sedimentation and also creates channels in which smaller particles (platelets and CVs) are pushed out. This creates a counter-flow due to which platelets and CVs accumulate in the plasma above erythrocytes [32, 42]. However, this effect is temporary, as platelets and CVs in the erythrocyte-poor plasma reverse their flow and sediment as well. Plasma obtained by relatively low-speed centrifugation contains platelets as well as residual erythrocytes

**Figure 1.**

*A: Blood plasma observed by SEM. B: CVs isolated from blood as observed on the inner wall of the tube by SEM. A: From [31]. B: From [47].*

and leukocytes (**Figure 1A**). Furthermore, some blood cells shed CVs during the processing. For further elaboration of the sample, different protocols have been proposed, differing in the amount of the required isolate and its purpose. Centrifugation is still the most widely used method as it does not induce changes in the chemical composition of the sample, is relatively simple, time effective and low cost, and enables the simultaneous elaboration of multiple samples. Different methods were suggested for the isolation of CVs from blood [43]. Centrifugation protocols may differ in time and speed of centrifugation as well as in other parameters (e.g., temperature, the type of laboratory material used, up-gradation by technologically advanced procedures) [44–46].

**Figure 1B** shows the interface of the isolate with the tube wall. The tube was cut and the sample was prepared directly on the piece of the tube for imaging with the scanning electron microscope (SEM). Smooth shapes of the particles in the isolate can be noted.

CV isolates from blood shown in this chapter were obtained by repetitive centrifugation and washing of samples with phosphate- and citrate- buffered saline (PBS). We used different centrifugation protocols. Unilamellar phospholipid vesicles were prepared by electroformation in sugar solution and rinsed into the observation chamber by solution of a lighter sugar with the same osmolarity.

Giant phospholipid vesicles exceed in size several micrometers and can therefore be observed live by optical microscope. Therefore, the comparison between theoretically predicted and experimentally observed shapes is straightforward. For submicron-sized CVs light microscopy does not provide sufficient resolution. The samples can be observed by electron microscope which requires more or less aggressive processing. To observe them by scanning electron microscope, samples are dried and sputtered with heavy metal. For cryo-electron microscopy, they are frozen in thin ice (about 100 nm of thickness) which deforms soft particles larger than this dimension and may cause their degradation. Interpretation of the images of processed samples is not always straightforward.

**Figure 2** shows SEM images of CVs found in isolates from blood (a-d), an erythrocyte of a healthy human in physiological *ex vivo* conditions (e), and optical microscope images of a giant phospholipid vesicle (f-i). The corresponding theoretically obtained contours that were obtained by the solution of the variational problem are also given (a-i). A rigorous solution of the system of differential equations was sought. Two sequences of shapes are given, representing the transformation of vesicles

### *Morphology and Formation Mechanisms of Cellular Vesicles Harvested from Blood DOI: http://dx.doi.org/10.5772/intechopen.101639*

composed of a single type of constituents with fixed relative volume *v* and changing < *h* >. The sequence a-d starts with a discocytic shape which with decreasing < *h* > transforms into a stomatocyte with a wide dimple. In continuation of the process, the dimple grows inwards while the neck at the top shrinks. The sequence f-i starts with a prolate shape which with increasing < *h* > transforms into a pear shape. The neck shrinks up to a point in which it becomes infinitesimal.

It can be seen that the shapes of the erythrocyte (**Figure 2e**) and the CV (**Figure 2a**) are the same although the size scale of the CVs is 10 times smaller. Mammalian erythrocytes have no nucleus and also no internal cytoskeleton and their shapes are likewise determined by the minimum of the free energy of the membrane (underlayed with membrane skeleton). Consideration of the membrane skeleton however requires additional assumptions which are important also in describing the formation of CVs.

