# Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip

^{1}

^{2}

^{3}

^{*}

## Abstract

**:**

_{q}gives insight into the correlation between the cells’ abilities to adhere and withstand shear flow and the topography of the substrates, finding a local optimum at R

_{q}= 22 nm. We use shear stress induced by acoustic streaming to determine a measure for the ability of cell adhesion under an external force for various conditions. We find an optimum of cell adhesion for T = 37 °C and pH = 7.4 with decreasing cell adhesion outside the physiological range, especially for high T and low pH. We find constant detachment rates in the physiological regime, but this behavior tends to collapse at the limits of 41 °C and pH 4.

## 1. Introduction

## 2. Materials and Methods

#### 2.1. The De-Adhesion Number Investigator (DANI)

_{3}chip and a circular substrate of arbitrary material (here: titanium) with adhered cells on top. We fastened the whole setup using a brass bridge, which is also thermally connected in order to heat the system using a heat bath. Here, the heat bath temperature is chosen about ΔT = 7 °C lower than the desired temperature. Together with the small SAW-generated temperature increase of the sample, this results in the temperatures given below. By applying a radio frequency signal to the interdigital transducer (IDT) on the piezoelectric substrate, surface acoustic waves were generated. These caused acoustic streaming leading to a fluid flow towards the cells under an angle of α = 21° relative to the surface normal [19].

#### 2.2. Scanning Particle Image Velocimetry

^{®}, Polysciences Inc., Hirschberg an der Bergstraße, Germany) as tracer particles to the fluid and recorded videos with a high-time resolution using a high-speed video camera (FASTCAM 1024PCI, Photron, Pfullingen, Germany). We determined the flow field in x–y direction in the plane as close as possible to the sample surface, since this is the relevant region to appraise its influence on the cells. For our experiments, we applied a power of p = 28 dBm to the IDT, which results in an average shear rate of $\dot{\gamma}=4314\text{}{\text{s}}^{-1}$. To investigate cell adhesion as a function of surface roughness and determine the ideal topography, we applied a milder shear flow by reducing the power to p = 25 dBm, which results in a shear rate of $\dot{\gamma}=2157\text{}{\text{s}}^{-1}$. This corresponds to a shear stress of about 2 Pa and is particularly of interest, as it is known from literature that, for example, endothelial cells show high response to very low shear stress between 0.1 and 0.8 Pa [25].

#### 2.3. Sample Preparation

_{q}= 3.76 µm, which is determined by profilometric measurements (Dektak 8 Advanced Development Profiler, Vecco Instruments Inc., Oyster Bay, NY, USA). To investigate the influence of the surface roughness on cell adhesion, we polished the discs to yield seven different R

_{q}. Therefore, we embedded the substrates in Technovit

^{®}5071 (Heraeus Kunlzer GmbH, Wehrheim, Germany) and polished them using the auto-grinder and polisher AutoMet

^{®}250 (Buehler, Illinois Tool Works Inc., Esslingen am Neckar, Germany) with abrasive paper of different granulation (60, 320, 1000, 2500, and 4000), followed by a polycrystalline diamond polish (grain size 1 µm) and a chemo-mechanical polish (grain size 40 nm). We analyzed the surface topography with an atomic force microscope (NanoWizard

^{®}AFM, JPK Instruments AG, Berlin, Germany ) and found R

_{q}= 640 nm, 150 nm, 70 nm, 30 nm, 22 nm, 10 nm, and 2 nm, respectively. We cleaned all samples in an ultrasonic bath for 10 min in a 30% water in ethanol solution and finally sterilized them in an autoclave at 120 °C for 20 min.

#### 2.4. Cell Culture Lines

_{3}and 1.0 g/L d-glucose, adding 50 mL fetal bovine serum (FBS Superior), 10 mL HEPES 1 M, 5mL l-glutamine 200 mM, 5 mL MEM vitamins 100× (all reagents from Biochrom GmbH, Berlin, Germany), and 1 mL primocin (ant-pm-2, Invitrogen™, Thermo Fisher Scientific GmbH, Dreieich, Germany) in humidified air containing 5% CO

_{2}at 37 °C. We harvested the confluent cells for our experiments, following the standard trypsinization procedure using 1 mL Trypsin/EDTA solution and PBS (w/o Ca

^{2+}, w/o Mg

^{2+}) (Biochrom GmbH, Berlin, Germany). By centrifugation and discarding of the supernatant with subsequent resuspension in media, we adjusted the cell density to 300,000 cells/mL.

