Real-time and quantitative measurement method for cell traction force

Abstract

A real time and quantitative method of measuring traction force of living cells include the following procedures. Place AT-cut and BT-cut quartz crystals of the same frequency, surface morphology and/or modified with the same cell adhesion molecules in petri dishes or detection cells; add the cells to the petri dishes or detection cells, the cell traction force at arbitrary time t during adhesion of the cells or under different internal/external environmental stimulations is estimated by the following equation: ΔS.sub.t=(K.sub.AT−K.sub.BT).sup.−1[t.sub.q.sup.ATΔf.sub.t.sup.AT/fr.sup.AT−tq.sup.BTΔf.sub.t.sup.BT/fr.sup.BT]. The method can be used to track the dynamic changes of cells generated force during the adhesion of cells and under different internal/external environmental stimulations, such as the effects of drugs. The drugs can be added before or after the adhesion of the cells. This method is suitable for all adherent cells, including primary cells and passage cells.

Claims

1. A real-time and quantitative measurement method for cell traction force, comprising the following steps: (1) placing an AT-cut quartz crystal and a BT-cut quartz crystal in culture dishes or detection cells, wherein the AT-cut quartz crystal having the same frequency, surface morphology and/or modified surface adhesion molecules as those of the BT-cut quartz crystal; and (2) adding cells to be tested to the culture dishes or the detection cells, and measuring the cell traction force ΔS.sub.t of the cells at an adhesion time t by the following formula:
ΔS.sub.t=(K.sub.AT−K.sub.BT).sup.−1[t.sub.q.sup.ATΔf.sub.t.sup.AT/fr.sup.AT−tq.sup.BTΔf.sub.t.sup.BT/fr.sup.BT]  (1), wherein K.sub.AT=2.75×10.sup.−12 cm.sup.2 dyn.sup.−1 and K.sub.BT=−2.65×10.sup.−12 cm.sup.2 dyn.sup.−1 are stress coefficients of the AT-cut quartz crystal and the BT-cut quartz crystal respectively; fr.sup.AT is the resonant frequency of the AT-cut quartz crystal, fr.sup.BT is the resonant frequency of the BT-cut quartz crystal, tq.sup.AT is the thickness of the AT-cut quartz crystal, tq.sup.BT is the thickness of the BT-cut quartz crystal, and all of which are constants; Δf.sub.t.sup.AT and Δf.sub.t.sup.BT are the frequency shifts of the AT-cut and BT-cut quartz crystals at any time t relative to their reference points respectively; when ΔS.sub.t is negative, it indicates that the stress on the cells is a compressive stress, the cells are contracted, and the corresponding extracellular matrices are subjected to a tensile stress equal and opposite to the compressive stress; when ΔS.sub.t is positive, the stress on the cells is a tensile stress, the cells are spread, and the corresponding extracellular matrices are subjected to a compressive stress equal and opposite to the tensile stress.

2. The method according to claim 1, wherein the cell adhesion molecules comprise extracellular matrix molecules capable of interacting with transmembrane proteins and integrins; extracellular matrix biomimetic molecules capable of interacting with transmembrane proteins and integrins; molecules capable of interacting with cell surface receptors; and molecules interacting with the surface of cells to promote cell adhesion.

3. The method according to claim 2, wherein the extracellular matrix molecules capable of interacting with transmembrane proteins and integrins are fibronectin, laminin, vitronectin or collagen; the extracellular matrix biomimetic molecules capable of interacting with transmembrane proteins and integrins are RGD adhesion sequence polypeptides; the molecules capable of interacting with cell surface receptors are molecules capable of interacting with cell surface cadherin; and the molecules interacting with the surface of cells to promote cell adhesion are poly-l-lysine.

