Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

All animal experiments described in this method were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine.

1. Preparation of Mouse Embryonic Day 10.5 Pancreatic Progenitors

Note: This protocol does not need to be followed under sterile conditions until step 2, however it is optimal to sterilize dissection tools and spray with 70% ethanol prior to use.

1. To set up for dissection, fill an ice bucket and place a container of phosphate buffered saline (PBS) in the ice. Clean two fine-tipped forceps and dissection scissors with 70% ethanol. Set near the dissection area, a container with at least three borosilicate capillary tubes, a mouth pipette, a lighter, and filtered 1,000 µL pipette tips.
   1. Prepare at least 1 mL of 1.25 mg/mL cold dispase diluted in sterilized water and place in the second well of a 12 well plate on ice. Put PBS in the first, third, and fourth wells of the 12 well plate. Place 1 mL of 0.05% Trypsin in a 1.5 mL tube on ice. Pre-warm a shaking incubator to 37 ^oC.
2. Euthanize e10.5 timed-pregnant mice in accordance with IACUC guidelines. Spray the abdomen with 70% ethanol to clean the region before making a V-shaped incision at the genital area of the mouse using scissors and forceps and continue it up to the diaphragm.
   ​NOTE: Appearance of a vaginal plug in this protocol indicates day 0.5. Using 5 – 6-week-old mice, a weight gain of more than 2 g acts as a secondary measure of successful impregnation.
   1. 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 it in a 10 cm Petri dish with the cold PBS.
      NOTE: It is possible to obtain organoids from e11.5 dissociated cells, however unlike the e10.5 pancreatoids, these organoids are not yet characterized and thus may not retain the capacity to differentiate into all three major pancreatic lineages.
3. 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 embryos by grasping the yolk sac gently with forceps and place in a 10 cm Petri dish with fresh, cold PBS. Place the Petri dish on ice.
   NOTE: It is important to heedfully remove embryos, preferably maintained within the yolk sac, as forceful removal can pull the umbilical cord, ripping tissue of the embryo and compromising the structural integrity.
4. Place multiple PBS drops on a 10 cm Petri dish lid. Use these droplets to transfer the embryo during dissection as extraembryonic tissues are removed to ensure visibility. Transfer an embryo to a PBS drop and gently remove from the yolk sac using forceps, while the remaining embryos stay in PBS on ice (Figure 1A). Remove the head before moving the embryo to a new PBS drop using forceps to lightly scoop the embryo up, to not damage the tissue (Figure 1B).
   ​NOTE: The tissue might need to be transferred more frequently to fresh PBS droplets than described here, depending on the opacity of the liquid.
   1. In a new PBS drop, remove the forelimb buds using forceps (Figure 1B). Place forceps into the opening where limb bud was present and gently tear only the most external tissue anteriorly (Figure 1B). Rotate embryo and repeat in the posterior direction, stopping at the hindlimb bud. At this point, the gastrointestinal tract should be visible (Figure 1B).
   2. The posterior region of the gastrointestinal tract has a slight bend (Figure 1F). Insert forceps behind this bend in the opening between the gastrointestinal tract and the spinal region of the body wall. Detach the gastrointestinal tract, working slowly upwards until the most anterior region is reached where the cardiac region connects (Figure 1B-E).
      OPTIONAL: Remove the primordial heart and the liver buds from the ventral regions of the gastrointestinal tract, leaving only the continuous gut tube. The first few times this protocol is performed it might be easier to leave the heart and liver as ventral landmarks until the morphology of the gastrointestinal tract and the dorsal pancreatic bud are more familiar (Figure 1C and D).
   3. Transfer the gastrointestinal tract to the fresh PBS droplet.
   4. Take forceps and gently pinch the tissue beneath the protruding bud and the intestine and slightly lift the outer tissue off (Figure 1D-F).
      NOTE: The dorsal pancreatic bud is located between the stomach and intestine (Figure 1C-G). The pancreatic bud beneath should look like a round, knobby structure (Figure 1G).
   5. Place forceps where the bud connects to the intestine and pinch upward to loosen the bud (Figure 1G). Wash one time in a new PBS bubble before transferring the bud into the first well of the 12 well plate in cold PBS on ice. Repeat until all buds are collected from embryos and placed in the first well of the 12 well plate.
5. Place the 12 well dish under the light dissection microscope. Count the number of pancreatic buds successfully dissected to later calculate the split ratio.
   1. Place a filtered 1,000 µL pipette tip into the mouth pipette with a 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 buds to the second well containing cold dispase solution for 2 min before transferring to the third well with the clean PBS (Figure 1G).
      NOTE: While transferring buds from dispase to PBS, avoid touching the tissue to the tip of the capillary tube. If the tissue gets stuck at the tip of the tube, pipette up and down or shake in the clean PBS solution until loosened.
   2. Pipette buds up and down, and transfer to the fourth well with clean PBS. Finally, using the capillary device transfer buds to the 1.5 mL tube with 0.05% Trypsin. Place the tube in the pre-warmed 37 ^oC shaker and shake at 1,500 rpm for 4 min.
   3. Vortex for approximately 10 s then immediately place the tube in a centrifuge and spin for 5 min at 200 g. Remove all but approximately 50 µL of solution cautiously as to not disturb the centrifuged cells (Figure 1G).