#### 2.5. Cell Adhesion and Fluorescence Imaging

_{inc}= 60 min allowing them to subside and adhere on the substrate. About 50% of the suspended cells adhere to the substrate under standard conditions (T = 37 °C, pH = 7.4, R

_{q}= 3.76 µm). In the incubation step, we either set pH = 7.4 = const. and incubated at different temperatures T = 27, 33, 37, 39, 41, 42 and 47 °C, or we set the temperature to T = 37 °C = const. and incubated at different pH = 4.0, 4.5, 5.5, 6.5, 7.4, 8.0, 9.0, 10.0. To adjust the pH, we added HCl or NaOH to the culture medium to generate acidic or alkaline conditions, respectively. After incubation, we gently replaced the supernatant by a cell-free medium to remove the cells that did not adhere. Before starting the experiment, we then needed about 10 min to mount the device on the microscope and to connect the heating system.

_{tot}= 3.48 mm × 2.65 mm = 9.22 mm

^{2}(compare Figure 2). We recorded micrographs of the cells before applying a shear flow (static measurement, $t=0\text{min}={t}_{0}$), and one micrograph every 5 min with applied shear flow for a period of 60 min ($0\le {t}_{i}\le 60\text{min}$) and quantified the amount of adhered cells as the fraction of the total field of view covered by cells

_{c,t}time-dependently, we converted the recorded micrographs into an 8-bit black and white format using the software ImageJ [26]. For each experiment, we set an individual but constant threshold to distinguish adherent cells from the background. The intensity is then inverted and the area covered with cells (black) on the free area (white) is quantified using the ImageJ particle analysis function and correlated with the starting value ${A}_{c,{t}_{0}}$ to determine the detached cells under flow.

_{0}) and their detachment (area covered with cells at t

_{0}but not at ${t}_{i}>{t}_{0}$) time-dependently, ending up with ${A}_{\mathrm{c},(t\text{}=\text{}60\text{min})}={A}_{c,60\text{}\mathrm{m}\mathrm{i}\mathrm{n}}$. Figure 2 visualizes this image analysis process. The magnified images show the initial and final state as well as their superposition created with the colocalization finder macro for ImageJ [26]. In the superposition, the yellow cells remain at their positions throughout the whole experiment, while spots where cells have been detached are labelled with red and cells that appear in the final image but not in the initial one and thus must have moved on the substrate are shown in green.

## 3. Results and Discussion

#### 3.1. Time-Dependent Cell Detachment

_{q}= 3.76 µm to investigate the time-dependency of cell adhesion under static and dynamic conditions for T = 37 °C and pH = 7.4.

_{c,t}in time steps of 5 min for up to 60 min under static conditions. We normalize A

_{c,ti}to the initial value for the cell covered area A

_{c,t}= 31% ± 3% and plot it over time, as shown for a typical example in Figure 3a. We find a linear decrease of A

_{c,t}down to A

_{c,60 min}= 0.94 ± 0.03 * A

_{c,0 min}meaning that gravity and bleaching result in a decrease of A

_{c,t}of approximately 6% within 60 min. The experiments are repeated at least five times to compensate for deviations of the initially seeded cell number and cell viability.

_{c,t}down to A

_{c,60 min}= (0.79 ± 0.03) * A

_{c,0 min}. To decouple gravity and bleaching from the influence of shear, we now normalize the results of the dynamic experiment using the linear fit function from the static measurement as baseline (used in all later dynamic measurements). The resulting graph (as shown exemplarily in Figure 3b) finally shows only the cell detachment due to shear forces.

_{c,t}at given time t. The calibrated, shear-induced decay now leads to A

_{c,60 min}= (0.85 ± 0.03) * A

_{c,0 min}.

#### 3.2. Influence of Temperature, pH and Surface Roughness on Cell Adhesion

_{c,0 min}as well as the dynamic results in terms of the final adhesion A

_{c, 60 min}and the normalized final adhesion A

_{c,60 min}/A

_{c}

_{,0 min}as a function of temperature (Figure 5a,d,g), pH (Figure 5b,e,h) and surface roughness (Figure 5c,f,i), respectively.

_{q}= 3.76 µm.

_{c}over T with a clear maximum of A

_{c,0 min}= 31% ± 3% for T = 37 °C. For lower temperatures, A

_{c,0 min}decreases only slightly to A

_{c,}

_{0 min}= 26% ± 5% at T = 33 °C at the lower end of the physiological range. Even for a temperature as low as T = 27 °C, A

_{c,0 min}= 24% ± 3% and thus about 78% of its maximum value at 37 °C, while it decreases strongly for T ≥ 37 °C, down to A

_{c,0 min}= 22% ± 4% for T = 41 °C, just below the upper physiological limit. A significant change can then be observed for T = 43 °C, where the cell-covered area falls to A

_{c,0 min}= 13% ± 5% (corresponds to 32% of the maximum). The highest temperature T = 47 °C only shows traces of cell adhesion with A

_{c,0 min}= 3% ± 1%.