4. The method according to claim 1, wherein in the formula of step (2), t.sub.q.sup.AT=0.1661/fr.sup.AT; and t.sub.q.sup.BT=0.2536/f.sub.r.sup.BT.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings illustrate one or more embodiments of the present invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

(2) FIG. 1 is a schematic diagram of cell structure-mechanics and QCM acoustic detection;

(3) FIG. 2 shows two configurations for cell traction force detection;

(4) FIG. 3 shows two configurations for simultaneously measuring cell traction force, cell morphology, and focal adhesion information;

(5) FIG. 4: example: dynamic QCM adhesion and force response curves under adhesion of 20,000 H9C2 rat cardiomyocytes (added at the first arrow) to AT-cut and BT-cut bare gold electrodes, and under the actions of 125 nM positive inotropic drug isoprenaline and 25 nM negative inotropic drug verapamil (the final concentration added at the second arrow). (A) Frequency shift and dynamic resistance change curve under adhesion and isoprenaline action, AT-cut; (B) Frequency shift and dynamic resistance change curve under adhesion and isoprenaline action, BT-cut; (C) Frequency shift and dynamic resistance change curve under adhesion and verapamil action, AT-cut; (D) Frequency shift and dynamic resistance change curve under adhesion and verapamil action, BT-cut; (E) Dynamic cell traction force change curve under adhesion and isoprenaline action; (F) Dynamic cell traction force change curve under adhesion and verapamil action;

(6) FIG. 5: example: QCM frequency shift, dynamic resistance change and traction force dynamic response curves during adhesion of 20,000 human umbilical vein endothelial cells to 9 MHz AT-cut and BT-cut quartz crystal gold electrodes modified at different KRGD concentrations. (A) Frequency shift response of RGD modified AT-cut crystal; (B) Frequency shift response of RGD modified BT-cut crystal; (C) Dynamic resistance change of RGD modified AT-cut crystal; (D) Dynamic resistance change of RGD modified BT-cut crystal; (E) Dynamic change in cell traction force of RGD modified crystal;

(7) FIG. 6: example: QCM frequency shift, dynamic resistance change and traction force dynamic response curves of adhesion of 20,000 human umbilical vein endothelial cells to 9 MHz AT-cut and BT-cut quartz crystal gold electrodes modified at different fibronectin concentrations. (A) Frequency shift response of fibronectin modified AT-cut crystal; (B) Frequency shift response of fibronectin modified BT-cut crystal; (C) Dynamic resistance change of fibronectin modified AT-cut crystal; (D) Dynamic resistance change of fibronectin modified BT-cut crystal; (E) Dynamic change in cell traction force of fibronectin modified crystal;

(8) FIG. 7: example: QCM frequency shift, dynamic resistance change and cell traction force dynamic response curves in adhesion of 20,000 human umbilical vein endothelial cells to 9 MHz AT-cut and BT-cut quartz crystal gold electrodes modified at the concentration of 50 μg/mL KRGD, and under the action of 1.22 μM blebbistatin drug. (A) Frequency shift and dynamic resistance response of AT-cut crystal; (B) Frequency shift and dynamic resistance response of BT-cut crystal; (C) Dynamic change in cell traction force;

(9) FIG. 8: example: QCM frequency shift, dynamic resistance change and cell traction force dynamic response curves in adhesion of 20,000 human umbilical vein endothelial cells to 9 MHz AT-cut and BT-cut quartz crystal gold electrodes modified at the concentration of 20 μg/mL fibronectin, and under the action of 0.5 μM nocodazole drug. (A) Frequency shift and dynamic resistance response of AT-cut crystal; (B) Frequency shift and dynamic resistance response of BT-cut crystal; (C) Dynamic change in cell traction force;

(10) FIG. 9: example: dynamic QCM adhesion and force response curves under adhesion of 20,000 human umbilical vein endothelial cells (added at the first arrow) to AT-cut and BT-cut bare gold electrodes, and under the actions of 0.1 unit/mL vascular endothelial barrier function destruction drug thrombin and 0.5 μM endothelial barrier function protection drug Y-27632 (the final concentration added at the second arrow). (A) Frequency shift and dynamic resistance change curve under adhesion and thrombin action, AT-cut; (B) Frequency shift and dynamic resistance change curve under adhesion and thrombin action, BT-cut; (C) Frequency shift and dynamic resistance change curve under adhesion and Y-27632 action, AT-cut; (D) Frequency shift and dynamic resistance change curve under adhesion and Y-27632 action, BT-cut; (E) Dynamic cell traction force change curve under adhesion and thrombin action; (F) Dynamic cell traction force change curve under adhesion and Y-27632 action;