**Figure 3** shows calculated shapes representing the swelling and budding of a membrane-enclosed structure. The swelling was simulated by an increase of *v* and budding was simulated by an increase of average mean curvature <*h*>. Vesicles were composed of one type of constituents. Calculation of the sequence is conveniently performed within a chosen class of shapes. For a membrane composed from a single type of constituents that favor flat shape, the pear shape sequence (**Figure 2f**-**i**) is energetically more favorable than the lemon shape sequence (**Figure 3B**-**D and E-G**) since the formation of the neck is energetically unfavorable for constituents that favor positive (evaginated) and flat regions. The geometrical constraints limit the power of the set of possible shapes. For relative volume 1, there is only one possible shape (e.g., sphere) and this is the shape that attains the biggest possible volume at the given surface area (*v* cannot exceed 1). With the decrease of *v*, the set of possible shapes increases. It can be seen that already small decrease of *v* (e.g., to 0.98 (**Figure 3B**-**D**))

#### **Figure 2.**

*Experimental: a-d: SEM of CVs found in isolates from blood, e: SEM of a discocyte at physiological ex vivo conditions, f-i: Optical microscope images of giant phospholipid vesicles. Theoretical contours derive from the solution of the variational problem by rigorously solving a system of differential equations. Within sequences a-d and f-i, A and V were fixed while < h > was changing: The parameters of the calculated shapes were hm* ¼ *dm* ¼ 0*, (a, e): v* ¼ 0*:*6, <*h* > ¼ 1*:*040, <*d*> ¼ 1*:*812*, (b, c): v* ¼ 0*:*6, <*h* > ¼ 0*:*650, <*d*> ¼ 1*:*167*, (d): v* ¼ 0*:*6, <*h* > ¼ 0, 435, <*d*> ¼ 0*:*235*, (f): v* ¼ 0*:*9, <*h* > ¼ 1*:*050, <*d*> ¼ 0*:*729*, (g): v* ¼ 0*:*9, <*h* > ¼ 1*:*105, <*d*> ¼ 0*:*697*, (h): v* ¼ 0*:*9, <*h* > ¼ 1*:*155, <*d*> ¼ 0*:*577*, (i): v* ¼ 0*:*9, <*h*> ¼ 1*:*240, <*d*> ¼ 0*:*163*. Adapted from [34].*

**Figure 3.**

*Shapes corresponding to the minimum of the membrane free energy calculated by rigorously solving a system of differential equations. The parameters of the calculated shapes are hm = dm = 0, A: v = 1, < ℎ > = 1, B: v = 0.98, < ℎ > = 1.0088, C: v = 0.98, < ℎ > = 1.011, D: v = 0.98, < ℎ > = 1.0224, E: v = 0.90, < ℎ > = 1.05035, F: v = 0.9, < ℎ > = 1.0634, G: v = 0.9, < ℎ > = 1.354.*

can induce visible changes in shape with respect to the sphere (*v* = 1). If the vesicle loses 10% of its relative volume, the shape is visibly elongated (**Figure 3E**-**G**).

The particles that are essentially membrane-enclosed fluid interior deform and eventually undergo fragmentation at the thin necks. As the tearing area is minute the membrane is likely to seal. Smaller fragments are thus created. Fragmentation of residual cells takes place in particular at the interface with the tube wall where the shear force is the highest. Centrifugation at high centripetal accelerations of the rotor was shown to induce the formation of CV aggregates composed of a mixture of CVs highly heterogeneous in size and number of associated CVs [32].

Characteristics of membrane-enclosed vesicles composed of constituents that are not directly interacting are the smoothness of the shape. Good agreement between the calculated and the observed shapes indicates that the particles in the samples are vesicles (membrane-enclosed fluid interior). There are no additional methods needed. According to this principle, **Figure 3B** shows particles that can be identified as CVs deriving from blood cells. It is, however, not clear from this point what is the origin of the CVs, as the material may undergo formation and re-formation of vesicles during the processing, and the constituents of the CVs may come from different cells as well as from the surrounding solution. Such vesicles are colloidal in their nature and their identity depends on the properties of the cells as well as on the processing of the samples (e.g., centrifugation parameters, the composition of the suspension,

temperature). They can be considered an artifact; however, this artifact can have clinical significance [30].