_{c,60 min}= 27% ± 3%. Low temperatures show a final adhesion of A

_{c,60 min}= 23% ± 3% for T = 27 °C and A

_{c,60 min}= 26% ± 5%. Even for 39 °C and 41 °C with A

_{c,60 min}= 21% ± 3% and A

_{c,60 min}= 19% ± 2% there is no significantly increased detachment under shear flow. Only at high temperatures with T ≥ 43 °C the final adhesion drops down to A

_{c,60 min}= 5% ± 1%. For T = 47 °C no adhered cells can be found after shear flow exposure.

_{c,t}with time. Applying our rate model (Equation (2)) for temperatures inside the physiological range (33 °C ≤ T ≤ 41 °C), we can calculate the detachment rate R and ${\mathit{A}}_{\infty}$, whereas going to extreme conditions (T = 27 °C and T ≥ 43 °C), the rate model collapses. At T = 27 °C we find only minor cell detachment in the beginning and an increase of cell detachment at t = 40 min as well as a final cell covered area of ${\mathit{A}}_{\infty}=$ 0. This is no reasonable result and is due to the flat slope and the concomitant large error of the fit. At T = 43 °C A

_{c,t}shows an almost linear decrease and, after t = 40 min, an abrupt decline. For T = 47 °C we find the same effect but with a stronger decline and no remaining cells on the substrate for t = 60 min. The abrupt change of the detachment rate prohibits to fit Equation (2) to the data.

_{c,60 min}and normalized final adhesion A

_{c,60 min}/A

_{c,0 min}instead of ${\mathit{A}}_{\infty}$. Doing so, we find the maximal cell adhesion for T = 33 °C (A

_{c,60 min}/A

_{c,0 min}= 0.92 ± 0.09) and an equally high value of A

_{c,60 min}/A

_{c,0 min}= 0.91 ± 0.6 for T = 27 °C. Both values are higher than the one for T = 37 °C (A

_{c,60 min}/A

_{c,0 min}= 0.85 ± 0.07), although the values overlap clearly and are about equal to A

_{c,60 min}/A

_{c,0 min}= 0.92 ± 0.05 for T = 39 °C. Obviously, temperatures just below the physiological range and close to the normal body temperature do not have significant impact on cell adhesion. However, for temperatures of T ≥ 41 °C, which approach the physiological limit, we find a decrease of A

_{c,60 min}/A

_{c,0 min}down to A

_{c,60 min}/A

_{c,0 min}= 0.72 ± 0.04. Here, the detachment rate is increased by a factor of 1.2 compared to T = 37 °C. Exceeding the physiological limits (T = 43 °C) leads to even higher detachment rates, leaving only a fraction of 40% of the initially attached cells with A

_{c,60 min}/A

_{c,0 min}= 0.45 ± 0.05 on the substrates (about half of the according value for T = 37 °C). For T = 47 °C, no cells are left on the substrate after 60 min. This behavior of A

_{c,60 min}/A

_{c,0 min}as function of temperature is qualitatively similar compared to the static measurements (see Figure 5a,g).

_{q}= 3.76 µm.

_{c,60 min}to quantify the cell adhesion under dynamic conditions.

_{c,0 min}as a function of the pH. Qualitatively, we find an asymmetric distribution of A

_{c,0 min}with a stronger cell detachment for acidic pH than in the alkaline range. A more quantitative analysis shows a maximum of A

_{c,0 min,max}= 31% ± 3% at pH = 7.4. In the acidic direction, we observe a strong decrease at the border of the physiological range (pH = 6.5) down to A

_{c,0 min}= 17% ± 3% = 0.54 * A

_{c,0 min,max}. For even lower pH, the final cell adhesion decreases strongly and we find only A

_{c,0 min}= 4% ± 1% for pH = 4.5. As a limit of the cells’ adhesion capability, we identify pH = 4, where the cells do not adhere to the substrate at all. In an alkaline environment (pH = 8.0) we find A

_{c,0 min}= 22% ± 4% = 0.7 * A

_{c,0 min,max}. A stronger alkaline pH of 9.0 results in A

_{c,0 min}= 11%. ± 2% = 0.35 * A

_{c,0 min,max}and we find the limit for cell adhesion in alkaline media to be for pH ≥ 10.

_{c,60 min}= 27% ± 3% for pH = 7.4 as depicted in Figure 5e. For the acidic environment we find A

_{c,60 min}= 15% ± 3% for pH = 6.5 and only about A

_{c,60 min}= 3% ± 1% remaining cells at the lower end with pH = 4.5. Deviations from ideal pH towards alkaline milieu result in stronger detachment of cells, leaving a remaining A

_{c,60 min}= 17% ± 3% for pH = 8 and A

_{c,60 min}= 8% ± 5% for pH = 9.