(11) FIG. 10: example: dynamic QCM adhesion and force response curves under adhesion of 50,000 human umbilical vein endothelial cells to AT-cut and BT-cut bare gold electrodes, and under the action of EGTA of different concentrations (the final concentration added at the second arrow). (A) Frequency shift and dynamic resistance change curve under adhesion and 1 mM EGTA action, AT-cut; (B) Frequency shift and dynamic resistance change curve under adhesion and 1 mM EGTA action, BT-cut; (C) Frequency shift and dynamic resistance change curve under adhesion and 10 mM EGTA action, AT-cut; (D) Frequency shift and dynamic resistance change curve under adhesion and 10 mM EGTA action, BT-cut; (E) Frequency shift and dynamic resistance change curve under adhesion and 50 mM EGTA action, AT-cut; (F) Frequency shift and dynamic resistance change curve under adhesion and 50 mM EGTA action, BT-cut; (G) Dynamic cell traction force change curve under adhesion and 1 mM EGTA action; (H) Dynamic cell traction force change curve under adhesion and 10 mM EGTA action; (I) Dynamic cell traction force change curve under adhesion and 50 mM EGTA action; (J) Dynamic cell traction force change curve comparison under different EGTA concentrations.

DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

(12) The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments of the present invention are shown. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure is thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like reference numerals refer to like elements throughout.

(13) FIG. 2 shows two configurations for quantitatively measuring the cell traction force using the AT-cut and BT-cut double resonator technology. In FIG. 2A, the AT-cut and BT-cut crystals are in two different culture dishes or detection cells. At this time, the AT-cut and BT-cut crystals have the same frequency and surface morphology and/or the same surface adhesion molecules modified. After the same number and quality of cells of the same batch are added to the two detection cells, the cell traction force can be quantitatively measured from formula (1) by monitoring the frequency changes of the two crystals in real time. In FIG. 2B, the AT-cut and BT-cut crystals are in the same culture dish or detection cell, and the two crystals are also required to have the same frequency and surface morphology and/or the same surface adhesion molecules modified, and after a certain number of cells are added to the detection cell, the cell traction force can be quantitatively measured from formula (1) by detecting the frequency changes of the two crystals in real time. The measurement of the cell traction force requires no microscopy, so the metal electrode and the modified molecules and materials on the surface of the quartz crystal are not required to be transparent, and can be any material, which is another advantage of the method. Specifically, the surface of the quartz crystal may be covered by metal gold or non-metal SiO.sub.2 or the like which is biocompatible with cells.

(14) Ideally, in order to acquire dynamic information such as cell morphology and focal adhesions accompanying the change in the cell traction force, the QCM crystal can be used in conjunction with an optical or fluorescence microscopy. In this case, an optically transparent QCM electrode, such as an ITO electrode, is required. Similarly, two different configurations can be used, as shown in FIGS. 3A and 3B.

(15) The real-time and quantitative measurement method for cell traction force includes the following steps:

(16) (1) placing an AT-cut quartz crystal and a BT-cut quartz crystal in two separate culture dishes or detection cells, the AT-cut quartz crystal having the same frequency, surface morphology and/or modified surface adhesion molecules as the BT-cut quartz crystal; and
(2) adding cells to be tested to the culture dishes or the detection cells, and measuring the cell traction force ΔS by the following formula:
ΔS.sub.t=(K.sub.AT−K.sub.BT).sup.−1[t.sub.q.sup.ATΔf.sub.t.sup.AT/fr.sup.AT−tq.sup.BTΔf.sub.t.sup.BT/fr.sup.BT],

(17) in which, ΔS.sub.t is the traction force of cells at the adhesion time t; K.sub.AT=2.75×10.sup.−12 cm.sup.2 dyn.sup.−1 and K.sub.BT=−2.65×10.sup.−12 cm.sup.2 dyn.sup.−1 are stress coefficients of the AT-cut quartz crystal and the BT-cut quartz crystal of given crystal orientations, respectively; fr.sup.AT and fr.sup.BT are the resonant frequencies of the AT-cut and BT-cut quartz crystals, respectively; tq.sup.AT and tq.sup.BT are the thicknesses of the AT-cut and BT-cut quartz crystals, respectively. All the above are constants. Δf.sub.t.sup.AT and Δf.sub.t.sup.BT are respectively the frequency shifts of the AT-cut and BT-cut quartz crystals at any time t relative to their reference points (e.g., a stable value in a medium).

