Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.
This method could help answer key questions in the pancreatic field by using these pancreatoids to understand how endocrine cells form in an architecturally and cellularly complex environment. The main advantage of this technique is that the pancreatoids develop endocrine cells due to the inclusion of the surrounding mesenchyme. Generally, individuals new to this method will struggle because the pancreas is very small at e10.5.
And the dissection is crucial to the formation of pancreatoids to get enough non-contaminating tissue. To set up for dissection, fill an ice bucket and place a container of phosphate buffered saline in the ice. Clean two fine-tip forceps and dissection scissors with 70%ethanol.
Near the dissection area, set a container with at least three borosilicate capillary tubes, a lighter, and filtered 1000 microliter pipet tips. Next, pipet at least one milliliter of 1.25 milligram per milliliter cold dispase diluted in sterilized water in the second well of a 12-well plate on ice. Pipet PBS in the first, third, and fourth wells of the 12-well plate.
Also, pipet one milliliter of 0.05%trypsin in a 1.5 milliliter tube on ice. After euthanizing e10.5 timed pregnant mice in accordance with IACUC guidelines, spray the abdomen with 70%ethanol to clean the region. Then, make a V-shaped incision at the genital area of the mouse using scissors and forceps and continue the incision up to the diaphragm.
Carefully excise the uterus by making an incision at the uppermost lateral portions of the uterus, pulling the organ upwards away from the mouse and following this incision down to the genital region before repeating on the opposite side. Place the excised uterus in a 10 centimeter petri dish with the cold PBS. Under a light dissection microscope, place the petri dish and carefully open the uterine tissue by placing two forceps between each embryo and peeling tissue away from the yolk sac.
Transfer the embryos by grasping the yolk sac gently with forceps and placing them in a 10 centimeter petri dish with fresh, cold PBS. Place the petri dish on ice. Next, place multiple PBS drops on the 10 centimeter petri dish lid.
Use these droplets to transfer the embryo during dissection as extra-embryonic tissues are removed to ensure visibility. Transfer an embryo to a PBS drop and gently remove it from the yolk sac using forceps while the remaining embryos stay in PBS on ice. Remove the head before moving the embryo to a new PBS drop using forceps to lightly scoop the embryo without damaging the tissue.
In a new PBS drop, remove the four limb buds using forceps. Place the forceps into the opening where limb bud was present and gently tear only the most external tissue anteriorly. Rotate the embryo and repeat in the posterior direction, stopping at the hind limb bud.
At this point, the gastrointestinal tract should be visible. The posterior region of the gastrointestinal tract has a slight bend;insert forceps behind this bend in the opening between the gastrointestinal tract and the spinal region at the body wall. Detach the gastrointestinal tract, working slowly upwards until the most anterior region is reached, where the cardiac region connects.
In the following steps, it is critical to avoid the surrounding tissue in the culture, so as to not contaminate the pancreatoids with non-pancreatic tissue. However, if you do not grab the whole pancreatic bud, then the number and size of the pancreatoids will be impaired. Transfer the gastrointestinal tract to the fresh PBS droplet.
Continue to remove the outer tissue surrounding the gastrointestinal tract. Once this outer tissue is removed, the pancreatic bud can be visualized posterior to the liver buds, in between the stomach and intestine. Then, take forceps and gently pinch the tissue beneath the protruding bud in the intestine and slightly lift the pancreatic bud off.
After pinching the region where the bud connects to the intestine, the bud is separated. Wash one time in a new PBS bubble before transferring the bud into the first well of the 12-well plate and cold PBS on ice. Repeat until all of the buds are collected from the embryos and placed in the first well of the 12-well plate.
Next, place the 12 well dish under the light dissection microscope. Count the number of pancreatic buds successfully dissected to later calculate the split ratio. Place a filtered 1000 microliter pipet tip into the pipet with the capillary tube attached.
Flame sterilize the capillary tube and create a bend in the tube for the ease of use. Use the capillary to transfer the buds to the second well containing cold dispase solution for two minutes before transferring to the third well containing clean PBS. Pipet the buds up and down and transfer them to the fourth well containing clean PBS.
Finally, using the capillary device, transfer the buds to the 1.5 milliliter tube with 0.05%trypsin. Place the tube in the pre-warmed 37 degrees celsius shaker and shake at 1, 500 RPM for four to five minutes. After vortexing for approximately 10 seconds, immediately place the tube in the centrifuge and spin for five minutes at 200g.
Cautiously remove all but approximately 50 microliters of solution as to not disturb the centrifuged cells. For every bud collected at 400 microliters of organogenesis media to the centrifuged cells, pulse vortex three times and plate 100 microliters per well of a low-attachment 96 well plate, splitting buds at a one to four ratio. Check under the microscope to visualize dissociated cells freely floating.
Then place the culture dish in a 37 degrees celsius incubator with 5%carbon dioxide on a rocker at medium speed. Careful dissection of mouse embryos at e10.5 from the uterine horn should yield undamaged embryos in PBS for further dissection. The gastrointestinal tract can be efficiently removed from the embryo, permitting discernment of the dorsal pancreatic bud at the junction of the intestine and stomach.
In organogenesis media, these free-floating scaffold-free cells self-assemble and organize into three-dimensional pancreatoids that grow and persist for at least 10 days in culture. The application of a protein kinase C activator at high concentrations alters the morphology of developing pancreatoids, leading to loosely associated epithelial cells and increased branching. Additionally, the use of transgenic animals allows visualization of cells as they differentiate in real time.
To assess the association of pancreatic mesenchyme tissue with pancreatic epithelial cells, immunostaining can be performed for markers of each tissue. Immunostaining of a duct marker with an endocrine marker and nuclei, reveal multi-lineage formation in pancreatoids. A mesenchyme marker co-stained with a pancreatic progenitor marker shows that the mesenchyme envelops the pancreatoid.
Beta cell development is revealed by staining for the epithelial marker and the beta cell marker. Using quantitative PCR analysis, transcripts of progenitors and differentiating cells can be assessed, such as progenitor genes and differentiated markers. Once mastered, this technique can be done in approximately one hour, including prep time, if it is performed properly.
After watching this video, you should have a good understanding of how to perform the dissection to obtain pancreatic tissue from 10.5 mice to generate pancreatoids. While attempting this procedure, it's important to remember to carefully remove the GI tract from the embryos, otherwise it will be very difficult to find the pancreatic bud. Following this procedure, other methods like GSIS and ELISA can be performed in order to answer additional questions, like how much insulin or C-peptide is produced after glucose stimulation.
After its development, this technique paved the way for us to explore the morphological consequences of PKC activation using small molecules. A way of further implementing this system to study the effect of transcription factor of expression and even to perform important epistasis experiments.
