Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The aim of the following experiment is to measure the contractile activity of cultured muscle fibers in response to stimulation in a controlled in vitro environment. This is achieved by plating dissociated muscle cells on microscale silicon cantilevers, and inducing the fusion of these mono nucleated cells to form fibers termed myo tubes. These cantilever chips supporting differentiated muscle cells are then transferred to an electrophysiology microscope modified to support a laser and photo detector used to measure cantilever deflection in response to muscle contraction after placement of the chip in the rig.

The laser is positioned so that it focuses on the tip of the cantilever and the photo detector is likewise aligned so that it catches the deflected beam Results are obtained that show the degree of cantilever deflection, which is caused by muscle contraction in response to electrical or chemical treatment, providing a direct measurement of the contraction profile of the seeded cells in real time. Ultimately, cantilever displacement can be processed to provide a readout of force. This system is multiplexed to provide a potential high throughput test bed for assessment of changes in contractile function in response to drug treatment, disease, state, or exercise protocols.

The main advantage of this technique compared with existing methods such as patch clamp electrophysiology, is that this method for functional analysis is non-invasive and easily adaptable for high throughput studies. This method can help answer key questions in the drug development field, such as identification of appropriate dose response concentrations prior to clinical evaluation. The Implications of this technique extend toward therapy of multiple muscular diseases because it provides means to investigate the functional performance of cells from different pathological phenotypes In real time, though, this method can provide insight into skeletal skeletal muscle pathologies.

It can be also be used for other tissue systems such as cardiac and smooth muscle Sterilized, previously prepared cantilever chips and 13 F cover slips in 70%ethanol solution and allow to air dry in a flow hood. Then place individual cantilever chips on top of 13 F cover slips. Inside a standard 12 well plate coat the cantilevers with the biopolymer or surface modification optimized for the cell type being used According to standard cell culture protocols, surface coating takes 30 minutes using our preparation method, but maybe altered depending on the investigator's specific culture protocol.

Reese has been the cells in their specific growth medium to the desired concentration, then hundred microliters of the cell suspension onto the cantilever chip surface, ensuring the bubble of medium covers the cantilever windows entirely. Transfer the plate containing the chips to an incubator and allow the cells to adhere for at least one hour after this plating period. Use sterile forceps to transfer the chip to a clean well without a 13 F cover slip and add one milliliter of growth medium to each.

Well return the plate to the incubator, maintain cells according to their standard protocol for in vitro maintenance. On cover slips, place a heated culture dish onto the stage of an upright electrophysiology microscope. Add three milliliters of the current cell feeding medium to the heated microscope.

Stage mount stainless steel electrodes on the inside of the heated culture dish at a separation distance of 15 millimeters. Connect them to a pulse generator capable of producing field stimulation, pulses of varying intensity, frequency and waveform to allow the system to produce field stimulation of cells when appropriate. Bolt a helium neon laser mounted on XY translational stages to the underside of the microscope table.

Then adjust the laser so that the laser beam is directed through the base of the heated culture dish at a 30 degree angle relative to the plane of the cantilever. Next bolt, a quadrant photo detector module mounted on XY translational stages to the underside of the microscope stage. Adjust its position so that the reflected laser beam lands in the center of the four quadrants.

Write a software program to control the linear actuators that scan across the cantilevers with reference to the flow chart provided in the text protocol. Turn on the cantilever analysis hardware and associated software. Insert the heated stage ther mrta into the medium and wait for it to read 37 degrees Celsius.

Next, insert the cantilever chip into the stage with the cantilevers oriented towards the right hand side of the stage. Turn on the microscope light source. Focus the microscope to bring the edges of the cantilevers into view and use the laser photo detector control software to position the laser beam on the tip of cantilever one, assuming the cantilevers are orientated to the right of the stage cantilever.

One is the one positioned in the top left of the array and the numbers run down to 16. In the bottom left. Cantilever 17 is in the top right position and runs to 32.

In the bottom right press play on the recording software. Position the photo detector so that the signal reads zero in both the x and y frames by adjusting the stepper motors controlling the photo detector. Then set the cantilever one position in the laser photo detector control software.

Next, move the laser to the tip of cantilever 16. Repeat the positioning of the photo detector and set the cantilever 16 position in the laser photo detector control software. Now move the laser to the tip of cantilever 32.

Repeat the positioning of the photo detector and set the cantilever 32 position in the laser photo detector control software. Finally, move the laser to the tip of cantilever 17. Repeat the positioning of the photo detector and set the cantilever 17 position in the laser photo detector control software.

Turn off the microscope light source and then the overhead light in the laboratory. Press record on the recording software. Set the pulse generator hardware to 40 millisecond and five volt pulses, a frequency of one hertz.

Then turn the machine on Using the laser photo detector control software, set the hardware to scan across the 32 cantilever array. Stopping for five seconds at each one. When the scan of the 32 cantilevers is complete, turn off the stimulator.

Then stop the recording software and bring up the data file. Examine the recorded trace from each cantilever. For evidence of contractile activity.

A contraction is defined as a peak. If the deflection is at least 0.1 volts above the baseline, make a note of each cantilever with positive responses. Remove any non-responsive cantilevers from the scan protocol on the laser photo detector control software.

The active cantilevers can then be re-scanned without stimulation in order to get a reading of the cell spontaneous contractile activity. Next, add a therapeutic compound to the medium to observe its effect on the functional output of the cultured cells. Following the addition of compound run scans with or without broad field electrical stimulation carry out fatigue assessments by electrically stimulating the cells for extended periods, followed by scanning levels of contractility to measure how long it takes for peak force to drop below a specific threshold.

In experiments where motor neurons are maintained in co-culture with muscle measure neuromuscular junction formation through treatment of motor neuro myo tube cantilever co cultures with a neuronal stimulant or synaptic inhibitor and scanning for increases and decreases in spontaneous activity, proceed to analyze cantilever deflection data as detailed in the text protocol. Successful culture of contractile cells on cantilevers is a relatively straightforward procedure using standard cell culture techniques. Standard electrophysiological software can be used to analyze the raw data facilitating calculation of relevant functional properties, such as peak force time to peak force, and time to half relaxation.

Extended stimulation protocols provide the means to assess rates of fatigue in cultured cells, thus broadening the level of physiological data obtainable from this system. The cantilever culture system can be modified to include motor neurons in the culture system with skeletal muscle myo tubes, so as to allow assessment of neuromuscular synapse formation in vitro. In such cultures, rates of spontaneous contractile activity are compared to rates of contraction in response to treatment with a neuron specific stimulant such as glutamate.

Any observed glutamate induced increases in contraction rates suggests the activation of cultured neurons leading to acetylcholine release and subsequent myo tube activation Treatment with synaptic inhibitors such as the acetylcholine receptor blocker, dtu rine that lead to cessation of glutamate induced activity provides further evidence for the presence of functional neuromuscular synapses in these cultures. After watching this video, you should have a good understanding of how to use microscale silicon cantilevers to assess the functional properties of cultured skeletal muscle.