_{c,60 min}/A

_{c,0 min}= 0.85 ± 0.03) to pH = 6.5 with A

_{c,60 min}/A

_{c,0 min}= 0.87 ± 0.07, although the standard deviation of these values overlap strongly. To the alkaline side, we observe only a slight decrease of cell adhesion down to A

_{c,60 min}/A

_{c,0 min}= 0.79 ± 0.07 at pH = 9. In contrast, towards the acidic side, we find the cell detachment to be increased (A

_{c,60 min}/A

_{c,0 min}= 0.67 ± 0.09 for pH = 4.5). However, the effect of these extreme deviations of the pH from its optimum is less pronounced than the influence of temperature.

^{+}into the cell due to the internally negative membrane potential [38]. Therefore, cells have evolved several methods for pH regulation, e.g., Na

^{+}/H

^{+}antiporters and Na

^{+}dependent HCO

_{3}

^{−}/Cl

^{−}exchanger for acidic regulation, as well as the Na

^{+}independent HCO

_{3}

^{−}/Cl

^{−}exchanger in alkaline pH [39]. It is expected that the cell metabolism is affected by intracellular (cytoplasmic) pH, which is influenced by Na

^{+}/H

^{+}exchangers acting on the enzyme phosphofructokinase, the rate-controlling enzyme in the glycolytic pathway. Irreversible cytoplasmic acidification starts at pH 6.8 and below as here the pH leads to an enzyme inactivity [38] and a decrease in adhesion molecule expression which is responsible for unspecific binding processes [40].

_{c,0 min}decreasing as the expression of adhesion molecules will decline. Nevertheless, the unspecific binding mechanisms are still able to withstand shear flow.

_{c,0 min}= 31% ± 3%), is reached at the sandblasted surface roughness of R

_{q}= 3.76 µm, whereas the use of polished surfaces leads to lower cell adhesion of A

_{c}

_{,0min}≈ 15%, nearly independent of the surface roughness.

_{c,60 min}shows a maximum of adherent cells at R

_{q}= 22 nm (A

_{c,0 min,max}= 14% ± 4%) as can be seen in Figure 5f and therefore a remaining fraction of A

_{c,60 min}/A

_{c,0 min}= 0.96 ± 0.04 normalized to the initial adhesion (see Figure 5i). Above and below this value, we find a decrease of A

_{c,60 min}. However, we still find A

_{c,60 min}= 8% ± 7% and respectively A

_{c,60 min}/A

_{c,0 min}= 0.84 ± 0.08 for the smoothest surface with R

_{q}= 2 nm. Eying the rough surfaces, we find a decline in remaining cells with increasing roughness with a minimum for R

_{q}= 640 nm with A

_{c,60 min}= 10% ± 3% and respectively A

_{c,60 min}/A

_{c,0 min}= 0.68 ± 0.12 = (0.8 ± 0.14) * A

_{c,0 min,max}. For the untreated standard sample with R

_{q}= 3.76 µm the maximum in adhered cells can be found again with A

_{c,60 min}= 27% ± 3%. Although normalized to the initial adhesion, the detachment increases to A

_{c,60 min}/A

_{c,0 min}= 0.85 ± 0.03 =(0.90 ± 0.03) * A

_{c,0 min,max}and therefore shows a medium adhesion behavior compared to all substrates.

_{q}= 22 nm.

_{a}≤ 1.20 µm, with R

_{a}= 150 nm showing the best cell adhesion and spreading compared to smoother (R

_{a}≤ 70 nm) or rougher (R

_{a}≥ 330 nm) substrates. We, too, see a strong increase in cell adhesion with increasing roughness up to R

_{q}= 150 nm, but no further increase for 640 nm. Therefore, we conclude that an appropriate surface roughness can produce beneficial mechanical interlocking at the initial adhesion stage and foster further cell adhesion with its ideal spacing at R

_{q}= 150 nm [47].

_{q}. This observation can be explained by shear flow-induced activation of focal adhesions, leading to an enhancement of stress fibers [7]. Integrin receptors function as mechano-sensors and induce an activation on signaling proteins which act on the cytoskeletal anchorage [48]. It has been reported that surface topography correlates with binding of focal adhesion and thus formation of stress fibers. Ideal spacing for focal contacts was found to be 30 nm [6], which is in good agreement with our results that show maximal cell adhesion under dynamic conditions for R

_{q}= 22 ± 5 nm. Along the same lines, we compare our results to the ones reported by Cavalcanti-Adam et al. [49], who used nano-patterned surfaces to investigate the influence of ligand spacing to cell adhesion under static conditions. They found the initial attachment of cells to be independent of the surface, but for the formation of stable focal adhesion and persistent spreading they found a critical ligand density (interligand spacing < 70 nm). Our findings indicate that we see a similar effect, although we are varying the sample topography and not the ligand density.