(18) Cell Traction Force Double Resonator Technology Experiment

(19) The steps for measuring the cell traction force by using bare gold electrode AT-cut and BT-cut quartz crystals are as follows:

(20) 1) dripping 1 drop of Piranha solution (80° C. 1:3 (v:v) 30% H.sub.2O.sub.2:H.sub.2SO.sub.4) to the center of each quartz crystal gold electrode for about 30 s, then rinsing with distilled water, drying with nitrogen, and repeating this step by 3 times;

(21) 2) assembling the crystals in a Teflon well cell;

(22) 3) cleaning the Teflon cell twice with distilled water, then adding about 300 μL of sterilized water, and putting into a 5% CO.sub.2 incubator at 37° C.;

(23) 4) checking to make sure that the 8-channel QCM instrument QCA922 has crystal resonant frequency and dynamic resistance outputs, connecting detection cells in turn, determining that each detection cell (e.g., two AT-cut crystal detection cells, two BT-cut crystal detection cells) works, and starting the software to acquire data;
5) removing the sterilized water after the data corresponding to each channel is stable, cleaning twice with sterilized water, then cleaning with PBS, add 52 μL of DMEM medium containing fetal bovine serum, and acquiring QCM resonant frequency (f) and dynamic resistance (R) data for 2 h; adding 250 μL of medium containing a certain number (e.g., 20,000) of H9C2 rat cardiomyocytes or human umbilical vein endothelial cells (HUVECs), continuously acquiring f and R data for about 20 h. The QCM relative frequency shift Δf and dynamic resistance change ΔR of each channel caused by the adhesion of cells at different adhesion time are determined by subtracting the corresponding QCM stable values in media of the channel at the time (t).
6) After the experiment, collecting the medium, gently washing with PBS, adding trypsin for digestion, and counting the cells in the collected fraction with a cytometer;
7) Quantitatively measuring the dynamic change ΔS in the cell traction force during the cell adhesion process according to the frequency shifts Δf.sub.t.sup.AT and Δf.sub.t.sup.BT of the paired AT-cut and BT-cut quartz crystals at the time t:
ΔS.sub.t=(K.sub.AT−K.sub.BT).sup.−1[t.sub.q.sup.ATΔf.sub.t.sup.AT/fr.sup.AT−tq.sup.BTΔf.sub.t.sup.BT/fr.sup.BT]  (1),

(24) in which, ΔS.sub.t is the traction force of cells at the adhesion time t; K.sub.AT=2.75×10.sup.−12 cm.sup.2 dyn.sup.−1 and K.sub.BT=−2.65×10.sup.−12 cm.sup.2dyn.sup.−1 are stress coefficients of the AT-cut and BT-cut quartz crystals of given crystal orientations, respectively, and are constants. fr.sup.AT and fr.sup.BT are the resonant frequencies of the AT-cut and BT-cut quartz crystals, respectively; tq.sup.AT and tq.sup.BT are the thicknesses of the AT-cut and BT-cut quartz crystals, respectively, and are constants. Therefore, the surface stress or traction force applied to the crystal by cells in the adhesion process or under the action of a drug can be quantitatively measured according to the frequency shifts Δf.sub.t.sup.AT, Δf.sub.t.sup.BT (in Hz) of the AT-cut crystal, the BT-cut crystal at any time t relative to its reference point (e.g., a stabile value in the medium or a stabile value before dosing) based on formula (1). The frequency of the quartz crystal is a digital signal, which can be easily, quickly and continuously acquired or measured by a frequency counting device or a QCM special instrument. The crystal frequency used in the experiment of the present disclosure is 9 MHz, where tq.sup.AT=0.0185 cm, and tq.sup.BT=0.0282 cm. Thus, formula (1) can be simplified as:
ΔS.sub.t=2.058×10.sup.4(0.0185Δf.sub.t.sup.AT−0.0282Δf.sub.t.sup.BT)  (2)

