Protocol to use de-epithelialized porcine urinary bladder as a tissue scaffold for propagation of pancreatic cells

Summary Ex vivo organ culture can be a useful alternative to in vivo models, which can be time-, labor-, and cost-intensive. Here we describe a step-by-step protocol to use de-epithelialized porcine urinary bladders as scaffolds in air-liquid interface in vitro culture systems for a variety of pluripotent stem-cell-derived and patient-derived pancreatic cells and organoids. The scaffold can trigger cell maturation and enable cell-cell interaction and invasion capacity studies. However, this model is limited by the lack of functional vasculature. For complete details on the use and execution of this protocol, please refer to Melzer et al. (2022),1 Breunig et al. (2021),2 and Breunig et al. (2021).3


SUMMARY
Ex vivo organ culture can be a useful alternative to in vivo models, which can be time-, labor-, and cost-intensive. Here we describe a step-by-step protocol to use de-epithelialized porcine urinary bladders as scaffolds in air-liquid interface in vitro culture systems for a variety of pluripotent stem-cell-derived and patient-derived pancreatic cells and organoids. The scaffold can trigger cell maturation and enable cell-cell interaction and invasion capacity studies. However, this model is limited by the lack of functional vasculature. For complete details on the use and execution of this protocol, please refer to Melzer et al. (2022), 1  , 2  . 3

BEFORE YOU BEGIN
The protocol below describes the specific steps to employ a porcine urinary bladder (PUB) as an organ culture model for different pancreatic cell types. The protocol was initially established using human embryonic stem cell (hESC)-derived pancreatic progenitor cells (PPs) and pancreatic duct-like organoids (PDLOs). In a next step, we have successfully implemented the PUB ex vivo model to investigate human pancreatic cancer patient-derived organoids (PDOs), pancreatic stellate cells, and pancreatic cancer cell lines.
The in vitro differentiation of human pluripotent stem cells (hPSC) into PPs and PDLOs 2,3 as well as propagation and maintenance of PDOs 4,5 has been described in detail (e.g., step-by-step protocols 3,6 ). Pancreatic stellate cells and cancer cell lines were cultured using standard media conditions. 7,8 Institutional permissions All experiments were performed in accordance with the institutional and governmental regulations of Ulm University and the state of Baden-Wü rttemberg in Germany. PUBs were obtained as commercial byproduct of the food industry and thus, did not need further specific approval. However, such approval may be necessary in certain countries and should be obtained before starting the procedure. 6. GFR-Matrigel.
a. To thaw original Matrigel vials, place them on ice and transfer the container with the ice into a 4 C-8 C environment for 10-16 h. b. Pre-cool tips and reaction tubes at À20 C before handling the Matrigel. c. Prepare 1 mL aliquots and store at À20 C for up to 2 years (check certificate of analysis for each batch). d. Thaw the required number of aliquots on ice for 30 min to 1 h before use. e. Avoid repeated freeze-thaw cycles of aliquoted Matrigel.
Note: In this protocol, we have used Matrigel with a protein concentration ranging from 8.8 to 11.3 mg/mL and an endotoxin content of less than 1.5 EU/mL. 7. ROCK inhibitor Y-27632 (10 mM).
a. Volumes are calculated for a molecular weight of 338.28 g/mol. b. Original vials must be centrifuged before opening. c. Dissolve 10 mg in 2.956 mL of sterile, double-distilled water by pipetting. d. Store 100 mL aliquots at À20 C for no more than 6 months.
Note: Specific molecular weight may be variable between batches. Therefore, volumes of the dissolvent must be adjusted accordingly. 8. Collagenase/dispase solution for enzymatic harvesting of organoids (Stock: 100 mg/mL, working solution: 1 mg/mL). a. Dissolve 500 mg of collagenase/dispase powder in 5 mL dH 2 O. b. Mix by pipetting up and down. c. Prepare aliquots of 1 mL each and store at À20 C. d. To prepare working solutions, thaw one aliquot and dilute the stock solution in a 1:100 ratio in standard DMEM/F12, GlutaMAX. e. Filter the solution through a 0.22 mm filter. f. Always prepare working solution freshly. g. After defrosting, stock solution may be stored at 4 C for up to 4 weeks. 9. Supplemented Advanced DMEM/F- 12. a. Prepare 485 mL of Advanced DMEM/F-12 by removing 15 mL of Advanced DMEM/F-12 from a 500 mL medium bottle in a laminar flow bench. b. Add 5 mL of penicillin/streptomycin (1% P/S, equal to a final concentration of 100 IU/mL of penicillin and 100 mg/mL of streptomycin) to the medium. c. Add 5 mL of 1003 GlutaMAX supplement (200 mM) to the solution. d. Add 5 mL of 1 M sterile HEPES to the medium. e. Store at 4 C for up to 3 months. 10. Collagenase-II solution.
a. Dissolve 1 g of collagenase-II in 50 mL of supplemented Advanced DMEM/F-12 to prepare a 20 mg/mL stock solution. b. Filter collagenase-II solution (20 mg/mL) sterile through a 0.22-mm filter. c. Store the collagenase-II stock solution aliquoted in dark reaction tubes at À20 C for up to 3 months. d. Prepare working solution by diluting the stock solution 1:5 for a final concentration of 4 mg/mL collagenase-II in standard (not Advanced) DMEM/F-12, GlutaMAX supplemented with 10 mM Y-27632. Boundary rings for seeding cells on the PUB Prepare rings for seeding cells by cutting 0.2-mL PCR-tubes to rings with a height of approximately 3 mm and a diameter of around 5 mm.
Autoclave (121 C 20 min) rings before placing on PUBs.
Note: Equivalent rings can be produced from different types of PCR tubes.