## 4. Conclusions and Outlook

_{q}= 3.76 µm under physiological standard conditions of T = 37 °C and pH = 7.4. Applying shear flow reveals an even more pronounced decrease of cell adhesion for deviations from these conditions. High temperatures and low pH are especially critical. Under extreme conditions we find our rate model collapses due to cell death during the experiment. Regarding surface roughness, we found a local optimum of adhesion at an average roughness of R

_{q}= 22 nm. Future studies should also account for the influence of cell density on the de-adhesion rate, since, for example, cells are shielded from the flow by cells positioned further upstream or adhesive forces between cells may appear. A quantitative correlation between local shear rates and time-dependent detachment of cells might give further insight into the shear sensitivity of arbitrary cell-substrate-combinations. Interesting systems thereby include novel metal ion releasing implant materials [53] and osteoblasts. On the other hand, cell biologists may apply the DANI-setup to study the internal response of adaptive processes of adherent cells to shear flow. Such processes are, for example, the mobility of adhesive bridges, the creation of reactive oxygen species, the structure and the orientation of the cytoskeleton as well as gene expression. A comparison of the results with in vivo experiments could then highlight the biological relevance of such studies.

## Acknowledgments

## Author Contributions

## Conflicts of Interest

## Abbreviations

DANI | De-Adhesion Number Investigator |

SPIV | scanning Particle Image Velocimetry |

SAW | Surface Acoustic Waves |

IDT | Inter Digital Transducer |

## References

- OECD Indicators. Health at a Glance 2015; OECD Publishing: Paris, France, 2015. [Google Scholar]
- Kurtz, S.; Ong, K.; Lau, E.; Mowat, F.; Halpern, M. Projections of Primary and Revision Hip and Knee Arthroplasty in the United States from 2005 to 2030. J. Bone Jt. Surg.
**2007**, 89, 780–785. [Google Scholar] [CrossRef] [PubMed] - Anselme, K.; Bigerelle, M.; Noel, B.; Dufresne, E.; Judas, D.; Iost, A.; Hardouin, P. Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses. J. Biomed. Mater. Res.
**2000**, 49, 155–166. [Google Scholar] [CrossRef] - Anselme, K. Osteoblast adhesion on biomaterials. Biomaterials
**2000**, 21, 667–681. [Google Scholar] [CrossRef] - Khalili, A.; Ahmad, M. A Review of Cell Adhesion Studies for Biomedical and Biological Applications. Int. J. Mol. Sci.
**2015**, 16, 18149–18184. [Google Scholar] [CrossRef] [PubMed] - Burridge, K. Focal Adhesions: Transmembrane Junctions between the Extracellular Matrix and the Cytoskeleton. Annu. Rev. Cell Dev. Biol.
**1988**, 4, 487–525. [Google Scholar] [CrossRef] [PubMed] - Shyy, J.Y.J.; Chien, S. Role of integrins in endothelial mechanosensing of shear stress. Circ. Res.
**2002**, 91, 769–775. [Google Scholar] [CrossRef] [PubMed] - Pellegrin, S.; Mellor, H. Actin stress fibres. J. Cell Sci.
**2007**, 120, 3491–3499. [Google Scholar] [CrossRef] [PubMed] - Nine, M.J.; Choudhury, D.; Hee, A.C.; Mootanah, R.; Osman, N.A.A. Wear Debris Characterization and Corresponding Biological Response: Artificial Hip and Knee Joints. Materials
**2014**, 7, 980–1016. [Google Scholar] [CrossRef] - Hirashima, Y.; Ishiguro, N.; Kondo, S.; Iwata, H. Osteoclast induction from bone marrow cells is due to pro-inflammatory mediators from macrophages exposed to polyethylene particles: A possible mechanism of osteolysis in failed THA. J. Biomed. Mater. Res.
**2001**, 56, 177–183. [Google Scholar] [CrossRef] - Pye, A.D.; Lockhart, D.E.A.; Dawson, M.P.; Murray, C.A.; Smith, A.J. A review of dental implants and infection. J. Hosp. Infect.
**2009**, 72, 104–110. [Google Scholar] [CrossRef] [PubMed] - Physiologie des Menschen, 22nd ed.; Schmidt, R.F.; Thews, G. (Eds.) Springer: Berlin/Heidelberg, Germany, 1997.
- Ducommun, P.; Ruffieux, P.-A.; Kadouri, A.; von Stockar, U.; Marison, I. W. Monitoring of temperature effects on animal cell metabolism in a packed bed process. Biotechnol. Bioeng.
