In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The overall goal of this procedure is to expand associated multipotent pancreatic progenitors in a 3D Matrigel scaffold where they can differentiate and self-organize. This is accomplished by first isolating the pancreas from embryonic day 10.5 mouse embryos. The second step is to remove the mesenchyme and dissociate the epithelium to single cells and small clumps.

The final step is to suspend the cells in matrigel and incubate the polymerized gel for expansion. Ultimately, this leads to self-organizing branched pancreatic organoids, similar to the mouse embryonic pancreas that can be visualized live or at endpoint by microscopy. The major advantage of this technique over existing methods, like all pancreas explan, is that one starts from a well-defined and less complex cell population.

Pancreatic progenitors are better maintained in 3D than in 2D cell cultures. This method can help us answer some critical questions within the field, such as cell cell interaction or cell interaction with the matrix. This will ultimately lead to understanding of how polarization emerges, and therefore also how cell diversity emerges.

The implications of this technique extend to diabetes therapy after adaptation in human, because we can expand pancreas that will give rise to better cells that produce insulin. Visual demonstration of this method is critical as the removal step of the amazing time is difficult to perform and the dissociation step is important for organ organoid formation. To begin place a mouse embryo that was previously isolated at embryonic day 10.5 into a clean 35 millimeter Petri dish in PBS.

Next, use thin forceps approximately 0.05 millimeter wide to remove the fore limb. Gently insert the forceps in the opening to detach the digestive tract from the spinal cord region. Locate the stomach, liver, and intestine on the embryo.

Then use the needles to isolate the gastrointestinal tract from the stomach to the intestine. Place the gastrointestinal tract in cold DMM on ice. Repeat the previous step to isolate every individual gastrointestinal tract.

Keep the individual gastrointestinal tracts in different wells. In a 24 well plate with cold DMM on ice, it is only necessary to keep them separated if the genotype is of importance. Place one gastrointestinal tract in a clean 33 millimeter Petri dish in cold PBS and visualize on a dissection microscope using the illumination from below as brightfield.

Once the gastrointestinal tract is in focus, dissect the dorsal pancreatic bud using electrolytic sharpened tongues than needles or 20 gauge syringe needles. Isolate the pancreatic bud with as little mesenchyme as possible around the epithelium. Place the isolated bud into a Petri dish and incubate in a cold one point 25 milligram per milliliter to space solution for two to three minutes.

From this point when transferring the bud, use a flame pulled 50 microliter glass capillary attached to a mouth controlled flexible tube. Place the bud into the solution while under microscopic control. Place a pancreatic bud back in PBS.

Then clean the isolated pancreatic bud from the mesenchyme using the needles and gentle aspiration with the glass capillary. Once the entire mesenchyme is removed, rinse the pancreatic bud in cold PBS and transfer each bud to individual wells. In a 60 well plate containing 10 microliters of cold DMM per well.

To rinse the dissected pancreatic buds, pipette each bud into individual conical wells and a 60 well mini tray containing 10 microliters of PBS per. Well then transfer the pancreatic bud into a well containing 10 microliters of 0.05%trypsin and incubate for four minutes. At 37 degrees Celsius, inactivate the trypsin by transferring the bud into a well containing 10 microliters of DMM with 10%fetal calf serum pipee the cell suspension through a thin capillary pulled with a pipette puller to dissociate the cells.

Partial dissociation is recommended because pancreatic organoids optimally start from small groups of five to 15 cells. Pool the cells from several embryos into an einor tube to minimize differences that result from individual processing. Dilute the cell suspension at a one to three ratio in chilled matrigel.

Aliquot the diluted cell suspension into a four well plate, or a 96 well plate by pipetting. Eight microliters per well. Incubate the plate at 37 degrees Celsius for five minutes to allow the matri gel to thicken.

Fill the micro well with culture medium and place the plate in a humidified environment at 37 degrees Celsius containing 5%CO2 and 95%air. Replace the medium every fourth day and monitor the growing pancreatic colonies daily. Document the growth process using time-lapse microscopy, dorsal pancreatic progenitor cells from mice at embryonic.

Day 10.5, grown in organoid medium, displayed expansion of cell clusters throughout the seven day culture period and started branching after four days. Single cells did not expand and lost PDX one expression. While the large clusters retained pdx.

One expression, pancreatic progenitor cells grown in sphere. Medium expanded more frequently than in organoid. In addition, Illumina was detected at day two to three and further expansion led to mono layered hollow spheres with occasional local multilayered areas.

Histological analysis of a seven day organoid confirmed composition of pancreatic progenitors as shown by positive staining for H NF one B and PDX one differentiated cells were also identified by staining for the exocrine marker amylase and the endocrine marker insulin. The spheres more homogenously expressed. The pancreatic progenitor markers hnf one B and PDX one and differentiated less than organoids.

While attempting this procedure, it is important to remember to not fully dissociate the cells Following this procedure. You can treat these organoids just as it was a small organ. Therefore, you can perform normal QPCR.

You can perform single cell QPCR, do live imaging. You can do immunohistochemistry, and by doing all these methods, you can have an, you can get an understanding of the cellular dynamics going on in this, in this small organ like structure, you can incubate with small chemicals and therefore interact with the different pathways of interest. This technique will pave the way for researchers to identify the mechanisms of pancreas development and better cell formation at the moment in mouse, but in the future, starting from human e ESLs or IP S cells.

After watching this video, you should have a good understanding of how to make a small pancreas that mimics the normal pancreas development from a few progenitors in 3D cultural environment.