STEP-BY-STEP METHOD DETAILS
Part I: Preparation of porcine urinary bladders for organ culture In a first step, PUBs were washed and de-epithelialized to enable the subsequent culture of engrafted pancreatic cells. De-epithelialization was achieved by enzymatic and mechanical treatment of the PUBs. PUBs were then either sterilized for long-term storage in PBS at 4 C or were immediately used.
CRITICAL: The use of sterile (autoclaved) forceps, scissors, and beakers at any time is mandatory to avoid handling-associated microbial contamination.
1. Obtaining porcine urinary bladders. a. Collect fresh porcine urinary bladders from the local slaughter-house within 3 h after isolation. Transportation on wet ice is recommended, however not obligatory. b. Ensure that PUBs are not frozen before processing.
Note: PUBs should be processed as soon as possible after isolation to reduce microbial growth.
CRITICAL: Frozen PUBs should be discarded as scaffold integrity cannot be guaranteed. (Figure 1). a. Transfer PUB inside the laminar flow benches. Ensure the handling of PUBs under strict sterile conditions. b. Using sterile forceps, transfer PUBs from the transportation bag to a 15-cm cell culture dish, filled with DPBS. c. Adjust orientation of the PUB so that the urethra faces the operator ( Figures 1A and 1B). d. Remove any adjacent tissue including fat, urethra, penis, vagina, or ureter ( Figures 1A and  1B). e. Transfer the PUB into a new 15-cm dish, filled with DPBS ( Figures 1B and 1C). f. After removal of the urethra, grab the bladder neck with forceps and use scissors to open it along the middle line ( Figure 1C). g. Unfold the bladder and discard any remaining urine by washing the PUB in DPBS ( Figures 1D  and 1E). h. Perform a detailed visual inspection and check for bleedings, signs of infection, tumors, or other abnormalities ( Figures 1E-1G).