**2002**, 77, 838–842. [Google Scholar] [CrossRef] [PubMed] - Weiss, L. Cell contact phenomena. In Vitro
**1970**, 5, 48–78. [Google Scholar] [CrossRef] [PubMed] - Usami, S.; Chen, H.-H.; Zhao, Y.; Chien, S.; Skalak, R. Design and construction of a linear shear stress flow chamber. Ann. Biomed. Eng.
**1993**, 21, 77–83. [Google Scholar] [CrossRef] [PubMed] - Bussonnière, A.; Miron, Y.; Baudoin, M.; Bou Matar, O.; Grandbois, M.; Charette, P.; Renaudin, A. Cell detachment and label-free cell sorting using modulated surface acoustic waves (SAWs) in droplet-based microfluidics. Lab Chip
**2014**, 14, 3556–3563. [Google Scholar] [CrossRef] [PubMed] - Stamp, M.E.M.; Brugger, M.S.; Wixforth, A.; Westerhausen, C. Acoustotaxis—In vitro stimulation in a wound healing assay employing surface acoustic waves. Biomater. Sci.
**2016**, 4, 1092–1099. [Google Scholar] [CrossRef] [PubMed] - Hartmann, A.; Stamp, M.; Kmeth, R.; Buchegger, S.; Stritzker, B.; Saldamli, B.; Burgkart, R.; Schneider, M.F.; Wixforth, A. A novel tool for dynamic cell adhesion studies—The De-Adhesion Number Investigator DANI. Lab Chip
**2014**, 14, 542–546. [Google Scholar] [CrossRef] [PubMed] - Frommelt, T.; Gogel, D.; Kostur, M.; Talkner, P.; Hänggi, P. Flow Patterns and Transport in Rayleigh Surface Acoustic Wave Streaming: Combined Finite Element Method and Raytracing Numerics versus Experiments. IEEE Trans. Ultrason. Ferroelectr. Freq. Control
**2008**, 55, 2298–2305. [Google Scholar] [CrossRef] [PubMed] - Strobl, F.G.; Breyer, D.; Link, P.; Torrano, A.A.; Brauchle, C.; Schneider, M.F.; Wixforth, A. A surface acoustic wave-driven micropump for particle uptake investigation under physiological flow conditions in very small volumes. Beilstein J. Nanotechnol.
**2015**, 6, 414–419. [Google Scholar] [CrossRef] [PubMed] - Thielicke, W.; Stamhuis, E.J. PIVlab—Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB. J. Open Res. Softw.
**2014**, 2, e30. [Google Scholar] [CrossRef] - Thielicke, W.; Stamhuis, E.J. PIVlab—Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB (Version: 1.4). 2015. Available online: http://pivlab.blogspot.com/ (accessed on 19 October 2016).
- Thielicke, W. The Flapping Flight of Birds—Analysis and Application. Ph.D. Thesis, Rijksuniversiteit, Groningen, The Netherlands, 2014. [Google Scholar]
- Lindken, R.; Rossi, M.; Große, S.; Westerweel, J. Micro-Particle Image Velocimetry (µPIV): Recent developments, applications, and guidelines. Lab Chip
**2009**, 9, 2551–2567. [Google Scholar] [CrossRef] [PubMed] - Davies, P.F.; Dewey, C.F.; Bussolari, S.R.; Gordon, E.J.; Gimbrone, M.A. Influence of hemodynamic forces on vascular endothelial function. In vitro studies of shear stress and pinocytosis in bovine aortic cells. J. Clin. Investig.
**1984**, 73, 1121–1129. [Google Scholar] [CrossRef] [PubMed] - Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods
**2012**, 9, 671–675. [Google Scholar] [CrossRef] [PubMed] - Kaufmann, H.; Mazur, X.; Fussenegger, M.; Bailey, J.E. Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. Biotechnol. Bioeng.
**1999**, 63, 573–582. [Google Scholar] [CrossRef] - Goergen, J.L.; Marc, A.; Engasser, J.M. Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release. Cytotechnology
**1993**, 11, 189–195. [Google Scholar] [CrossRef] [PubMed] - Wust, P.; Rau, B.; Gellermann, J.; Pegios, W.; Löffel, J.; Riess, H.; Felix, R.; Schlag, P.M. Radiochemotherapy and Hyperthermia in the Treatment of Rectal Cancer. Recent Results Cancer Res.
**1998**, 146, 175–191. [Google Scholar] [PubMed] - Hildebrandt, B.; Wust, P.; Ahlers, O.; Dieing, A.; Sreenivasa, G.; Kerner, T.; Felix, R.; Riess, H. The cellular and molecular basis of hyperthermia. Crit. Rev. Oncol. Hematol.
**2002**, 43, 33–56. [Google Scholar] [CrossRef] - Holme, T.A. Denaturation. Available online: http://www.encyclopedia.com/topic/denaturation.aspx (accessed on 21 July 2016).
- Konings, A.W.T.; Ruifrok, A.C.C. Role of Membrane Lipids and Membrane Fluidity in Thermosensitivity and Thermotolerance of Mammalian Cells. Radiat. Res.
