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

Abstract

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.

Keywords: Cell Differentiation; Cell culture; Material sciences; Stem Cells; Tissue Engineering.

Graphical abstract

Figure 1

Cleaning of porcine urinary bladders (A) PUB in correct orientation in a 15-cm cell culture dish. Urinary bladder is marked in black. Adjacent tissue is marked in blue. (B) Urinary bladder after removal of adjacent fat tissue. Urinary bladder is marked in black. Urethra is marked in blue. (C) Urinary bladder after removal of fat tissue and urethra. The black line indicates opening of the bladder along an imaginary middle line vertical to the urethra. (D) PUB after sectioning along such middle line. Arrows indicate the direction to flap the two parts for complete opening. (E) Urinary bladder from the inner side displaying the urothelial layer. (F) Example of a small bleeding within the urinary bladder. After removal of the site of bleeding, the bladder can be still used. (G) Zoom-in to the small bleeding area presented in (F). (H) Urinary bladder after turning it upside down now displaying the outer wall. The square indicates one ureteral ostium which should be removed. (I) Zoom-in to the ureteral ostium. (J) Example of a large bleeding area on the outer side of the urinary bladder. The whole bladder should be discarded. (K) Example of a completely submerged bladder in wash solution. Scale bars indicate 1 cm.

Figure 2

Example of a microbial-contaminated PUB (A) 6-well plate with two microbial contaminated PUBs in the two middle wells depicted from the top view. (B) View from below of the two contaminated wells in the middle. (C) View from top on the contaminated wells without the PUBs.

Figure 3

Preparation of fixed PUB samples for histological analysis Tissue outside of the implantation site can be removed (indicated by rectangle). If PUBs are used to investigate invasion they should be divided in the middle of the ring (indicated by the dashed line). The image on the right presents an example of a PUB after cutting the middle of the implantation site for vertical sectioning. Scale bars indicate 1 cm.

Figure 4

Examples of successful engraftment and sectioning orientation (A) Example of horizontal sectioning of a tumor cell engraftment after 21 days. Dispersed tumor cells are found within the scaffold indicative of potential invasion. Stars indicate examples of suspected invasive cancer cells. However, invasion in this orientation should be carefully considered. (B) Example of tumor cells invading the PUB as investigated by vertical sectioning. Arrows indicate tumor cell infiltration.

Figure 5

Workflow for isolation of cells from PUBs The upper panel is a schematic representation of the workflow and timing necessary to isolate cells from the PUBs. The middle panel shows the transfer of PUBs from cell strainers to 12-well plates filled with DPBS. Tissue not adjacent to the rings is removed with scissors. After shaking PUBs in DPBS and careful removal of the rings, PUBs are transferred into new wells, filled with collagenase-II working solution. After a 1–3 h incubation on a shaker, the supernatant, containing the cells of interest, is collected in a centrifugation tube, PUBs are additionally rinsed with neutralization solution, and neutralization solution is added to the centrifugation tube. After centrifugation and washing in DPBS, cells are incubated in accutase for 30 min to achieve singularization of the cells. PUB-derived cells are then stained with specific antibodies to perform flow cytometry.

Figure 6

Representative flow cytometry results after isolation of cells from the PUB PUBs without prior cell seeding (scaffold) and PUBs with seeding of human pancreatic progenitor cells and cultivation for 6 weeks were treated with collagenase-II. Subsequent singularization was performed with accutase. Cells were stained for DAPI to exclude dead cells and with a human-specific HLA-ABC-APC antibody according to manufacturer`s recommendations. Presence of viable cells expressing HLA-ABC was only detected in samples with prior seeding of human cells.

Figure 7

Representative results of DNA and RNA yields after isolation of cells from PUB (A) Bar graphs of total DNA yield after digestion of PUBs with cells of interest and scaffold only samples as measured with a Nanodrop. Mean ± S.E.M. (B) Bar graphs of total RNA yields of PUBs with cells of interest and scaffold only samples as measured with a Nanodrop. Of note, the Nanodrop device does not differ between fragmented and non-fragmented nucleic acids. Mean ± S.E.M. (C) Gene expression is displayed as 40-Ct-values of cells harvested from PUBs (Cells of interest vs. scaffold only vs. cDNA H2O control sample). If threshold was not reached Ct-values were set to 40. Line indicates mean. n = 2 independent PUBs.

Figure 8

Examples for de-epithelialized and re-epithelialized PUBs (A) Example of a native porcine urinary bladder with intact urothelial cell layer (indicated by red line) and sub-epithelial stroma (indicated by black line). (B) Example of a successful de-epithelialization with no remaining urothelial cell layer. Stroma is indicated by the black line. (C) Example of a de-epithelialized, sterilized, and re-epithelialized urinary bladder. The black line indicates the stromal compartment where no or only few nuclei and intact cells are found. The blue line indicates the cells of interest that were seeded onto this scaffold 2 weeks before histological examination. (D) Example of a good engraftment after proper de-epithelialization on a non-sterilized urinary bladder. Human cells are stained by an anti-human nucleoli antibody (Abcam, ab190710) and are indicated by the green line. (E) Example of a poor engraftment of human cells on a non-properly de-epithelialized porcine urinary bladder. Human cells are stained by an anti-human nucleoli antibody and are indicated by the green line. Next to the human cells, remnants of the porcine urothelial cell layer are present. Scale bars indicate 100 μm.