Cleaning of PUBs
Note: Small bleedings ( Figures 1F and 1G) can be trimmed away. In case of other abnormalities, discard the bladder. When signs of pus or infection are evident within the bladder, immediately discard the PUB, remove any contaminated tools, and rigorously clean the laminar flow bench before processing fresh bladders.
i. Turn the bladder upside down and inspect the outer wall of the PUB for the same criteria as the inner side ( Figures 1H-1J). l. Incubate for at least 5 min at room temperature and transfer to a new beaker with bladder wash solution. m. Repeat this step three times.
Note: It is crucially important to completely cover the PUBs with wash solution. Extending the washing steps to up to 60 min/step was tested and may result in fewer microbial contaminations.
3. De-epithelialization of PUBs (Methods video S1), a. Transfer PUBs to a beaker filled with the dispase-II working solution.
Note: It is important to ensure permanent contact of dispase-II working solution with the urothelial layer of the PUBs. The urothelial layer is located on the inner, highly folded side of the PUB. We typically use around 10 mL dispase-II working solution per bladder.
b. Incubate the PUBs overnight at 4 C. c. Transfer one PUB at a time to a new 15-cm cell culture dish filled with DPBS. d. Lift the PUB with forceps above the cell culture dish. e. Strip off the epithelial cell layer which was loosened by dispase-II treatment by using a second pair of forceps. f. Repeat this step from different angles of the bladder until complete removal of the epithelial layer.
Note: Make sure to keep the PUB sterile while stripping off the urothelial layer. Take long sterile forceps (>15 cm) to avoid any direct contact between the PUB and the operator's fingers.
CRITICAL: Sterility is crucial to ensure smooth experimental procedure.
g. Transfer de-epithelialized PUBs to a beaker filled with DPBS and sway PUBs to remove any remaining urothelial cells. h. Repeat step g in a new beaker with fresh DPBS. Troubleshooting 1. 4. Alternative 1: Taking PUBs directly into culture.
Note: After cleaning and de-epithelization, PUBs can be taken directly into culture. Especially if interactions with the host, e.g., paracrine signaling, cell-cell communication with porcine cells etc., are of interest, the cultivation of non-sterilized PUBs may be beneficial. Good results for pancreatic cell culture on the PUB have been achieved by this method (please refer to Melzer et al. 1 ). However, PUBs are degrading without additional PAA sterilization thereby limiting the culture period to a maximum of four weeks. Also, dropouts due to microbial contamination can be prevented by additional sterilization.
Note: Always use sterile instruments to handle the PUBs.
a. Transfer one PUB at a time into a new 15-cm cell culture dish filled with PBS. b. Cut the PUB into pieces of approximately 2 cm 2 with sterile scissors. c. Add 6 mL of HBSS to each well of a 6-well plate.
Note: To investigate potential contaminations, no antibiotics or antimycotics are added to the medium during this step. However, during cultivation of cells, media are supplemented with 3% antibiotic-antimycotic solution. Note: To reduce media to 3 mL per well of a 6-well plate, wells can be precoated with 3 mL of a 2% Agarose/PBS solution (sterilized by autoclavation). After solidification of the Agarose, medium can be filled up to 6 mL and cell strainers can be placed on top as described below. Agarose-filled plates can be also prepared in advance and stored for around two weeks at 4 C.
d. Place one 40-mm cell strainer into one well of the 6-well plate. Ensure that the cell strainer is being laid up the rim of the well and the mesh on the bottom is in contact with the medium.
Note: Only the lower part of the cell strainer should be in contact with the medium. The strainer must not be placed completely into the medium and formation of air bubbles below the cell strainer should be avoided. Depending on the manufacturer of the plates and cell strainers, the volume of HBSS per well might need to be adjusted to fulfill these requirements. Be aware that not all manufacturers provide cell strainers that are suitable for this kind of application.
e. Place a piece of PUB into each cell strainer of the 6-well plates. Ensure that the former urothelial side (tunica mucosa) is directed toward the air-phase (up) and the outer wall (tunica serosa) faces the medium (down). Incubate in a cell culture incubator for 24 h at 37 C with a 5% CO 2 and 21% O 2 atmosphere.
Note: This period can be extended to 48 h, which might ease the detection of microbial contamination. However, non-sterilized bladders will lose viability and start to degrade. Therefore, longer pre-incubation times are not recommended for the direct culture method.
f. Check for any signs of microbial contamination by visual and microscopical inspection of the wells. Proceed with the next steps in case of no sign of contamination.
Note: Contamination-free setting is critical for further experiments. Macroscopic signs of infections include cloudy medium and significant color changes of the medium. However, microbial contamination may be more discrete and should be also carefully investigated with a microscope. An example of contaminated and non-contaminated PUBs is presented in Figure 2.
g. Prepare new 6-well plates containing 6 mL of the respective cell culture medium, including 3% antibiotic-antimycotic solution and in case of PSC-derived cultures 10 mM Y-27632.
Note: A variety of different media, cytokines, and other substances were used in previous PUB experiments. While none of the conditions had an obvious impact on the PUB itself