(25) The Steps for Measuring the Cell Traction Force with AT-Cut and BT-Cut Quartz Crystals Modified with Specific Cell Adhesion Molecules RGD and Fibronectin are as Follows:

(26) 1) cleaning with anhydrous ethanol and Millipore water, and blowing AT-cut and BT-cut 9 MHz crystals with nitrogen;

(27) 2) dripping 1 drop of Piranha solution (80° C. 1:3 (v:v) 30% H.sub.2O.sub.2:H.sub.2SO.sub.4) to the quartz crystal gold electrode for treating 30 s, rinsing with Millipore water and anhydrous ethanol, blowing with nitrogen, and repeating 3 times. Dripping the anhydrous ethanol onto the electrode to stand for a few minutes, rinsing with sterile water, and blowing with nitrogen;
3) installing the surface treated AT-cut and BT-cut quartz crystals into Teflon well cells;
4) adding a mixed anhydrous ethanol solution of 20 mM 3-mercaptopropionic acid and 1 mM triethylene glycol mono-11-mercaptoundecyl ether to the Teflon cell at room temperature, and standing overnight in the dark;
5) taking out the solution, and rinsing with sterile water; adding a PBS buffer solution (pH=5.5) with 150 mM EDC and 30 mM NHS dissolved therein, and standing for about 30 min;
6) taking out the solution, and rinsing with PBS buffer solution (pH=5.5) and sterile water; adding a PBS solution of KRGD or fibronectin of different concentrations, and standing for 1-2 h (RGDK) or overnight (fibronectin);
7) taking out the solution, and rinsing with sterilized PBS and sterile water to obtain KRGD or fibronectin modified gold electrodes. Adding 20,000 HUVEC or H9C2 cells, and starting QCM for monitoring;
8) collecting the medium after the experiment, gently wash with PBS, adding trypsin for digestion, and measuring the cells in the collected fraction with a cytometer; and
9) Quantitatively estimating the dynamic change ΔS in the cell traction force during the cell adhesion process according to the frequency shifts Δf.sub.t.sup.AT and Δf.sub.t.sup.BT of the AT-cut and BT-cut quartz crystals modified with the same concentration of RGD or fibronectin at the time t based on formula (2).

(28) Experimental Steps for Effects of Cardiovascular Stimulating Drug Isoprenaline (ISO) and Inhibitory Drug Verapamil (VRP) on Traction Force of H9C2 Rat Cardiomyocytes

(29) Take four Teflon well cells, two identical 9 MHz AT-cut gold electrode crystals and two identical 9 MHz BT-cut gold electrode crystals. Based on the aforementioned steps of measuring the cell traction force with bare gold electrode AT-cut and BT-cut quartz crystals, add 20,000 H9C2 cells to the four Teflon cells respectively, culture cells for 20 h, then take 5 μL of the culture solution out from the four Teflon cells respectively, add 5 μL of 10 μM ISO (final concentration 125 nM) and 5 μL of 2 μM VRP (final concentration 25 nM) to the two AT-cut and BT-cut crystal detection cells respectively, continuously monitor for 20 h, and collect data.

(30) Experimental Steps for Effects for Vascular Endothelial Barrier Function Destruction Drug Thrombin and Protective Drug Y-27632 on Traction Force of Human Umbilical Vein Endothelial Cells

(31) Take four Teflon well cells, two identical 9 MHz AT-cut gold electrode crystals and two identical 9 MHz BT-cut gold electrode crystals. Based on the aforementioned steps of measuring the cell traction force with bare gold electrode AT-cut and BT-cut quartz crystals, add 300 μL DMEM medium to the four Teflon cells respectively, and collect data for about 2 h; add 300 μL of mediums containing 20,000 human umbilical vein endothelial cells respectively, collect data for about 24 h, then add the drugs thrombin and Y-27632 to the final concentrations, and continue to collect data for about 24 h.

