Cell generated mechanical forces are essential to proper function in various organs throughout the body. Plus the intestines bladder, heart, and others. These organs must generate stable patterns of cell contraction and relaxation, to maintain the internal hemostatic state.
Abnormal smooth muscle cell contraction can lead to the onset of various disorders, including intestinal dysmotility characterize abnormal patterns of intestinal with muscle contraction, as well as conditions like overactive or underactive bladder. And in the airway, some muscle cells that contract any irregular patterns can trigger asthmatic hyper responsiveness. Clearly, contractile mechanisms within cells and tissues can lead to diseases that require treatment options.
And as these can conditions directly stem from dysfunctional contractile behaviors of cells, it becomes both logical and necessary to measure cell contractile function itself, when screening potential drug candidates. Following recent advances in micro technology we developed a user friendly micro plate based assay that enables quantitative measurements of single cell contraction. In hundreds of thousand of cells called flex, also known as fluorescently labeled elastomeric contractable surfaces.
In this approach, we embed fluorescent protein micro patterns, and so soft films, which deform and shrink when cells apply traction forces to them. Importantly, the protein micro patterns constrain cell position, shape and spread area. The uniform test conditions and allow simple measurements based only on their dimensional changes.
Overall, the platform, which includes a browser based image analysis module, enables straightforward analysis of contractable cell force without requiring delicate handling procedures or registration of fiduciary markers and can be operated by any researcher with the basic cell cultures and simple fluorescent microscope with low magnification. This technology be designed with the end user in mind, and aims to reduce the barrier to entry for any laboratory scientist to study cellular force biology Begin by adding 20 milliliters of media into a chronical tube, obtain a 24-well plate designed to assay cell contractility. Set your pipette to 500 microliters and obtain a cell strainer for cell pathogen.
Remove the lid on the 24-well plate, hold up the plate as so, then proceed to gently take off the layer of plastic on top of the plate. Carefully set the plate back down. Aspirate just top layer PBS from each well to prevent any spillage.
Now, one row at a time, remove the remaining PBS from the well and quickly fill with 500 microliters of cell media. Shake the plate gently, and tap on its side to ensure the entire bottom of the well is covered with solution. Once all the Wells have been filled with media, set the plate to the side.
At this point, retrieve your cell culture from the incubator. We will be conducting a solid association protocol. Purpose of this protocol is to create a suspension of 50, 000 cells per milliliter.
Prior to seeding the cells make sure to strain your cells once through the strainer, in order to break up clumps of cells into single cells, carefully place 500 microliters of your cell solution into each well. After cell seeding, it is important to let the plates sit for an hour so, that the cells can settle directly onto the patterns. After an hour has passed, place the plate into an incubator overnight.
The second portion of the experiment consists of adding the drugs to the 24-well plate and fluorescent imaging of the plate. For this portion of the protocol we have two important requirements to ensures robust cell viability and response. The first is that the final concentration of DMSO in the wells with cell is no more than 1%meaning that we need to make a 100 full dilution from our drug stocks.
The second is that we cannot add DMSO so directly to the wells because it will crash to the bottom of that mixing, and then harm the cells with a high level concentration. Instead, we must create an intermediate solution of DMS0 drug and media, that we can thoroughly mix together to avoid high local DSSO exposure to the cells. In this protocol, we will be doing a six step eightfold dilution that covers a range from 40 micromolar down to one Nano molar Create the six step eightfold dilution series by transferring 30 microliters of stock drug solution into consecutive 210 microliter volumes of DMSO and thoroughly mixing between each transfer step In our experiment, we will be evaluating the effect of blebbistatin.
on human bladders through the muscle cells. In order to meet both requirements, we will first create an intermediate drug DMSO and media solution. That yields a 16.7 fold dilution of DMSO.
Then we will add this intermediate drug solution to our cells, using proportions yielding an additional sixfold dilution, resulting in a total 100 full dilution and the final concentration of 1%DMSO To accomplish this, for each stock drug solution, mix 30 microliters of drug into 470 microliters of cell culture medium, then transfer 200 microliters of this intermediate solution to the appropriate well on the 24-well plate, which contains 1000 microliters in each well. This collectively yields a 1%final concentration of DMSO Incubate for the appropriate amount of time. In our experiment, we incubate for 30 minutes, when you're ready to image add a live nuclear stain all over your Wells add a one to 10, 000 dilution from the stock.
Allow it to incubate for an additional 15 minutes. When you're ready to image, you will need a fluorescent microscope that is equipped to image the fluorescent channels for both the labeled cell nuclei and the fluorescent dye. The micro patterns are labeled with which in our example is the tri C channel.