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(concerning morphology of the scaffold), pre-testing of specific media formulations should be considered for new conditions and cell types.
Note: To enhance cell viability of pluripotent stem cell-derived pancreatic cell types, add 10 mM Y-27632 for cell seeding.
h. Transfer non-contaminated PUBs with their respective cell strainers into new 6-well plates by using sterile forceps, always ensuring that no air bubbles are present below the nylon meshes and PUBs are not covered by medium but only in contact on the lower site. Troubleshooting 2. Troubleshooting 3. 5. Alternative 2: Sterilization of PUBs.
Note: Follow these steps directly after step 3 as an alternative to step 4. Sterilization of PUBs is achieved by treatment with peroxy-acetic acid as initially described in Rosario et al. 11 a. Prepare a beaker with around 350 mL sterilization solution.
Note: Ensure coverage of all PUBs with sterilization solution. Typically, we use 350 mL sterilization solution for a maximum of 5 bladders.
CRITICAL: Sterilization with PAA reduces the likelihood of PUB-derived contamination. However, extreme precaution must be maintained when handling the PUBs to prevent operator-derived contamination.
b. Transfer PUBs from DPBS to the sterilization solution with sterile forceps. c. Incubate the PUBs in sterilization solution for 24 h at 4 C-8 C. d. Repeat the sterilization for another 24 h with new sterilization solution. e. Wash PUBs twice with DPBS for at least 5 min.
Note: After washing, PUBs can be stored in DPBS at 4 C-8 C for up to 3 months. Longer storage periods were not tested.
Pause point: At this point, PUBs can be stored at 4 C-8 C in DPBS before continuing the procedure.
Troubleshooting 4. f. Transfer one PUB at a time to a 15-cm cell culture dish filled with DPBS. g. Cut the PUB into pieces of approximately 2 cm 2 with sterile scissors. h. Prepare 6-well plates containing 6 mL of the respective cell culture medium, including 3% antibiotic-antimycotic solution and in case of PSC-derived cultures also 10 mM Y-27632.
Note: See part I-4c for the use of 3 mL 2% Agarose/PBS-coated plates to reduce culture media.
i. Place a cell strainer into each well as indicated in step 4. j. Transfer PUB pieces into cell strainers, ensuring that the former urothelial side is facing the air and the tunica serosa is in contact with the medium. k. Incubate in a cell culture incubator for 24-72 h at 37 C.
Note: The medium may rapidly turn yellow due to sudden pH change which is caused by the ongoing PAA-evaporation in case of little residual PAA. The yellow medium color switch alone should not be considered as a sign of contamination as long as no other signs are Note: No decline of quality was observed between 24-72 h, but 72 h is recommended in order to allow exclusion of contaminated bladders. Longer periods were not tested but are likely possible. However, when working with temperature-sensitive media or supplements such as cytokines that are degraded over time, a media change may be necessary before proceeding with the cell seeding to avoid loss of cell viability or differentiation status. l. Check for any signs of contamination before proceeding with the cell seeding.
Note: Although sterilization has been performed, microbial contamination cannot be totally excluded. Handling of the PUBs is challenging and comprises many steps prone to contamination. However, according to our experience, the rate of microbial contaminations drops to less than 5% of the PUBs if handled properly. Examples of contaminated PUBs are depicted in Figure 2.
CRITICAL: Take quality control samples for histological examination of the de-epithelialization. Samples should include a native PUB control and a de-epithelialized PUB control after dispase-II treatment and also after PAA-treatment. Antibodies specific for the markers of your cells of interest should be used to discriminate them from porcine cells in case the de-epithelialization is not complete. Prior to cell seeding, small rings will be placed on the PUBs serving as a boundary during seeding and subsequent culture. The herein described workflow worked robustly for different cell lines and cell numbers and across single cells, whole and fragmented organoids as indicated in Table 1. However, the optimal number of cells should be adjusted according to particular applications.
6. Preparing bladders for seeding of cells.
a. Place two rings on each bladder using sterile forceps.
Note: Rings should be placed straightly on PUBs to prevent cell suspension spilling during seeding.