(32) Experimental Steps for Verification of the Established Methods with Drugs Blebbistatin and Nocodazole are as Follows:

(33) Based on the aforementioned steps of quantitatively measuring the cell traction force with RGD and fibronectin modified AT-cut and BT-cut quartz crystals, use the 9 MHz AT-cut and BT-cut quartz crystals modified by 50 μg/mL KRGD and 20 μg/mL fibronectin, then add 20,000 human umbilical vein endothelial cells, detect the adhesion process by QCM for about 17 h, add 1.22 μM blebbistatin or 0.5 μM nocodazole (final concentration) to the AT-cut and BT-cut crystal detection cells respectively, continue to monitor for about 10 or 5 hours, collect data, and obtain the change characteristics of the cell traction force in the cell adhesion process and under the actions of blebbistatin and nocodazole drugs.

(34) Experimental Steps for Effects of Different Concentrations of EGTA on Traction Force of Human Umbilical Vein Endothelial Cells

(35) Based on the aforementioned steps of measuring the cell traction force with bare gold electrode AT-cut and BT-cut quartz crystals, clean the 9 MHz AT-cut and BT-cut gold electrode crystals, and install the crystals into Teflon well cells. Add 400 μL of serum-free DMEM medium to the four Teflon cells respectively, and collect data for about 2 h. Add 200 μL of mediums containing 50,000 umbilical vein endothelial cells respectively, collect data for about 24 h, then add EGTA dissolved into PBS to the final concentrations of 1 mM, 10 mM and 50 mM, and continue to collect data for about 2-15 h.

(36) Dynamic Changes of Cell Traction Force During Adhesions of Rat Cardiomyocytes and Under the Treatments of Cardiovascular Inotropic Drugs

(37) Given below are dynamic QCM responses during the adhesion of rat myocardial H9C2 cells followed by the treatments of positive inotropic drug ISO and negative inotropic drug VRP detected with bare gold 9 MHz AT-cut and BT-cut quartz crystals. The results are shown in FIG. 4. The bare gold electrode achieved non-specific adhesion to H9C2 cells by adsorbing the adherent factor contained in 10% fetal bovine serum in DMEM. As shown in FIG. 4A and FIG. 4B, with the addition of ISO, the f (frequency) of the two bare gold electrodes deposited AT-cut and BT-cut crystals decreased, and R (dynamic resistance) increased. The results under the action of negative inotropic drug VRP are shown in FIG. 4C and FIG. 4D, wherein as VRP is added, QCM f increased, and R decreased. The results are consistent with those of the previous cell adhesion tests and drug experiments obtained with AT cut crystals. In addition, the dynamic changes of the surface stress or traction force ΔS applied to the quartz crystal by cells in the cell adhesion process and under the action of the drug were quantitatively determined from double resonator AT-cut and BT-cut frequency shifts based on formula (2) (see FIG. 4E, FIG. 4F). The results of formula (2) show that when ΔS is negative, the force borne by the cells is compressive stress (during cell contraction or positive inotropic effect); and when ΔS is positive, the force borne by the cells is tensile stress (during cell spreading or negative inotropic effect). Due to the limited adhesion of cells to the bare gold electrode, the result of FIG. 4 shows that ΔS fluctuates around 0, indicating that the cells are not well spread on the bare gold electrode under the experimental condition, and the cells are still contracted to some extent. Under the action of the positive inotropic drug ISO, the cell contraction is strengthened, so the cell traction force decreases and changes negatively. Under the action of the negative inotropic drug VRP, the cells are relaxed and spread, and the cell traction force increases and changes positively.

(38) Changes in Cell Traction Force Accompanying Adhesion of Human Umbilical Vein Endothelial Cells to KRGD Modified Gold Electrodes

