The aim of the following experiment is to generate aligned functional myocardial tissue through micro contact printing. This is achieved by first micro contact printing fibronectin onto poly dimethyl siloxane substrates. As a second step, double transgenic.
Embryonic stem cell lines are differentiated in vitro and isolated by means of fluorescence activated cell sorting, which allows highly purified populations of strongly cardiogenic progenitors to be obtained. Next, isolated cardiac progenitors are seeded onto micro patent PDMS substrates to make them malign and take a cardiac myocyte like rod shape. Results are obtained that show the successful isolation and an isotropic growth of cardiac progenitors based on facts and immunofluorescence analysis.
Generating an isotropic myocardial tissue can help advance in the fields of stem cell biology and tissue bioengineering by raising the possibility of developing disease specific cell resay for drug development and discovery. And by laying the foundation for cardiac regenerative medicine. First IL guard, 180 4 poly dimethyl alane elastomer at a 10 to one based acuring agent ratio and mix well.
Degas the assembly using a desiccate to eliminate any air bubbles. Pour the PDMS into a previously fabricated master with 20 micron wide and two micron tool ridges separated by 20 micron Spacing, then cure for two days in a desiccate at room temperature to obtain micro textured PDMS stamps. After rendering the PDMS stamp surfaces hydrophilic with a plasma cleaner.
Sterilize the stamps with 70%ethanol for one minute. In a biological safety cabinet, dry quickly with compressed air. Cover the sterile stamp surfaces with fibronectin solution to absorb for at least 10 minutes after absorption.
Shake off the fibronectin solution from the stamps and dry quickly with compressed air. Establish conformal contact between stamp and previously prepared PDMS coated glass cover slips as described in the text protocol for two minutes and press firmly. Rinse micro pattern substrates with distilled water.
Store them in distilled water for a maximum of two weeks of four degrees Celsius unless they're to be used immediately. Coat six well plates with sterile 0.1%gelatin in distilled water and let it absorb for 15 minutes 37 degrees Celsius During this time, prepare the mouse embryonic fibroblasts from ahor aliquot by adding them to 50 milliliters of PBS in a conical tube. Centrifuge the meth at 1000 RPM for five minutes and re suspend them in meth medium.
Once the incubation time has elapsed, aspirate the excess gelatin and add the mes into the wells. Allow the MES one day to attach. Prepare the embryonic stem cells or ES cells from a thought aliquot by adding them to 50 milliliters of PBS in a conical tube Centrifuge.
The ES cells 1000 RPM for five minutes and Resus suspend them in ESC medium. Add the E ES cells to the wells containing myths and maintain in ESC medium until confluence is reached to massage cofluent ES cells. First aspirate the ESC medium and wash the cells once with sterile PBS.
Then aspirate the PBS and add 500 microliters of trips into the, well. Incubate the plate for three and a half minutes at 37 degrees Celsius. Pipette the TRIPSIN solution up and down several times.
First to break down the E es cell colonies. Then neutralize the tripsin solution by adding 500 microliters of ESC medium to the well. Massage the ES cells according to the required ratio here the E ES cells are passaged at a ratio of one to 30, which allows them to reach confluence within three days.
Begin the in vitro differentiation when the ES cells have reached confluence. First coat 10 centimeter culture dishes with sterile 0.1%gelatin in distilled water and let it absorb for 15 minutes at 37 degrees Celsius. During this time, harvest the ES cells as before.
Once the 15 minutes have elapsed, aspirate any excess gelatin and add approximately one third to half of the E ES cell suspension into cultured dishes containing adaptation medium. After culturing the cells in adaptation medium for two days, harvest the adapted ES cells into a 50 milliliter conical tube containing PBS using 1.5 milliliters of trypsin. Centrifuge them at 1000 RPM for five minutes and re suspend them in differentiation medium.
Make embryo bodies or EBS of approximately 1000 cells per 10 microliter drop of differentiation medium in 15 centimeter culture dishes. Using a multi-channel pipette, invert the plates and culture. EBS is hanging droplets for two and a half days at 37 degrees Celsius.
After two and a half days, pull the EBS from six to eight 15 centimeter dishes into one using differentiation medium and culture them for further three and a half days. After a further three and a half days, collect the EBS in a 50 milliliter conical tube and wash the plate once with sterile PBS to collect all of the ebs. Then let them sink to the bottom for approximately five minutes.
Then remove the supinate and wash the EBS with sterile PBS. Let the EBS sink to the bottom again for approximately five minutes. Dissociate the EBS by adding 2.5 milliliters of trypsin and shake the tube softly in a water bath at 37 degrees Celsius for two minutes.
Then add 2.5 milliliters of sterile PBS to the tripsin solution and pipette up and down with the 10 milliliter serological Pipette approximately eight to 10 times to break down cell clusters. Neutralize the trypsin with 10 milliliters of fax buffer containing 7.5%E ES cell or differentiation grade FBS and 0.01%DPI in PBS centrifuge, dissociated EBS at 1000 RPM for five minutes. Then remove the supinate and add approximately one milliliter of fax buffer pipette up and down with a one milliliter pipette approximately eight to 10 times to break down cell clusters.
Transfer the cells to a sterile round bottom tube with a 35 micron cell strainer to obtain a single cell suspension. Sort differentiated ES cells using a aria flow cytometer and obtain purified populations of committed ventricular progenitors. See the text portion of the protocol for the detailed gating strategy.
Ate the micro pattern substrates with 1%onic F1 27 in distilled water for 10 minutes. Then wash them three times with sterile PBS. Attach PDMS gaskets around the pattern region and press firmly.
Then seed cardiac progenitors onto fibronectin micro pattern substrates. The following images are from a representative flow cytometry chart of day six in vitro differentiated ES cells. This image shows gating on forward scatter amplitude FSCA and side scatter amplitude SSCA.
This allows for the separation of whole cells from cellular debris. Here gating on FSCA and forward scatter width FSCW is seen. This allows for the isolation of cell singlets from doublets and other cellular aggregates.
This image shows gating on SSCA and scatter with SSCW. This increases the purity of cell singlets. This image shows gating on FSCA and DPI staining that allows for the isolation of viable DPI negative cells from non-viable cells.
Here gating for fico, rine, PE and PE cyan in DI seven PE SI seven is seen. This allows true red positive and true red negative cells to be obtained and autofluorescent red negative cells to be excluded. These images show gating for fluorescein isothiocyanate amplitude fit CA and PEA that allows true green positive and true green negative cells within red positive and red negative populations to be sorted.
Fax purification of in vitro differentiated ES cells reveals four distinct populations of progenitors as seen from this flow cytometry plot. The four distinct populations of progenitors are red, positive green, positive red, positive green, negative red, negative green positive, and red negative green negative. This representative immunofluorescence microscopy image shows micro patterned fibronectin substrates.
The scale bar represents 80 microns. This image shows representative immunofluorescence microscopy of embryonic derived facts, isolated progenitors in the top panels and ESL derived facts, isolated progenitors in the lower panels after an additional five days of culture on fibronectin micro patterns nuclei stained blue. S-M-M-H-C-A stained red sarcomeric alpha actinin appears green.
The scale bar is 40 microns. After watching this video, you should have a good understanding of how to generate anisotropic functional myocardial tissue from a renewable cell source of cardiac protus.