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Note: We typically use two rings on each bladder, however, depending on applications and available cell numbers one might prefer the use of a single ring per bladder.
b. Gently press the ring into the scaffold to ensure close contact of the ring with the PUB. c. Wait for at least 2 h before seeding cells. Note: We typically harvest organoids by digestion of the Matrigel with 1 mg/mL collagenase/ dispase for 2-4 h at 37 C in a cell culture incubator with 5% CO 2 (typically 500 mL per well of a 24-well plate). Using this kind of enzymatic digestion, we have observed a proper digestion of the Matrigel without a significant decline in cell viability after harvesting. We stop the reaction with neutralization solution (twice the amount of the collagenase/dispase) and centrifuge the organoids for 5 min at 200 g before proceeding with the next steps. If singularization of cells is necessary, we wash the organoids once in DPBS and resuspend them in accutase, before incubating them 30 min at 37 C in a waterbath. After 15 min and at the end of the incubation, we gently pipette the organoids up and down to enhance the enzymatic digestion. The reaction is stopped by adding twice the amount of neutralization solution and centrifugation for 5 min at 200 g before proceeding with the next step.
b. Count cell/organoid number. Cell/organoid numbers of different pancreatic cell types and cocultures that worked well in our hands as well as their tested cultivation periods are provided in Table 1. Adjust cell/organoid numbers according to the number of intended rings.
Note: Cultivation periods can be adjusted for specific purposes, e.g., time course of tumor development, invasion, maturation of pluripotent stem cell-derived pancreatic progenitors etc. So far, we did not culture cells longer than 4 weeks on non-sterilized bladders and did Note: Although low cell/organoid numbers were tested and led to a successful engraftment, the histological search for a small engraftment can be cumbersome. Hence, we advise adhering to recommended cell numbers giving rise to large and easily identifiable engraftments.
c. If co-cultures are intended, mix different cell types well by pipetting.
Note: The exact number and ratio of cells for co-culture experiments should be optimized for the specific application but the recommended numbers in Table 1 can serve as a starting point. Coculture approaches have proven to be a very useful tool to study, e.g., cell crosstalk between epithelial and stromal cells and to investigate stromal redrafting by tumor cells. Cell numbers should be adjusted to recommended cell numbers as initial starting point (recommended starting point 2,000 organoids per ring, 166,000 single cancer cells per ring, and respective amounts of stromal cells). However, testing of cellular composition is necessary to find the optimal ratio and number of each cell type for specific co-culture approaches of interest.
d. Centrifuge cell suspensions and remove the supernatant completely. e. Resuspend cells/organoids in the respective cell culture medium with a volume of 15 mL medium per ring. In case of PSC-derived cell cultures, add 10 mM Y-27632 to the medium.
Note: The seeding volume was optimized for our system including organoids with a maximum organoid size of around 150 mm. Bigger/smaller organoids may need adjustments for the organoid numbers or seeding volume. However, we do not recommend to significantly increase the seeding volume as organoids will be mostly located then in the fragile Matrigel/medium layer rather than in contact with the PUB.
Note: Using the same media formulation in ex vivo PUB experiments as in the in vitro culture of the cell type of interest, is a good starting point and led to robust and good results in our experiments. However, we observed that on-PUB propagation allowed removal of certain supplements such as FBS, cytokines, e.g., EGF, FGF-10 etc. Examples are given in Table 2. However, head-to-head comparisons should be performed for the specific application before depleting any supplements from the original in vitro cell culture medium.
f. For every ring, add 15 mL GFR-Matrigel to the cell suspension to reach a final concentration of 50% GFR-Matrigel. g. Add 30 mL of cell suspension to each ring. h. Carefully check for any potential spilling outside the rings and mark respective rings on the cell culture plates.
Note: The likelihood of successful engraftment is very low if cell suspension spills outside of the ring.
Note: Seeding without Matrigel was tested successfully for murine cancer cells.
i. Place the cell culture plates in a cell culture incubator at 37 C until the next medium change. Troubleshooting 4. 8. Ongoing culture.
a. Once to twice a week, prepare new 6-well plates containing 6 mL of the respective culture medium for the cell types seeded on the PUBs.
Note: Frequency of medium changes depends on the presence of heat-labile molecules, e.g., cytokines, in your medium. If heat-labile molecules are within the medium, more frequent media changes might be necessary for your application.
Note: See Part I/4c for the use of 3 mL 2% Agarose/PBS-coated plates to reduce culture media.
b. Transfer PUBs from the old plate to the new plate using sterile forceps. 9. Treatment of PUBs with drugs or small molecules to investigate cytotoxicity and transgene expression.
Note: PUB can be used to investigate the effect of distinct compounds on the cellular behavior. For example, doxycycline can be added to the culture medium to modulate transgene expression with Tet-on systems (reviewed in. 13 ). In our case, treatment of PUBs with the same doxycycline concentrations (3 mg/mL) as used in the in vitro cell culture counterparts resulted in robust transgene (HA-tagged KRAS G12D -IRES-mCherry) expression. 1 In addition, the investigation of cytotoxic components and their effect on cancer cells or patient-derived organoids in this organ culture model is feasible. In our setting, we have tested the effects of olaparib (3.7 mM) and irinotecan (1.1 mM). 1 While doxycycline, olaparib and irinotecan have been added to the medium below the PUB requiring a penetration of the drug through the PUB scaffold, a direct treatment of cells can be also achieved by drug addition to the boundary rings. Thus, the use of other drugs and the duration of their application must be tested in respect to the specific application of interest as not all drugs might properly penetrate through the PUB scaffold. a. Add the respective drug or small molecule in the concentration of interest to the medium. b. Perform medium change as described in Part II-3a-b. c. Alternatively, add the drugs in the desired concentration in a small volume, e.g., 30 mL, to the rings.
Note: Treatment can also be directly started by adding the respective drug to the medium during seeding of the cells.
CRITICAL: Always use sterile forceps for handling PUBs. In this step, the harvesting of ex vivo on-PUB cultures for histological analysis is described in detail. For histological analysis, the orientation of the sample and the discrimination between porcine cells and human/murine cells is critical. 10. PUB processing to formalin-fixed paraffin-embedded tissue (FFPE). a. Fill the wells of a new 6-well plate with 3.7% formaldehyde.
Note: Formaldehyde is toxic and hazardous. Follow the manufacturer's indications on how to handle and store it. Wear personal protection equipment (gloves, clothing, eye, and face protection) and avoid exposure to it.
b. Take PUB pieces out of the cell strainers and transfer each to one well filled with formaldehyde. c. Drown the PUBs very carefully in the formaldehyde solution and ensure complete coverage.
Note: At this stage, rings should not be removed by force from the PUBs if they do not come off by themselves. Please ensure that no air bubbles are within the rings when covered by formaldehyde. Do not add the formaldehyde on top of the PUBs to avoid mechanical removal of cells. Be especially careful when working with samples from short-term experiments. The shorter the experiment, the more Matrigel will be present, and cells will be suspended in the Matrigel layer which is more sensitive to mechanical forces. d. Fix the cells overnight at 4 C-8 C. e. Take the PUBs out of the formaldehyde and carefully remove the rings to avoid the detachment of adherent cells. f. Remove any tissue distant to the rings from the PUBs with scissors or a scalpel (Figure 3).

