The overall goal of this procedure is to isolate human fetal pancreatic cell clusters. This is accomplished by first cleaning and coarsely chopping the pancreas. Next, the pancreatic tissue is digested with collagenase.
Then the digested aggregates are resuspended and plated in Petri dishes. Finally, the cells are plated on HTB nine matrix or incubated as aggregates, and an experiment is performed. Ultimately, R-T-P-C-R.
Western Blood Analysis or immunofluorescence microscopy are used to show changes in gene or protein expression or localization. The implications of this technique extend towards the therapy of Type one diabetes because a number of critical biochemical processes that regulate the transition of human pancreatic precursor cells into mature glucose responsive insulin secreting cells in vitro remains unknown. Generally, individuals new to this method will struggle because of incomplete or over digestion that leads to poor formation of fetal pancreatic cell clusters.
Demonstrating this procedure will be Lopez, a technician from my laboratory To begin dissecting the fetal pancreas aspirate liquid and transfer the pancreas to the 60 millimeter dish with forceps without HBSS and remove any visible splenic fat or connective tissue. Use about half a milliliter of HBSS to rinse the pancreas before using forceps to transfer it to a small sterile crucible and into an ice bucket. Place the crucible in a holder for a 50 milliliter centrifuge tube and with two pairs of scissors vigorously slice the pancreas for several minutes by holding one side of the scissors in a fixed position and moving only the opposite handles while resting the tips of the scissors on the bottom of the crucible at 1.5 milliliters of collagenase and the chopped up pancreas to a vial containing collagenase that was prepared.
According to the text protocol and place in a water bath shaker set to 37 degrees Celsius at 200 RPM for up to 10 minutes. Do not check more frequently than once during the first five minutes. Digestion time depends on the tissue quality as little as six minutes may be sufficient.
The endpoint is when the particles are small and uniform. To stop the collagenase activity. Use cold HBSS to fill the vial, then place on ice and allow the clumps to settle for 10 minutes.
After the incubation aspirate off the top layer, leaving it slightly less than half full. If strings of DNA are present due to cell death at 100 microliters of 10 milligrams per milliliter, DNAs to the solution, then transfer the cells to a 15 milliliter conical tube and add five milliliters of HBSS centrifuge for five minutes at 300 gs. After aspirating the HBSS re suspend the cells in five milliliters of medium containing RPMI 1640 glutamate, 10%normal human serum, 10 millimolar heaps, pH 7.2, penicillin streptomycin, and fungi zone.
Then plate the cells in a 60 millimeter Petri dish and incubate the cells in suspension at 37 degrees Celsius for 72 hours to aggregate and form iccs. After the incubation plate, the cells on the matrix of human cell line HTB nine as previously described, change the growth medium every 48 hours to carry out immunofluorescence on adherence cells. We remove the culture medium and wash the cells with PBS.
After washing the cells once with 10 milliliters of PBS, add 4%PFA to fix the cells for 20 minutes at room temperature and then use PBS to wash the cells twice. Next, after incubating the cells with 0.2%tritton X 100 in PBS for 10 minutes at room temperature and washing the cells twice with PBS, incubate the cells in blocking buffer for one hour, pipette 60 microliters of the primary antibody solution onto perfil that has been placed in a sealable container. Then remove the cover slip from the 12 Well plate and blot off extra blocking buffer before placing it on top of the primary antibody drop.
Incubate the sample for 90 minutes at room temperature. After washing the samples three times, place a drop of fluorescent secondary antibodies onto a piece of paraform with PBS. Rinse the samples and use tissue paper to blot the excess PBS before placing it on the drop of antibody solution.
Shield the sample from light and incubate for one hour at room temperature after washing the samples and blotting as before, add a drop of mounting gel and invert the cover slip onto a slide. Use aspiration to remove any excess mounting gel and the back of a 200 microliter pipette tip to gently tap out any air bubbles. Allow the samples to dry for 20 minutes and examine under a fluorescent or confocal microscope.
Careful preparation of fetal iccs is critical to the success of the protocol described in this video. This series of panels illustrates how the cells typically look at defined periods after plating. For example, after purification, the freshly plated cell aggregates appear as small, sparse clear clusters that contain few cells.
After the first 24 hours, many of the cell clusters become larger and numerous small clusters remain by 48 hours. The clusters have expanded significantly, but remain asymmetrical at 72 hours. The iccs should be clear, relatively uniform in size and shape, and can either be plated on matrices or allowed to grow for an additional 72 hours with or without chemicals or drugs to generate pancreatic endocrine cells.
These figures show a number of commonly used antibodies to assess either ICC proliferation or differentiation towards pancreatic endoderm. Here iccs were plated on HTV nine, grown for four days and stained for PDX one, a critical marker of pancreatic development. Although we routinely perform in vitro staining of iccs, more often human fetal iccs are transplanted under the kidney capsule of nude mice and allowed to proliferate and differentiate in vivo.
In this example, paraffin embedded kidney sections containing transplanted iccs were stained with the epithelial cell marker cytokeratin in green and KI 67 shown in red to assess cell proliferation. In another example, both human insulin in green and glucagon in red were detected after transplantation and maturation under the kidney capsule of a nude mouse. Once a master, this technique can be done in 90 minutes if performed properly.
While attempting this procedure, it's important to remember that each batch of collagenase differs and needs to be empirically optimized. After watching this video, you should have a good understanding of how to isolate culture and image. Human fetal pancreatic cell clusters.