(39) After the 9 MHz AT-cut and BT-cut quartz crystal gold electrodes are modified with different surface density of cell-specific adhesion polypeptides RGD at different KRGD concentrations (0 μg/mL, 25 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL), the QCM frequency shift response and dynamic changes of cell traction force during the adhesion of 20,000 HUVECs in DMEM media containing 2% fetal bovine serum are shown in FIG. 5, numbers 1-5 correspond to the above mentioned 5 RGD concentrations, respectively. The frequency shift response curves of AT cut and BT cut show that the quartz crystal modified at the medium KRGD concentration (50 μg/mL) has best adhesion to cells, maximum frequency shift (FIG. 5A and FIG. 5B), and maximum dynamic resistance change (FIG. 5C and FIG. 5D). The results of FIG. 5E show that the cell traction force ΔS is positive, and as time increases, ΔS rapidly increases to an extreme value at about 8 hours, and then decreases. Therefore, the cells are well spread on the RGD modified surface, the force borne by the cells is tensile stress, and ΔS is positive. Consistent with the QCM frequency shift response results, the RGD modified surface created at 50 μg/mL KRGD gives maximum cell traction force, so it is believed that the cells interact well with RGD and are well spread at the optimized RGD surface density. With the bare gold electrodes (0 μg/mL RGD concentration), the response of the sensor is minimum. The QCM responses produced by the cells on the RGD modified surface created at higher RGD concentrations (75 μg/mL and 100 μg/mL) are medium, which may be caused by the fact that the interaction of cells with the QCM sensor at higher RGD concentrations is weaker than that created at the 50 μg/mL RGD of the optimal adhesion effect on cells because the orientation of RGD is affected by steric hindrance.

(40) Changes in Cell Traction Force Accompanying Adhesion of Human Umbilical Vein Endothelial Cells to Fibronectin Modified Gold Electrodes

(41) FIG. 6 shows the QCM frequency shift and dynamic resistance response of 20,000 HUVECs cell adhesion and dynamic changes in cell traction force in DMEM media containing 2% fetal bovine serum, after the 9 MHz AT-cut and BT-cut quartz crystal gold electrodes were modified at different fibronectin (FN) concentrations (0 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL). FIG. 6 also shows the results contrasts of the bare gold electrodes, numbers 1-6 correspond to the above mentioned 6 FN concentrations, respectively. It can be seen that the QCM frequency shift and the dynamic resistance change caused by adhering 20,000 HUVECs to the bare gold electrode are minimum, indicating that the adhesion to cells is the weakest at this time, the cells are not well spread and are mainly contracted, and the cell traction force ΔS is negative. However, as the adhesion time increases, the surface of the electrode may adsorb adherent factors beneficial to cell adhesion and extracellular matrix factors secreted by the cells, so that ΔS changes positively, and the cells are gradually spread and are finally close to the value of the cell traction force on the gold electrode modified at the low FN concentration (10 μg/mL). After the gold electrode is modified with FN, the cell traction force ΔS is positive, indicating that the cells are spread well and apply compressive stress to the crystal, that is, contractile traction force. By comparing the responses of the gold electrode ΔS modified at the six FN concentrations (FIG. 6E), the results show that, like the RGD situations, ΔS is maximum and stable at about medium concentration (20 μg/mL), and the QCM frequency shift and dynamic resistance response are also maximum. At the low FN concentration (10 μg/mL) and the highest FN concentration (50 μg/mL) tested, the cell traction forces are close and the least, and the corresponding QCM frequency shift and dynamic resistance change response are also the least. The QCM frequency shift and dynamic resistance change responses at the higher FN concentrations (30 μg/mL, 40 μg/mL) are medium, and the corresponding ΔS response increases fastest at the beginning, but fluctuates with time and attenuates to some extent, so the final stable value is lower than the ΔS value at the medium concentration (20 μg/mL).

(42) Dynamic Responses of Cell Traction Force Under the Actions of Drugs Blebbistatin and Nocodazole

(43) In order to verify the established piezoelectric cell force sensing method, we investigated the QCM response under the action of a myosin II inhibitor blebbistatin by using the quartz crystals modified at the 50 μg/ml RGD concentration. FIG. 7 shows that the cell traction force ΔS decreases under the action of blebbistatin. Blebbistatin is a non-myosin type II atpase (ATP) inhibitor. The result here is consistent with the conclusion reported in other methods of the literature that blebbistatin reduces the cell traction force. In addition, we also investigated the effect of a microtubule inhibitor nocodazole on the mechanical properties of cells, indicating that under the action of 0.5 μM nocodazole, the cell traction force increases at the beginning and then decreases (FIG. 8). Microtubules, as a rigid structure in the cytoskeleton, exert compressive stress to cells, and determine the cell force balance together with cytoskeletal actin filaments exerting tensile stress to cells. The microtubule inhibitor nocodazole depolymerizes the microtubules, so the cell traction force increases in the initial phase. As the acting time of nocodazole increases, the intracellular rigid microtubules are further lost, cell contraction and focal adhesions decrease, resulting in a decrease in the cell traction force. This is consistent with the results reported by the cell traction force microscopy in the literature.