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Optional: Cut the piece of PUB in the middle of the former ring area, in case vertical sectioning and investigation of invasion into the former sub-epithelial stroma or muscle layer of the PUB is desired (Figure 3).
Note: Be careful when cutting into the middle of the implantation site as the seeded cells, especially in short-term condition, might be otherwise removed from the scaffold. Optional: To investigate the process of invasion, turn the two halves obtained from cutting the middle of the implantation site of one ring (in g) by 90 so that the sectioned side of the PUB faces the bottom during paraffin embedding.
Note: This vertical orientation will enable investigation of the interaction between the engrafted cells and the PUB scaffold. This enables evaluation of invasion and migration toward the former subepithelial stroma and muscle layer of the scaffold. While this is an excellent technique to analyze the cells with strong connection to the PUB scaffold, cells that are still located in the former Matrigel layer may be lost during dehydration or handling procedures. Hence, the number of detectable cells may be less than after horizontal sectioning.
i. Perform standard histological staining procedures and confirm identity of your cells by specific staining.
Note: Especially when working with non-sterilized PUBs, it becomes difficult to distinguish in H&E staining between urothelial cells of the pig and the cells of interest in case of incomplete removal of epithelial cells. The use of human-specific antibodies such as anti-human nucleoli, anti-human cytokeratine 19, as well as human specific Ki-67 (see also key resources table, and 1 supplemental information), however, enables a clear discrimination between human and porcine cells. No issues in distinguishing human or murine cells from other porcine cells after treating PUBs with PAA during the initial preparation arose during our investigations. CRITICAL: Careful handling is necessary to avoid removal of cells by mechanical forces. ''Invasive'' cells may also be observed when investigating sections prepared parallel to the surface (horizontal orientation). However, such observation should be regarded carefully as numerous foldings of the inner bladder wall may lead to false positive results if not sectioned vertically.
a. Fill the wells of a 6-well plate with DPBS. b. Transfer PUB pieces from cell strainers to the 6-well plate. c. Carefully submerge the PUBs in the DPBS and shake the plate to enable proper washing. d. Remove the rings from the PUBs very carefully.
Note: Especially in short-term conditions, cells may be located in the Matrigel/medium layer rather than being fully attached to the scaffold. This may lead to a certain degree of instability with the subsequent risk of losing cells of interest when removing the rings from the scaffold.
In that case, try to capture those Matrigel-embedded cell clusters with a pipette and further process them along with the PUBs in subsequent steps.
e. Prepare a new 12-well plate. Fill the wells with 2 mL of collagenase-II working solution.
Note: Other enzymatic digestion approaches may also work. In short-term experiments with cultivation of cells for up to 5 days, we achieved comparable RNA yield with either accutase (30-45 min incubation) only and or collagenase/dispase (2 h incubation) digestion only. However, in long-term conditions (>2 weeks), we observed a much better detachment of cells when using the collagenase-II solution for isolation of cells from the PUB scaffold.