**1985**, 102, 86–98. [Google Scholar] [CrossRef] [PubMed] - Majda, J.A.; Gerner, E.W.; Vanlandingham, B.; Gehlsen, K.R.; Cress, A.E. Heat Shock-Induced Shedding of Cell Surface Integrins in A549 Human Lung Tumor Cells in Culture. Exp. Cell Res.
**1994**, 210, 46–51. [Google Scholar] [CrossRef] [PubMed] - Coss, R.A.; Linnemans, W.A.M. The effects of hyperthermia on the cytoskeleton: A review. Int. J. Hyperth.
**1996**, 12, 173–196. [Google Scholar] [CrossRef] - Allgemeine und spezielle Pharmakologie und Toxikologie, 7th ed.; Forth, W.; Henschler, D.; Rummel, W.; Starke, K. (Eds.) Elsevier: Berlin, Germany, 1996. (In German)
- Punnia-Moorthy, A. Evaluation of pH changes in inflammation of the subcutaneous air pouch lining in the rat, induced by carrageenan, dextran and staphylococcus aureus. J. Oral Pathol. Med.
**1987**, 16, 36–44. [Google Scholar] [CrossRef] [PubMed] - McQueen, A.; Bailey, J.E. Effect of ammonium ion and extracellular pH of hybridoma cell metabolism and antibody production. Biotechnol. Bioeng.
**1990**, 35, 1067–1077. [Google Scholar] [CrossRef] [PubMed] - Tannock, I.; Rotin, D. Acid pH in tumors and its potential for therapeutic exploitation. Cancer Res.
**1989**, 49, 4373–4384. [Google Scholar] [PubMed] - Modenaar, H. Effects of growth factors on intracellular pH regulation. Annu. Rev. Physiol.
**1986**, 48, 363–376. [Google Scholar] - Serrano, C.V.; Fraticelli, A.; Paniccia, R.; Teti, A.; Noble, B.; Corda, S.; Faraggiana, T.; Ziegelstein, R.C.; Zweier, J.L.; Capogrossi, M.C. pH dependence of neutrophil-endothelial cell adhesion and adhesion molecule expression. Am. J. Physiol.
**1996**, 271, C962–C970. [Google Scholar] [PubMed] - Crouch, C.F.; Fowler, H.W.; Spier, R.E. The adhesion of animal cells to surfaces: The measurement of critical surface shear stress permitting attachment or causing detachment. J. Chem. Technol. Biotechnol. Biotechnol.
**1985**, 35, 273–281. [Google Scholar] [CrossRef] - Deligianni, D.D.; Katsala, N.D.; Koutsoukos, P.G.; Missirlis, Y.F. Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength. Biomaterials
**2000**, 22, 87–96. [Google Scholar] [CrossRef] - Meyle, J.; Gültig, K.; Wolburg, H.; von Recum, A.F. Fibroblast anchorage to microtextured surfaces. J. Biomed. Mater. Res.
**1993**, 27, 1553–1557. [Google Scholar] [CrossRef] [PubMed] - Boyan, B. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials
**1996**, 17, 137–146. [Google Scholar] [CrossRef] - Oakley, C.; Brunette, D.M. The sequence of alignment of microtubules, focal contacts and actin filaments in fibroblasts spreading on smooth and grooved titanium substrata. J. Cell Sci.
**1993**, 106, 343–354. [Google Scholar] [PubMed] - Huang, H.H.; Ho, C.T.; Lee, T.H.; Lee, T.L.; Liao, K.K.; Chen, F.L. Effect of surface roughness of ground titanium on initial cell adhesion. Biomol. Eng.
**2004**, 21, 93–97. [Google Scholar] [CrossRef] [PubMed] - Den Braber, E.T.; de Ruijter, J.E.; Smits, H.T.J.; Ginsel, L.A.; von Recum, A.F.; Jansen, J.A. Quantitative analysis of cell proliferation and orientation on substrata with uniform parallel surface micro-grooves. Biomaterials
**1996**, 17, 1093–1099. [Google Scholar] [CrossRef] - Schmidt, C. Mechanical Stressing of Integrin Receptors Induces Enhanced Tyrosine Phosphorylation of Cytoskeletally Anchored Proteins. J. Biol. Chem.
**1998**, 273, 5081–5085. [Google Scholar] [CrossRef] [PubMed] - Cavalcanti-Adam, E.A.; Volberg, T.; Micoulet, A.; Kessler, H.; Geiger, B.; Spatz, J.P. Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands. Biophys. J.
**2007**, 92, 2964–2974. [Google Scholar] [CrossRef] [PubMed] - Heitmann, S. Messung des Intraossealen Blutflusses zur Bestimmung der Klärfunktion im Gesunden und im Heilenden Knochen Mittels Laser- Dopplerflussmessung im Kaninchenmodell. Ph.D. Thesis, Tierärztliche Hochschule Hannover, Hannover, Germany, 2008. [Google Scholar]
- Laroche, M. Intraosseous circulation from physiology to disease. Jt. Bone Spine
**2002**, 69, 262–269. [Google Scholar] [CrossRef] - Cowin, S.C.; Cardoso, L. Blood and interstitial flow in the hierarchical pore space architecture of bone tissue. J. Biomech.
**2015**, 48, 842–854. [Google Scholar] [CrossRef] [PubMed] - Buchegger, S.; Vogel, C.; Herrmann, R.; Stritzker, B.; Wixforth, A.; Westerhausen, C. Antibacterial metal ion release from diamond-like carbon modified surfaces for novel multifunctional implant materials. J. Mater. Res.
**2016**, 31, 2571–2577. [Google Scholar] [CrossRef]

