Chunfei Wu

Chunfei Wu

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Materials characterisation - Solid – Structure and interaction analysis (1) - Atomic force microscopy (AFM) - Research application of AFM

Study of the Effect of Cell Prestress on the Cell Membrane Penetration Behavior by Atomic Force Microscopy

https://doi.org/10.3390/mi14020397

“In recent years, atomic force microscopes have been used for cell transfection because of their high-precision micro-indentation mode; however, the insertion efficiency of the tip of AFM into cells is extremely low. In this study, NIH3T3 mouse fibroblast cells cultured on a flexible dish with micro-groove patterns were subjected to various substrate strains at 5%, 10%, 15%, and 20%. It was found that the cell stiffness depends on the prestress of the cell membrane, and that the insertion rate of AFM tips into the cell membrane is proportional to the stiffness through the AFM indentation experiment. The finite element analysis proves that prestress increases the bending stiffness of the cytoskeleton, allowing it to better support the cell membrane, which realizes the stress concentration in the contact area between the AFM tip and the cell membrane. The results indicate that the prestress contributes to the mechanical properties of the cell and suggest that the insertion efficiency could be greatly improved with an increase of the prestress of the cell membrane.”

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3.1. Cell Alignment

The effects of the micro-pattern in a culture dish on cell alignment have been reported in many studies [22,23]. PDMS has been used to prepare micro-patterns in culture dishes due to its excellent combination of biocompatibility and flexible properties. Here, the size effect of the micro-pattern was investigated on the alignment of cells and it was found that micro-patterns with width between 15 μm and 30 μm allowed cells to grow more effectively along the pattern, while the growth direction of cells was random on the micro-patterns with smaller or larger width sizes. The growth of cells at different channel sizes is shown in Figure S3. In this study, the culture dish with a microgroove of 15 µm in width and a bridge of 30 μm in width was chosen to culture the cells. Figure 1a shows the AFM morphology of the yellow rectangular region in Figure 1b. The results show that the cells align well in the microgrooves of 15 µm. The growth process of the cells was recorded after seeding for 12 h, as shown in Figure 1b. The cells aligned well in 4 h, and the cell alignment phenomenon occurred not only in the groove but also on the bridge. Therefore, a flexible substrate with microgrooves of 15 µm in width was used as the cell culture dish in order to better unify the cell prestress.
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Figure 1. (a) AFM morphology of patterned substrate; (b) cells spread on the flexible culture dish with micro-patterns for 2 h, 4 h, 6 h and 12 h.

3.2. Apply Prestress to The Cells

The prestress was applied to the cells by stretching the substrate with microgrooves of 15 um. To verify that the stretching of the PDMS culture dish had been transferred to the cell, the changes of cell shape were tracked under an inverted microscope.

NIH3T3 cells were subjected to various strains in the stretching device. As soon as the stretching ratio reached the set value, the state of the cells was recorded immediately. A clear elongation of culture dish along the stretch direction was detected in Figure 2. The transmission from PDMS culture dish to cell membrane depends on the tightness of cell adherence to the collagen-coated PDMS culture dish. Ten cells were chosen to describe the relationship between PDMS strain and cell strain. The strain along the longitudinal direction can be calculated as

ε=(L1−L0)/L0�=(�1−�0)/�0

where L0 and L1 are the lengths of cells or substate before and after stretching, respectively. The stretching ratio of the cell increases with the increment of the stretching rate of the PDMS culture dish, as shown in Figure 2f. While the strains of PDMS are 5%, 10%, 15%, and 20%, the corresponding strains of the cells are 4.28%, 7.93%, 13.25%, 13.72%, respectively. According to Kozaburo’s report [24], fibroblast cells can be stretched up to four times the original length without breaking. Therefore, the cellular activity cannot be impaired when they are stretched by 13.72%. This assumption is confirmed from the subsequent culture after stretching. The cell stretch ratio is slightly smaller than the stretch ratio of the PDMS culture dish when the PDMS strain does not exceed 15%. However, compared with the cell strain when PDMS stretching ratio is 15%, the cell strain does not increase significantly when the PDMS stretching ratio reaches 20%. This suggests that there is an interfacial slipping between the cell and the substrate. This is mainly because large strains, such as focal adhesions, disrupt the cell–matrix junction.

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Figure 2. Cell stretching in PDMS culture dish with micro-patterns. The cells were stretched at 0% (a), 5% (b),10% (c), 15% (d), and 20% (e) strains of the substrate. (f) The transmission of PDMS culture dish stretching to the cells (scale bar = 50 µm).

3.3. Cell Stiffness

3.3.1. Measurements of Cell Stiffness

The spherical tip of the AFM probe was used to measure the stiffness of cells. The morphology of the tip is characterized by scanning electron microscope (SEM), as shown in Figure 3a. The large contact area between the spherical tip and the cell prevents damage to the cell even when the tip presses deeply into the cell, thus enabling the detection of the mechanical properties of its entire structure. The representative force–displacement curves at different strain states are plotted in Figure 3b. The average values of the stiffnesses were calculated, by Hertz fitting, to be 423 ± 72 Pa, 608 ± 43 Pa, 1321 ± 26 Pa, 1710 ± 54 Pa, and 1862 ± 69 Pa, which correspond to the respective PDMS strains of 0%, 5%, 10%, 15%, and 20%. The results show that the stiffness of the NIH3T3 cell increases with the increasing stretch ratio. However, when the strain attained 20%, the stiffness increased in a way that is not obviously compared with the substrate strain of 15%. All tests were completed in a very short period of time due to the way in which the strain and stress of the actin cross-linking network is dependent on time [17].
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Figure 3. The force–displacement curves under the indentation depth of 1.5 µm when using the spherical glass tip. (a) An SEM image of the spherical glass tip. (b) The representative force–displacement curves under different substrate strains.
To explain the increased stiffness of the cells by stretching the substrate, the cytoskeleton—which dominates the mechanical properties of the cell—needs to be introduced. It is known that the cytoskeleton is essential in maintaining cell shape, movement, and anchorage [25]. The cytoskeleton is composed of actin filaments—which play a major role in cell mechanical properties—intermediate filaments, and microtubules. The actin filament network is located below the cellular cortex, which supports and strengthens the cell membrane. This network allows cells to maintain their shapes. Additionally, the actin filaments together with myosin and other actin-binding proteins often form bundles known as actin stress fibers. F-actin shows a faster-than-linear strain hardening behavior [21]. Many of the stress fibers are arranged parallel to the long axis of the cell. The stress fibers are seen along the long axis of the cell in the AFM image, as shown in Figure 4a and which can also be confirmed from the immunofluorescent image in Figure 4b. These conduct force by connecting to the extracellular matrix through the focal adhesions on the cell membrane [26]. Therefore, when the substrate is stretched, both the cell membrane and the stress fibers are prestressed by the focus adhesion and the complex actin filament network.
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Figure 4. The finite element analysis model and simulation result. (a) AFM topography image of a single NIH3T3 cell. (b) Immunofluorescence image of NIH3T3 cells. (c) The schematic of a cell adhering to the substrate. (d) The finite element analysis model.

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