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Note: Also porcine cells of the scaffold PUB can be isolated by this method, which can become relevant for certain downstream applications.
f. Take PUBs from the DPBS and place them with the cell containing side up into the collagenase-II working solution.
Note: Ensure complete coverage of the PUBs with the collagenase-II sollution.
g. Incubate for 60 min at 37 C on an orbital shaker with 115 rotations per minute.
Note: Incubation on an orbital shaker outperformed the enzymatic digestion without a shaker.
h. Shake the plates carefully to allow cells to spread throughout the whole well. i. Check under the microscope if cells are floating around. In case of very few detached cells, prolong the incubation in the collagenase-II solution to up to 180 min.
Note: As the collagenase-II digestion leads to isolation of porcine cells as well, precautions have to be taken into consideration for further downstream analysis. There is a high chance of porcine DNA-contamination. RNA is likely degraded over time and during treatment with peroxy-acetic acid. However, even in sterilized samples, remnant porcine nucleic acid fragments including RNA were detected in scaffold-only samples. Hence, choosing human-specific (or murine-specific) primers is mandatory to avoid measuring porcine scaffold gene expression. Indeed, after reverse transcription and performing a qRT-PCR analysis, we hardly  r. Take cells from the waterbath and pipette up and down again until cells are homogeneously resuspended using a 1,000 mL pipette and respective tips. s. Add 2 mL of neutralization solution and mix well. t. Perform a centrifugation at 200-250 3 g for 5 min. u. Aspirate supernatant and resuspend in 2 mL DPBS. v. Subject the cells from the PUBs to standard flow cytometry staining and measurement.
Note: When subjecting cells to flow cytometry analysis, antibody specificity for human/murine cells should be tested. Representative flow cytometry results for cell viability and the expression of the pan-human marker HLA-ABC are demonstrated in Figure 6.