**Figure 1.**(

**a**) Computer animation of the De-Adhesion Number Investigator (DANI) setup showing the acoustic streaming in the chamber towards the substrate with adhered cells (indicated by the black dots) that is generated by the interdigital transducer (IDT) (gold, comb-like structure) (by courtesy of C. Hohmann, Nanosystems Initiative Munich (NIM)); (

**b**) Schematic drawing of the same setup. The IDT is located on the LiNbO

_{3}chip inside the polydimethylsiloxane (PDMS)-chamber, which holds the substrate 2 mm above the chip. The fluid flow induced by the SAW is directed towards the cell substrate under an angle of α = 21°.

**Figure 2.**Micrograph of the adhered cells on the substrate with magnification of (

**a**) initial state at t

_{0}= 0 min; and (

**b**) final state at t = 60 min; (

**c**) Superposition of both images using the colocalization finder macro for ImageJ [26]. Cells remaining fixed throughout the whole measurement are colored in yellow, detached cells are colored in red and moved or newly adhered cells are green.

**Figure 3.**(

**a**) Cell covered area over time under static and dynamic conditions for T = 37 °C, pH = 7.4 and R

_{q}= 3.76 µm. We fit a linear function to the static results (black line) and use it as calibration baseline to decouple the shear-induced detachment from the effects of gravity and bleaching. The data points and error bars show the mean and standard deviation respectively from n ≥ 5 measurements; (

**b**) Dynamic measurement from (

**a**) normalized to the linear fit extracted from the static experiments.

**Figure 4.**Temperature-dependent cell detachment with time and exponential fit for detachment rate in the physiological range of 27 °C ≤ T ≤ 39 °C.

**Figure 5.**Percentage of area covered with cells after an incubation time of 60 min under standard culture conditions but for different temperatures (

**a**), pH (

**b**) and surface roughness (

**c**); Physiological standard conditions of T = 37 °C and pH = 7.4 show the highest adhesion A

_{c},

_{0 min}while increasing roughness leads to higher A

_{c,0 min}. After applying shear flow for 60 min cell detach leading to a remaining adhesion A

_{c,60 min}for different temperatures (

**d**), pH (

**e**) and surface roughness (

**f**). While the effect of low temperatures hardly effect the cells, high temperatures and deviations from pH = 7.4 lead to strong detachment. For the influence of the topography, a peak in cell adhesion appears for R

_{q}= 22 nm, although the highest final adhesion can be found for R

_{q}= 3.76 µm. A

_{c,60 min}/A

_{c,0 min}denotes the fraction of the field of view that is covered with cells after 60 min exposure to shear flow. Similar to static conditions, the influence of temperature (

**g**), and pH (

**h**) inside the physiological range changes A

_{c,60 min}/A

_{c,0 min}only slightly, whereas high temperatures and low pH exhibit a strong effect; A

_{c,60 min}/A

_{c,0 min}as a function of surface roughness (

**i**) shows a maximum at R

_{q}= 22 nm. The data points and error bars show the mean and standard deviation respectively from n ≥ 5 measurements and the dashed lines are guides to the eye.

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

## Share and Cite

**MDPI and ACS Style**

Stamp, M.E.M.; Jötten, A.M.; Kudella, P.W.; Breyer, D.; Strobl, F.G.; Geislinger, T.M.; Wixforth, A.; Westerhausen, C.
Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip. *Diagnostics* **2016**, *6*, 38.
https://doi.org/10.3390/diagnostics6040038

**AMA Style**

Stamp MEM, Jötten AM, Kudella PW, Breyer D, Strobl FG, Geislinger TM, Wixforth A, Westerhausen C.
Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip. *Diagnostics*. 2016; 6(4):38.
https://doi.org/10.3390/diagnostics6040038

**Chicago/Turabian Style**

Stamp, Melanie E. M., Anna M. Jötten, Patrick W. Kudella, Dominik Breyer, Florian G. Strobl, Thomas M. Geislinger, Achim Wixforth, and Christoph Westerhausen.
2016. "Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip" *Diagnostics* 6, no. 4: 38.
https://doi.org/10.3390/diagnostics6040038