In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.
The overall goal of these protocols is to characterize the cell colonies grown from pancreatic progenitor cells isolated from adult mice using cultures assays that employ methylcellulose containing semi-solid medium. Our methods can help answer key questions in the pancreatic progenitor cell biology field, such as, what are the self renewal and differentiation capacities of progenitor cells in the adult pancreas? The main advantage of these techniques are that they allow the study of individual progenitor cells which is important for discerning their true biological potentials.
We first had the idea for these methods when we realized that the colony assays were studying hematopoietic stem cells could also be applied to pancreatic cell populations. Demonstrating the procedures will be Jacob Tremblay, a graduate student, Dr.Janine Quijano, a post doc, and Miss Angela Luo, a technician, all from my laboratory. As soon as possible after sacrifice, use scissors to make a vertical incision on the midline of the abdominal wall, and open the abdominal cavity.
Using forceps, gently lift the spleen, cutting the connective tissue between the spleen and the splenic lobe of the pancreas with scissors. Sever the connective tissues for the duodenal and gastric lobes in the same way, and place the pancreatic tissues in a Petri dish containing DPBS supplemented with 0.1%BSA and antibiotics on ice. Then, under a dissecting microscope, use fine tip forceps to remove the fat tissue.
Now transfer the pancreata to a tissue culture hood and rinse the tissue in three sequential 100 millimeter Petri dishes containing 10 milliliters of cold PBS plus BSA each. After the final wash, place the pancreata into a sterile dry Petri dish to remove as much PBS plus BSA as possible, and transfer the tissues to another dry Petri dish on ice. Next, use spring scissors to mince the pancreata for two to three minutes until fine pieces of tissue are generated, and suspend the tissue fragments in two to three milliliters of freshly prepared cold PBS BSA DNase I solution.
Using a 10 milliliter pipette, transfer the pieces to a 50 milliliter conical tube and add enough PBS BSA DNase I to bring the total volume up to 10 milliliters. Then add 350 to 450 microliters of collagenase B to the tissues. Place the tube in a 37 degree Celsius water bath for eight minutes, with swirling every three to four minutes.
At the end of the incubation, use a 10 milliliter syringe with a 16 and a half gauge needle to draw up and subsequently spray the tissue solution down the wall of the 50 milliliter tube at room temperature seven times. Then return the tissues to the water bath for another eight minutes with occasional swirling, followed by seven passes through the syringe as just demonstrated. After the last mechanical disruption, transfer a 10 microliter aliquot of the cell suspension onto a dry Petri dish and confirm the presence of single cells and small sized cell clusters under an inverted phase contrast light microscope with a 10x objective lens.
Next, place the tube on ice and bring the total volume up to 50 milliliters with PBS BSA DNase I.Centrifuge the cells, and use a P1000 pipette to re-suspend the pellet in five milliliters of cold PBS BSA DNase I.Then filter the cells through a 70 micron nylon mesh filter followed by a 40 micron nylon mesh filter into a new 50 milliliter polypropylene conical tube. Adjust the volume to 5 milliliters of PBS BSA DNase I, and count the cells. Then dilute the single cell suspension to a final concentration of two times 10 to the seven cells per milliliter in cold PBS BSA DNase I on ice.
To whole mount immunostain pancreatic colonies grown in methylcellulose containing semi-solid medium, first prepare a glass Pasteur pipette with a fine opening using a Bunsen burner. Next, attach the small end of a one milliliter pipette tip to one end of a thin walled rubber tube and a mouthpiece to the other end. Attach the glass Pasteur pipette to the large end of a pipette tip.
Then draw up 10 to 50 microliters of DMEM/F12 medium with one percent methylcellulose into the mouth pipette. Now place the dish of colonies on an inverted phase contrast microscope stage and use the 10x objective to find suitable colonies for picking. Then place the opening of the pipette next to the colony of interest, and slowly apply suction to the mouth to pick a single colony.
For an effective collection, it is important to make sure the colony of interest is in focus before placing the opening of the glass pipette next to the cell cluster. Once the colony has been harvested, place the pipette tip into one well of a 96 well plate containing 4%paraformaldehyde and deposit the colony into the well. After all of the colonies have been collected, fix the colonies overnight at four degrees Celsius with gently shaking.
The next morning, wash the colonies two times in PBS for 10 to 30 minutes at room temperature. From this point onward, pick up colonies using a plastic P10 pipette with a bent tip. After the second wash, store the fixed colonies overnight in 1.5 milliliter tubes filled with paraffin at four degrees Celsius.
Then transfer the colonies to individual wells of a 96 well black plate with a clear bottom containing 200 microliters of blocking buffer per well. Incubate the plate overnight at four degrees Celsius with gentle shaking. The next morning, transfer the colonies to new wells containing 200 microliters of the primary antibody of interest for another overnight culture at four degrees Celsius with gentle shaking.
The next morning, transfer the colonies into three consecutive wells containing 200 microliters of PBS plus 0.1%Tween 20 for 10 minute washes at room temperature. After the third wash, transfer the colonies to clean wells containing 200 microliters of the appropriate secondary antibody, and incubate the plate at room temperature for two hours in the dark. At the end of the incubation, wash the colonies three times in PBS Tween as just demonstrated, and transfer the colonies into new wells of DAPI for five minutes at room temperature.
Then pool the immunostained colonies in a 35 millimeter glass bottom Petri dish and cover them with a cover slip. Finally, image the colonies on a confocal microscope. Adult pancreatic progenitor cells can be enriched by flourescence activated cell sorting using the appropriate gating parameters, as the colony forming capability is found only within CD133 Sox9/EGFP positive ductal cells.
The pancreatic colonies can then be classified according to their morphologies by inverted phase contrast light microscopy. After hand picking, the colonies can also be analyzed by microfluidic qRT-PCR analysis for gene expression, or by whole mount immunostaining for protein expression. Indeed, many individual colonies express lineage markers for duct, endocrine, and acinar cells, for example, indicating that the majority of the initiating pancreatic colony forming progenitors for these colonies are tri-potent.
Once mastered, the dissection, dissocation, cell staining and sorting, and cell plating into methylcellulose containing semi-solid medium procedures can be completed in six to seven hours if they are done properly. Following the colony formation, other methods like quantitative RT-PCR, western blot, or insulin secretion assays can be performed to answer additional questions about the gene expression or functional abilities of the colonies. After watching this video, you should have a good understanding of how to dissociate an adult murine pancreas and how to characterize pancreatic progenitor cell generated colonies in methylcellulose containing semi-solid cultures.