EXPECTED OUTCOMES
The most powerful feature of the ex vivo PUB model is the formation of tissue-like structures from a variety of different pancreatic cell systems. Hence, histological analysis including immunohistochemical and immunofluorescence staining allows a detailed analysis of cellular processes such as differentiation, atypia, dysplasia, invasiveness, and cellular interactions. In this respect, the PUB model did not only serve as scaffold for developmental maturation of PSC-based differentiations but also for the study of oncogenic transformation capacity. Examples for histological readouts of cultures on non-sterilized and sterilized PUBs are presented in Melzer et al. 1 as well as in Figure 4. The histological readout proved to be highly reliable if precautions are taken and cells are not spilled during seeding. Flow cytometry-based readouts can identify the target population when a broadly expressed marker such as HLA-ABC is chosen ( Figure 6). From around 300,000 pancreatic progenitor cells that were seeded per ring in two rings per bladder, between 5,000-20,000 human, living cells were detected in flow cytometry analysis. In addition, around 500 ng total DNA per bladder could be retrieved (Figure 7). RNA isolation typically yielded between 250 ng and 400 ng in total per bladder.

LIMITATIONS
The major drawback of using non-sterilized urinary bladders is the potential risk of microbial contamination. Together with ongoing degradation limiting the use of non-sterilized PUBs to 3-4 weeks, we strongly recommend implementing PAA sterilization, if the intended application does not require interaction with host cells. After sterilization, the contamination rate was below 5% and long-term culture for at least 8 weeks was possible (no longer timepoint was investigated but seems likely to be feasible). While histological analysis of PUB samples works very robustly, the application of further downstream applications, e.g., flow cytometry -, RNA-, or DNA-analysis yields rather few cells and low amounts of nucleic acids requiring sensitive isolations kits. In addition, contamination with porcine nucleic acids makes it necessary to carefully design and choose human-/murine-specific primers. Although reproducibility is high for histological readouts, the yield and purity of RNA/DNA may vary from experiment to experiment. To overcome potential drawbacks and variance of DNA and RNA analysis, it is recommended to work at least in PUB triplicates (hence, typically 6 rings per condition). Also, the enrichment of cells of interest by sorting should be considered as option to overcome such hindrances.
Albeit we tested many different cell types and media formulations in the context of pancreatic development and carcinogenesis, the implementation of not yet tested cell systems might need additional optimization.
During the preparation of the PUBs, incomplete de-epithelialization might occur (step 3). This will result in poor engraftment and difficulties to distinguish host cells from cells of interest. Examples for good and poor de-epithelialization and subsequent engraftments are displayed in Figure 8.

Potential solution
Prolong and deepen the mechanical stripping of the porcine urinary bladder as indicated in Methods video S1 (step 3). Consider treating the PUBs with PAA, as this results in a decline of viability and integrity of porcine cells leading to better de-epithelialization ( Figure 8C) (step 5). Always use de-epithelialized control samples without additional cell implantation for histological examination on the quality of de-epithelialization. Use species-specific antibodies to identify your engraftment. Include native porcine urinary bladders as negative controls.
Microbial contamination can occur due to two main reasons. 1. Contamination can be carried along from the beginning, as PUBs are not harvested under sterile conditions (step 2). 2. Sterile handling of PUBs and trans-well systems is more difficult than typical in vitro cultures (step 8).

Potential solution
Contamination carried along from the beginning: Carefully check freshly obtained bladders for any sign of contamination. In case of doubts, discard the bladder (step 2). Consider treating PUBs with PAA to sterilize them. This step leads to a highly reduced rate of microbial contaminations. This is especially relevant, as bacterial infections might only present at later stages after reaching a certain detection limit despite being present from the beginning (step 5). Increase antibiotic concentration in the medium. However, any influence on cell viability should be carefully controlled. Contamination during ongoing culture (step 8).
Always take sterile forceps and use a cleaned laminar flow bench for handling bladders. Ensure that cell culture incubators are cleaned and sterilized regularly. Transfer PUBs to new wells for each medium change.
Do not place forceps directly on the working area of laminar flows.

Problem 3
Degradation of PUB.

Problem 5
Low DNA/RNA yield.
Concentration and/or quality of isolated DNA/RNA might be poor (step 11).

Potential solution
Use isolation methods/kits for nucleic acids that are designed for high yields with low input material. Include replicate samples processed in parallel for histology to assess the size of the engraftment and its cell viability. Consider pooling several bladders from the same condition for increasing the input material.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Alexander Kleger, alexander.kleger@uni-ulm.de.

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not analyze/generate datasets/codes.