2. Culturing Dissociated Progenitors to Form Pancreatoids

Note: The following should be performed in a sterile atmosphere in a standard tissue culture hood, using standard sterile procedures.

1. For every bud collected, add 400 µL organogenesis media^16,17 (Table 1) to the centrifuged cells. Pulse vortex three times and plate 100 µL per well of a low attachment 96 well plate, splitting buds at a 1:4 ratio (Figure 1G).
2. Check under the microscope to visualize dissociated cells freely floating (Figure 1H).Place the culture dish in a 37 ^oC incubator with 5% CO₂ on a rocker at medium speed.
3. Monitor the progress of pancreatoids daily. Replace with 100 µL of fresh media every 3 days, carefully pipetting media under microscopic control in the hood to remove while leaving pancreatoid in the well. Alternatively, fresh 100 µL of media can be added to a new well and pancreatoid transferred using the borosilicate capillary tube attached to mouth pipette and 1000 µL filtered pipette tip.

3. Processing Pancreatoids for Immunofluorescent Imaging

1. To process pancreatoids for immunofluorescent images, first prepare 4% paraformaldehyde/PBS, pH7.4 (PFA) solution. Place 50 µL of 4% PFA in wells of a 96 well plate.
   1. Using the borosilicate capillary tubes and mouth pipette with the 1,000 µL filtered pipette tip, transfer pancreatoids from culture medium taking as little media as possible into the fresh well with 4% PFA. Set on the rocker at room temperature for 15 min.
   2. Transfer pancreatoids from 4% PFA into a well with 100 µL of fresh PBS, rock at room temperature for 5 – 10 min. Repeat for a total of three fresh PBS washes.
      NOTE: The protocol can be paused here, with pancreatoids stored in 100 µL of PBS at 4 ^oC for up to one week.
2. For wholemount imaging (recommended if antibodies are suitable, alternative approach in section 3.3. and microscopy in 3.4.), transfer from PBS into 50 µL 5% donkey serum in 1x PBS with 0.1% nonionic detergent (PBST) to block. Rock overnight at 4 ^oC or alternatively for 4 h at room temperature.
   1. Transfer pancreatoids to a new well containing primary antibodies diluted in 50 µL of 5% donkey serum in 1x PBST. Optimally, rock for 24 h at 4 ^oC (at least 12 h but up to 36 h).
      NOTE: Antibodies listed in the Table of Materials against Chga (1:100), DBA (1:400), Vim (1:400), and Pdx1 (1:100) are suitable for wholemount staining; other antibodies may require optimization.
   2. Transfer pancreatoids to a well containing 100 µL of fresh PBST to wash and rock for 30 min at room temperature. Repeat this step for a total of three fresh PBST washes.
   3. Transfer pancreatoids to a new well containing secondary antibodies diluted in 50 µL of 5% donkey serum in 1x PBST. Protect the plate from light and place in 4 ^oC, rocking for optimally 24 h (at least 12 h but up to 36 h).
      NOTE: All secondary antibodies listed in the Table of Materials and DAPI nuclear counterstain are suitable for wholemount staining.
   4. Transfer pancreatoids to a new well containing 100 µL of 1x PBST and rock at room temperature for 30 min. Repeat for a total of three washes in 1x PBST. In the third and final wash, add 300 nM DAPI.
   5. Finally, place into 100 µL of clean PBS and store at 4 ^oC protected from light for up to a week before imaging by confocal microscopy, although best results come from imaging immediately after staining. To prevent movement of pancreatoids during imaging but prevent drying out, place each pancreatoid into a 20 µL droplet of PBS. If pancreatoids do not remain still, reduce PBS volume.
