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ISOLATING PORCINE BLADDER TO EXPANDED MESENCHYMAL STEM CELLS

Create Type 1 Biosafety Cabinet from Acrylic sheets, order pre cut to size, then joined together, with front opening access
Drill holes and screw together Acrylic sheets, drill hole in rear top corner for 12 volt PC extract fan and container filter
Incubator for cell culturing set to 37 ℃, 5% CO2. Estabish a clear process for sanitation. Pressurising the biosafety cabinet air flow ,drawing air upwards, filtering the air, for the state of being clean and conducive to health, sanitary (free of germs) as by sterilising
Two 100% CO2 (carbon dioxide tanks with Regulator With Single Gauge and Adjustable Output Pressure set to one bar and clear hose)
Xenogeneic source cells from a fresh domestic pig (less than 10kg), stored on a tray of ice, collected from local farm. Begin process 15 minutes from execution, maximum 1 hour before Rigor mortis.
Start dissection from hind leg where there is loose skin, to expose Abdomen, carefull pulling skin away to preserve internal organs
Kidneys (bean shaped) are positioned at the back of Abdomen, follow Ureters to bladder (sack shaped). Either side of the urinary bladder is the two umbilical arteries that connect to the umbilical cord.
Remove bladder, which is full of growth factors.
Carefully remove small blood vessels.
Urinary bladder was trimmed to remove external connective tissues, including adipose tissue, cleaned with PBS of all residual urine, any thin layers of Epithelium
Schematic of a bladder and the different layers. The urothelium is the layer that lines the bladder lumen and forms the urine-body barrier. The urothelium is the primary barrier layer and is subject to insults, such as injury, inflammation, and infection, and requires continued maintenance and repair. The lamina propria is a connective tissue layer that contains nerves and vessels (blue line = basement membrane, red lines = blood vessels, black lines = nerves). The muscularis propria is the muscular layer that provides structural support to the bladder and facilitates its physiological functions of filling and emptying. The serosa is the outermost layer.
Cut along one side to open bladder to become flat.
Petri Dishes, 150 mm x 25 mm, Clean using PBS , then disect into 1mm to 2mm pieces,using Mayo Scissors and Tissue Forceps.
Finally drain all liquid using sterile 40 µ cell strainer leaving only tissue, plate on petri dish with trypsin for partial incubation at 37 ℃ and 5% CO2.
Plate pieces into flasks (Falcon Polystyrene Cell Culture Flask with Blue Plug-Seal Screw Cap), add some culture medium to dilute trypsin, drain using cell filters.
The disected tissue is distributed into a cell culture flask is suspended with adequate amounts of complete medium ( [DMEM]Culture Medium l‐glutamine, and sodium pyruvate), [FBS] (Fetal bovine serum).
Complete medium in the cultured flask was replaced every 2 days. After 1 week, nonadherent cells were washed off with phosphate‐buffered saline (PBS), and fresh medium was added. Equipment needing Pipettes, Ethanol spray for sterilising gloves, wiping bottles with sterile tissue roll and cleaning biosafety cabinet. Once passage 0 (P0) cells reached 70% confluency in the seeded flask, the colonies were trypsinised and seeding passaged cells are subcultured. The passaged cells suspensions were rinsed to remove traces of completed medium. After a final rinse with sterile PBS, the cells were carefully introduced into a new syringe. This was performed under aseptic conditions.
The third passaging of cells has the fastest cell outgrowth (Log Phase Growth)
Olympus CK2 Compound Inverted Phase Contrast Microscope, with Olympus ULWCD 0.30 phase contrast condenser
Olympus EA 100x Oil, NEA4x, NEA10x and NEA40x objectives and a pair of Olympus CWHK 10x/18L eyepieces.
Using cell microscopy (compound inverted phase contrast microscope) for live cell imaging, view cells dividing, Passage culture medium then begin standard Cell culture for Mesenchymal stem cell expansion of plastic adherent cells.
16 hour time lapse reduced to 14 seconds of Mesenchymal stem cells in cell culture.
Allow the tissue pieces to settle for 3 minutes, centrifuge, then carefully aspirate the supernatant conditioned medium (Containing vast quantities of Biomolecules, Extracellular vesicles (EVs), Microvesicles, Exosomes, and Cytokines for Paracrine signaling)


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