Insights Gained from Force Measurements

A considerable number of studies observed the behaviour of different primary cells when presented with various surroundings. In particular, the contractile force in dependency of the environment’s stiffness was of interest. For cells such as fibroblasts [69], endothelial cells [12], Jurkat T-cells [46], neutrophils [88] and also cancer cells [55], it was shown that the forces they exert on the underlying substrate increase with increasing stiffness. This adaptation demonstrates an ac-tive feed-back loop within the cells that first sense the stiffness and then regulates a corresponding response by,e.g., changing the internal cellular structure.

Other examples of an active adaptation process within the cells was observed in the speed of their contractile response. In Trichetet al.[120], it could be shown that the velocity of contraction increases with the stiffness. Simultaneously, the cells’ spread area also increased with the rigidity. Furthermore, the greater the cell’s area was, the higher the exerted force was measured to be [12, 35].

The different observation methods were adapted to also include smaller cells such as dictyostelium cells [20, 21] and T-cells [46]. Compared to larger cells such as e.g. fibroblasts or endothelial cells, it was shown that they exert considerably less force. Additionally, for dictyostelium cells, it was demonstrated that they not only exert forces in a planar direction but also perpendicular to the substrate surface.

On a molecular level, it was revealed by a combination of knock-down and inhi-bition experiments in combination with force measurements that both actin poly-merisation as well as myosin-driven contraction contribute to the exerted forces

Force Measurements of Contractile Cells 2.2 of a cell [11, 46]. Which of the two mechanisms contribute more to the

contrac-tion can, however, vary between different cell lines. As an example, while mouse embryonic fibroblast cell could be shown to be driven to 90 % by myosin activ-ity [11], the traction forces observed from T-cells decreased significantly when the actin polymerisation was inhibited [46].

For platelets, most of the different experimental methods were employed to measure the forces exerted by the activated, contracting cells. In particular, contin-uous PAA substrates [81, 107], AFM measurements [58], micro-post arrays [29, 65]

and, most recently, tension sensors [125] have been applied. Note that, due to their size, experiments conducted on micro-post arrays used small aggregates of platelets while the other experiments were conducted on single platelets. All of the different approaches aimed to answer the question which force a platelet can exert on the surrounding during contraction. The resulting forces varied considerably depending on the approach chosen.

In Schwarz Henriqueset al.[107], a substrate of stiffness 4 kPa was utilised and the development of the force studied over time. Here, the point of attachment was taken as the point of reference for the un-stressed substrate. A comparatively sparse bead density was used to measure the contractile forces. It was shown that the platelets, on average, yielded a force of about 34 nN. Further, the force devel-oped towards a force plateau that was reached within 25 min. The contraction was described as directed towards the cell centre and close to isotropic behaviour. It was also shown that the total force scaled with the total spread area.

In contrast to the continuous substrate used above, Lamet al.[58] used an AFM to measure the axial contraction. The stiffness of the set-up was calculated to be 12 kPa, 29 kPa and infinitely stiff for different experiments. For the single platelets, the maximum contraction force was determined to be between 1.5 nN and 79 nN, reached within 10 min to 15 min. Interestingly, the averaged maximal forces per stiffness were lower than the forces measured on the PAA substrates above, in particular about 18 nN. It was further observed that the platelets adapted to the rigidity of the substrate by exerting more force with increasing stiffness. Addition-ally, the adhesion force was measured to be about 70 nN. The platelet’s elasticity was calculated to be 10 kPa, with an increasing stiffness for cells that exerted a higher force. In a following work to this experiment, Myerset al.[81] exchanged the AFM to fibrinogen patterned PAA substrates. Contrary to the substrates used by Schwarz Henriques et al. [107], the platelets were only allowed to adhere be-tween two fibrinogen patches instead of an evenly coated substrate. The force

Chapter 2 STATE OF THE ART

was calculated by the changes in position of the protein patches. Although the PAA gel provided a two-dimensional base for the experiment, due to the protein patterns, the force was determined in a uni-axial manner, similar to the AFM ap-proach. The stiffness was tuned between 25 kPa and 100 kPa and the thrombin concentration varied from 0.1 u/mL to 5 u/mL. They found that the magnitude of forces not only depend on the stiffness of the substrate but also on the thrombin concentration with an optimum found for the stiffness at 75 kPa or a thrombin concentration of 5 u/mL. The average force ranged from the 15 nN to about 40 nN depending on the stiffness and thrombin combination.

Next, in Refs. [29, 65], micro-post arrays were used. Lianget al.[65] determined the stiffness of the micro-posts to be about 2.9 MPa. Here, different thrombin con-centrations were utilised to study the dependency of the contraction process on the amount of stimulus. It was shown that an increase in thrombin concentration yields a higher force response until a concentration of 3.5 u/ml after which no change was seen. At the same time, the clot volume also stagnates in size. Over time, with a constant supply of thrombin and platelets, the clot increased in size and contractile force. The force per platelet in the contracting clot was estimated to be about 2 nN, considerably less than the works conducted on PAA substrates or with an AFM. Feghhi et al.[29] used posts of a stiffness of about 2.5 MPa and determined the force exerted by the clot on each micro-post. They measured a force of about 14 nN per post and three to four posts per clot. Instead of deter-mining the total force per platelet, they wanted to test the influence of myosin IIa on the contraction process. By blocking different activation pathways for myosin, it was shown that this motor is essential for force generation and clot retraction.

Most recently, Wang et al. [125] developed a molecular tension sensor for the force transmitted by theαIIbβ3 integrin. This sensor was demonstrated to tunable between 10 pN and 60 pN. Using this sensor, they revealed that the force mag-nitude of platelets determines the force distribution. Integrins of low tension,i.e.

below 54 pN, were distributed in a ring-like pattern around the periphery with two to three spots of higher force. Integrins exhibiting higher tensions were only found in two to three focus points. The latter were co-localised with vinculin, a protein associated with focal adhesions. Lastly, they demonstrated that the lower forces develop directly at the beginning of the adhesion and spreading process while the higher, focused forces first appear at later time-points.

Microuidics in Biology and Cellular Forces Measured Under Shear Flow 2.3

2.3 Microuidics in Biology and Cellular Forces Measured Under Shear