The overall goal of the following experiment is in vitro monitoring of cardiac beading clusters derived from human embryonic stem cells in 3D real time and high spatial resolution. This is achieved by first culturing and expanding embryonic stem cells until they form Embry bodies. Next, the beating clusters are dissected and plated on POLYTETRAFLUOROETHYLENE or PTFE porous filters, which allows them to be maintained as long-term air.
Liquid cultures then beating clusters are labeled with harmonic nanoparticles in order to perform realtime multi photon microscopy. Results obtained allow the analysis of spatial elongation and frequency of cardiac contractions at the microscopic scale. So the main advantage of these nanoparticles is that you can use any wavelength, especially infrared, so you can go deeper in tissue and they are also very stable over time.
So we can monitor our long processes over long timeframes. This stem cell culture method can help to answer key questions in the field of cardiomyocyte functional contraction in three dimensions. In particular, cardiac maturation and tissue engineering.
Individuals new disease method should be aware that the labeling procedure with harmonic nanoparticles can only be used for multiphoton microscopy and cannot be transferred to other kind of imaging systems. We first had the idea of using harmonic nanoparticles in 2005. These nanoparticles present advantages with respect to fluorescent dyes and quantum dots.
In fact, their signal doesn't fade out over time and they can be used to monitor developing samples over long time periods. Visual demonstration of the sample preparation is critical for the efficiency and the reproducibility of the different steps, in particular when performing drug testing and could also be applied to different tissues or disease. Tissue modeling.
After culturing and expanding human embryonic stem cells or ESCs according to the text protocol and incubating them with warm collagenase, four for 10 minutes, collect the colony fragments in two milliliters of fresh differentiation, medium or DM and centrifuge at 115 times gravity for three minutes. Culture, the colony fragments in suspension in ultra low attachments. Six well plates for four days to allow them to form embryo bodies or ebs.
Then plate the newly formed EBS on 0.1%Gelatin coated 24 well plates for 15 to 30 days until the appearance of beating clusters. Remove the PBS and using acutus, dissociate the colonies into single cells after centrifuging and resus, suspending the cells in DM medium plate two milliliters per agro. Well chamber and incubate at 37 degrees Celsius for one day.
Collect the newly formed EBS and plate three per well on 0.1%gelatin coated 24 well plates after two to four weeks in culture. Identify clusters of beating cardiomyocytes and using an elongated sharply cut paste pipette, manually dissect them. Deposit four PTFE filters per insert and place each in a well of a six well plate.
After adding one milliliter of DM into each, well add one dissected beating cluster onto each PTFE filter. To label the clusters, transfer the PTFE filters with the beating clusters into a 3.5 centimeter glass bottom dish. Add one milliliter of second harmonic generation nanoparticles at 50 micrograms per milliliter and incubate for 30 minutes at 37 degrees Celsius to carry out non-linear optical imaging.
After transferring labeled HEBs or cardiac beating clusters to an imaging chamber, use wide field imaging under white light illumination to identify structures of interest within differentiated EBS or active beating clusters. Perform a fast low resolution three-dimensional scan to rebuild the overall morphology of the cardiac cluster and select an image plane within the cluster volume with several visible second harmonic generation nanoparticles to monitor in real time. The cardiac cluster contractions set the microscope optical scanner in the fastest mode, allowing multi hertz acquisition of two dimensional images.
Analyze the images using the MATLAB software according to the text protocol as demonstrated here prior to assessing the beating activity. Characterization of the nonlinear optical response of the PTFE filters is performed to ensure that the bare substrate to photon fluorescence is very weak and cannot prevent measuring the relevant biological samples and the emission from isolated. Second harmonic generation nanoparticles can be easily acquired by imaging through the substrate in epi detection mode.
In this figure, second harmonic generation nanoparticles labeled cardiac structures and EBS are displayed blade. The red, yellow and green colors correspond to NADH autofluorescence while the intense blue second harmonic spots stem from isolated second harmonic generation nanoparticles. Note the good optical contrast and the relatively sparse labeling as compared to quantum dots or up conversion nanoparticles.
This figure illustrates a slice view of a second harmonic generation nanoparticles labeled cardiac beating cluster. As shown in this movie, the 3D structures are recorded at high speed to monitor the contraction pattern In the second cell. Harmonic generation nanoparticles aggregate to maintain a high acquisition speed rate for resolving the NP motion.
The overall acquisition sensitivity was not sufficient to record cell autofluorescence with second harmonic emission from second harmonic generation. Nanoparticles image analysis performed on movie frames indicates that within the same cardiac cluster displacement are on the order of a few micrometers, and the frequency is constant for in and out of plain second harmonic generation nanoparticles motion. When master the cell preparation technique, the betting cluster dissection the air liquid culture and the lambing can be done in 24 to 36 hours if performed properly.
Especially with this technique, open air contacts have to be avoided to prevent contaminations. Following this procedure, one can apply other methods like electrophysiological recordings using multiple electrode arrays in order to answer additional questions like correlating action potential profiles with beating frequency in three dimension. Our novel technique based on the detection of the nonlinear signal by harmonic nanoparticles can be further developed for in vivo Studies infect the wavelengths used for imaging allow deeper penetration in tissues.
One possible development could be following the integration of stem cells derived structure for regenerative medicine applications.