3. For frozen sections, transfer pancreatoids from PBS into 30% sucrose solution overnight at 4 ^oC.
   1. Add embedding media in a bubble under a dissection microscope. Using forceps under microscopic control, soak pancreatoids by gently pushing through the embedding media several times to remove residual sucrose before transferring to a tissue block with frozen embedding media.
   2. Place the tissue block on dry ice until frozen and section on the cryostat at 8 µm.
      NOTE: Sections can be stored at -80 ^oC or immunostained immediately.
   3. Allow sections from -80 ^oC to thaw at room temperature for approximately 5 min until dry. Draw a hydrophobic barrier using the hydrophobic pap pen around the tissue and the block using 5% donkey serum diluted in 1x PBST for 30 min at room temperature.
   4. Remove media and replace immediately with primary antibodies diluted in 5% donkey serum diluted in 1x PBST overnight at 4 ^oC.
   5. Remove the primary solution and wash tissues three times with 1x PBST for 10 min each. Following this, add secondary antibody solution diluted in 5% donkey serum diluted in 1x PBST for 1 h at room temperature and protect slides from light.
   6. Remove the secondary solution and wash slides three times in 1x PBST for 10 min each. In the final wash, add 300 nM DAPI.
   7. Remove media and add mounting media and a coverslip. Store slides away from light in 4 ^oC until ready to image.

4. Isolation of RNA from Pancreatoids for Transcript Analysis

1. Collect pancreatoids using borosilicate capillary attached to a mouth pipette with a filtered 1,000 µL pipette tip for sterility and place into 500 µL of acid guanidinium thiocyanate-phenol-chloroform solution in a 1.5 mL tube. Store at -80 ^oC or proceed immediately to RNA isolation.
2. For RNA isolation, thaw samples on ice if necessary and add 100 µL of chloroform. Vortex well and place in 4 ^oC for 15 min. Pre-cool a centrifuge to 4 ^oC.
   1. Place tubes into centrifuge and spin for 20 min at 12,000 g at 4 ^oC.
   2. Carefully remove tubes to not disturb the separation of layers. Using a 200 µL pipette, collect the clear aqueous solution and place into a new 1.5 mL tube, leaving behind a small amount to buffer from the white protein layer and pink DNA layer. Do not touch the tip of the pipette to anything in this process and do not touch the walls of the 1.5 mL tube.
   3. Add 500 µL of 100% isopropanol to the new tube containing the aqueous layer and vortex. Let sit at room temperature for 20 min, longer on the ice, or for maximum precipitation leave overnight at -20 ^oC.
   4. Centrifuge at 12,000 g for 15 min at 4 ^oC to pellet the RNA. Remove the isopropanol without disturbing the pellet.
      NOTE: As pancreatoids are small, it is likely the pellet will not be visible.
   5. Add cold, 75% ethanol and invert the tube several times. Place in the centrifuge and spin at 9,000 x g for 10 min at 4 ^oC. Remove the ethanol and spin for 2 min at 9,000 x g.
   6. Use a 200 µL pipette to remove any remaining ethanol in the tube, without contacting the region the pellet resides in. Leave the cap open on the tube for 5 – 10 min at room temperature to ensure evaporation of residual ethanol.
   7. Resuspend pellet in by vortexing in 20 µL of clean, nuclease-free water.
      Optional: Heat the RNA at 65 ^oC for 3 min and immediately return to the ice. RNA can be stored at -80 ^oC or immediately used for reverse transcription and qPCR.