(44) Effects of Vascular Endothelial Barrier Function Modulation Drugs Thrombin and Y-27632 on Cell Traction Force

(45) FIG. 9 shows curves of changes in frequency, dynamic resistance and cell traction force of 9 MHz AT-cut and BT-cut crystals caused by vascular endothelial barrier destruction drug thrombin and protective drug Y-27632 acting on human umbilical vein endothelial cells. It can be seen that under the action of thrombin, the frequency shift of the crystals decreases and the dynamic resistance increases slightly, indicating that the cell adhesion is enhanced and the cell traction force increases. Under the action of Y-27632, the frequency shift of the crystals decreases and the dynamic resistance decreases, indicating that the cell adhesion is weakened and the cell traction force decreases. The vascular endothelial barrier destruction reagent thrombin is a cytoskeletal contraction agonist that increases the cell traction force and the cell permeability. The role of the vascular endothelial barrier protective agent Y-27632 is opposite. The Y-27632 is a Rho kinase inhibitor and a cytoskeletal relaxant, affects cortical myosin activity and decreases actin-myosin activity, and has the functions of reducing the cell traction force and maintaining the permeability. The results of the two reagents measured by the double resonator QCM technology are consistent with their functions and the results of cell traction force measurement reported in the literature.

(46) Effects of EGTA of Different Concentrations on Traction Force of Human Umbilical Vein Endothelial Cells

(47) FIG. 10 shows the dynamic QCM responses during adhesions of 50,000 human umbilical vein endothelial cells to bare gold 9 MHz AT-cut and BT-cut quartz crystals and the subsequent actions of different EGTA concentrations. It can be seen that after 50,000 HUVECs are added, the QCM frequency decreases and the resistance increases. After 24 hours, the QCM frequency decreases by 300 to 400 Hz. In all experiments, except for the initial phase, the frequency shift of the BT-cut crystal is always greater than the frequency shift of the paired AT-cut crystal at the same time, so the surface stress ΔS applied to the electrode by cells in the adhesion process is positive. As the cells are spread, ΔS increases rapidly and then becomes stable. After 24 hours, ΔS reaches 115,000 to 175,000 dyne/cm (FIGS. 8G-I). After different concentrations of EGTA are added, the overall cell traction force shows a tendency to decrease, indicating that the cells are desorbing at this time. The cell morphology simulation experiments show that the cell spreading area observed became smaller after the EGTA treatments for five minutes, and the cells were retracted into ellipses. With the increase of EGTA time, except for the low 1 mM EGTA concentration, the cell generated force decreases, and decreases more quickly at 50 mM EGTA concentration than 10 mM EGTA concentration. This is consistent with the result of cell morphology simulation showing that the cell adhesion area is reduced more quickly with the increase of the EGTA concentration. Under the action of the low 1 mM EGTA concentration, the cell traction force in the initial phase did not decrease but increase slightly, and then the decreasing trend became consistent with those of the other EGTA concentrations tested. The integrins for cell-matrix interactions and the E-cadherins for cell-cell interactions are closely related to Ca.sup.2+ concentrations. It is therefore expected that the EGTA chelated with Ca.sup.2+ affects the dynamic adhesion and force balance of cell-matrix and cell-cell. Under 50000 endothelial cells, the short intercellular distance causes strong intercellular force. Therefore, we speculated that at the low 1 mM EGTA concentration, the EGTA acts mainly between cells, initially causing a decrease in cell-cell interaction, which in turn leads to an increase in cell traction force between cells and sensor matrices, and then a decrease in cell traction force between cells and the substrate.

(48) The foregoing description of the exemplary embodiments of the present invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

(49) The embodiments were chosen and described in order to explain the principles of the invention and their practical application so as to activate others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.