Now, we will be fluorescent imaging blebbistatin nuclei in order to identify single cells, be sure to image the stain cell nuclei in the exact same position as you view the micro pattern so that they can be aligned during analysis. Upload the acquired images to your computer ensure that the images are labeled properly, such that the pattern channels end in underscore PT.And images in the nuclear channel end in underscore dapi. Now, we will be converting the tif images to PNG images, using image J Once image J is open, load in one channel at a time.
We will first be loading the pattern channel and adjusting the contrast, in order to maximize the signal, and to minimize the background. In addition, we'll be smoothing out the image. Once the image has been modified to our liking, we will then change the image type to eight bit.
Then export the image as a PNG type. Then check the box, use slice labels as file names. Create a new folder called PNGs.
Then save the PNG files there, To analyze the images, navigate to Biodock dot AI, create an account and contact the authors to gain free access to the analysis module. Log in once your account has been created. Here you'll be able to upload new images.
We will call this new batch, web experiment data JoVE. Now, we can import the images by dragging them and then dropping them, press OK.At this point, select your data. Then click analyze, scroll down to the module labeled contractility analysis then hit select.
Set the magnification to the same value that you used for imaging Once the data is complete, click on it then we can select download data. Once the data is downloaded, we can then see overlayed image pairs for every inputted image. We also have access to a summary statistic that will give us the average contraction for each set of cells.
The unit of measure for contraction is in pixels. Looking at the data we can see here that in the wells treated with DMSO only, there was a much higher contraction of cells in comparison to those cells that were treated with blebbistatin. We also have access to data for every single pattern, that was detected in any of the images.
This gives us detailed information regarding the location of the image, the image origin, the position type, the number of cells, either zero one or more than one, the size of the micro pattern, and the calculated contraction of the cells. This lay consists of every single identified pattern within the micro plate. Single cell patterns from this list were average to calculate the average contraction for the PNG image in the other file.
You can sort through and analyze the single cell data as needed for your experiment. For this portion of the video, we will be displaying representative data that are collectible from a 24-well plate showing those response data from bladder smooth muscle cells treated with two different drugs. These are images of wells treated with DMSO and blebbistatin respectively.
Here, we can see that the cells treated with DMSO only, display a large number of contract micro patterns but cells treated with blebbistatin were larger and open to noting that there is less contraction. The data in this histogram displays the variation cell contractility as a result of the different treatments to the cells. The center of distribution for cells treated with blebbistatin is lower than the center of distribution for cells that were treated with the DMSO.
This is consistent with the information displayed in the previous images. Because the cells that were treated with blebbistatin exhibited much smaller contraction. This denotes that treatment with blebbistatin significantly relaxes the cells.
Each of these sets of data contributes to a point on the dose response curve that is collectible from a single 24-well plate, following the protocols shown in the video. Here, we can see that cytochalasin D is more potent than blebbistan as shown by the lower values of contraction, given the higher concentrations of drug. If you wish to significantly scale your experiments, a 384 well plate version is available, and this can be used with automation to dramatically scale the experiments.
This data is collectible from a 384 well plate, rather than having six dilution steps for each drug on the 24-well plate, the 384 well-plate allows us to conduct 20 dilution steps for each drug with multiple replicates. Snowflakes technology is unique. It allows for visualization of single-cell contraction using microscopy, which has been impossible so far.
So, technology relies on the form of metrics that is patterned with fluorescently labeled protein. Systematic pattern is to form for cellular contraction and allows for precise measurement of the modulation of a contract of phenotype by any given drug. Of course, this technology is still being actively developed for applications and many different disease areas, such as asthma, cardiovascular diseases, inflammation immune oncology, and of course, any of our disease indication where abnormal cellular contraction plays a key role in the disease progression.
As we demonstrate this micro patterning based technology for measuring cell contractility offers a simplified alternative to traditional traction force microscopy, providing intuitive analysis, watching the pattern shrink, and providing single cell resolution in large cell populations. Some considerations, this method may pose challenges for use of cells that are either too small, such as T-cells and neutrophils, or cell types that do not adhere. In addition cells that weekly bind, bind to each other predominantly or that do not completely spread, will not produce measurable contractile signals.
Users of the technology must carefully evaluate different possible cell culture medium formulations for their particular cell type. as different components, growth factors, serum levels and pH sensitivities may drive variable behaviors. Optimization of the protocols should proceed scaling of any experimental workflows.
Ultimately if single cell resolution is not necessary, or if the target cell type has minimal spreading capacity, then traditional attraction force microscopy methods can be employed for such experiments. Otherwise we hope this tool provides an additional avenue for cell biologists to study cellular contraction and perform their studies.
