, Mg 2+ free Phosphate buffered saline (PBS)
, PBT solution: 0.5% Triton-X100 is diluted in PBS
, Blocking solution: 10% normal donkey serum and 1% Bovine Serum Albumin (BSA), Fraction V, are diluted in PBS
, Mounting medium: 70% glycerol diluted in PBS (see Note 5)
, Ethanol solutions: 70, 75, 85, 90, 95 and 100% histology grade ethanol (v/v) diluted in Milli-Q -purified water
, 5-ethynyl-2'-deoxyuridine (EdU) solution: EdU powder (#A10044, Thermo Scientific) is diluted at 10mg/mL in sterile PBS (see Note 6)
, , vol.7001
,
, Click-iT EdU Alexa Fluor 488 Imaging Kit (#C10337, Thermo Scientific)
,
, Antibody diluent: 1% BSA is diluted in PBS
, Antibodies and fluorescent counterstain (see Table I)
, Sacrifice mice in accordance with the approved IACUC animal protocol in place (Isoflurane inhalation followed by cervical dislocation) 8 to 12 weeks post-transplantation
, Cut the tHIO using a razor blade to expose the interior lumen (see note 23)
, Using 0.2 mm diameter minutien pins, secure the piece of tissue on a silicone-coated glass Petri dish filled with ice-cold DPBS
, Stretch and pin the tissue flat with the mucosal side facing up (see Note 24)
, Gently scrape the surface of the mucosa with curved forceps to remove villi and debris and any mucus that is present
, Wash the tissue 3-4 times with ice-cold chelation buffer to remove villi and debris (see note 25)
, Cover the biopsy with freshly prepared 2mM EDTA chelation buffer (see Note 26)
, Place the Petri dish on ice and shake gently for 30 min on a horizontal orbital shaker
, Wash the tissue with ice-cold chelation buffer without EDTA
, Gently scrape the mucosal layer to release the intestinal crypts using curved forceps, vol.27
, Gently remove the crypt suspension from the petri dish using a pipette and transfer it to a 15 mL conical tube (see Note 28)
, Filter the crypt suspension through a 150 µm nylon mesh (see Note 29)
, Centrifuge the crypt suspension 5 min at 50 ´g, 4°C and discard the supernatant
, Resuspend the pellet in 1 mL ice-cold chelation buffer
, Centrifuge the crypt fraction for 10 min at 150 ´g, 4°C and remove the supernatant
, Resuspend the crypt pellet in basement membrane matrix using pre-chilled pipet tips
, Apply 50 µl of crypt suspension in basement membrane matrix per well on the pre-warmed plate. Slowly eject the basement membrane matrix
, Replace the medium with fresh Intesticult® Human Organoid Growth medium every 2 days for 8-10 days (see Notes 30 and 31) (Figure c-e)
, Figure 2: Enteroids derived from transplanted Human Intestinal Organoids (tHIO)
, Example of an experimental setup to generate tHIO-derived enteroids. (b) Close-up picture on representative tHIO crypts picked up before embedding in Matrigel®. (c-d) tHIOderived enteroids in Matrigel® after 2 days in culture. (e) Immunofluorescence staining of tHIO-derived enteroids. DAPI is shown in blue
, Divide intestinal growth medium into 10 mL aliquots in 15 mL conical tubes and freeze at -20°C for up to 3 months. Store thawed aliquots up to 5 days at 4°C without loss of activity
, Males are preferably used for the kidney subcapsular transplantation as their kidneys are bigger and easier to access
, The chow diet is supplemented with antibiotics and given to the mice at least 14 days prior any surgeries. The antibiotics decrease inflammation and risk of infection
, The anesthesia system delivers an isoflurane and oxygen mixture that can be controlled and monitored to maintain the anesthesia during surgery. The extra anesthetic gas is collected and evacuated into a canister
, Aqueous mounting media can also be used to mount coverslips onto microscope slides
, Desiccated 5-ethynyl-2'-deoxyuridine (EdU) is resuspended in PBS and heated at 70°C for
, Intestinal growth medium supplemented with 100 ng/ml recombinant Wnt3a (1:1000 dilution of 100 µg/ml stock), 1 µg/ml R-spondin 1 (1:1000 dilution of 1 mg/ml stock, p.100
, dilution of 100 µg/ml stock), and 50 ng/ml EGF (1:10 000 dilution of 500 mg/ml stock) can be used to culture HIO-derived epithelial organoid. Alternatively, recombinant growth factors could be replaced by Wnt3a, R-spondin, and Noggin conditioned media
, Intestinal growth medium aliquots are thawed and can be kept up to 5 days at 4°C without loss of activity. Add the human recombinant EGF prior media change (1:10 000 stock dilution)
, HIOs can be transplanted from 20 to 40 days of culture in vitro. 10. Final anesthetic gas concentration is achieved by delivering 2% Isoflurane with 2, pp.5-8
, Analgesia provisions are most effective at reducing the intensity of painful stimulation when given prior to the surgery. Any animal showing evidence of pain should be provided analgesia
, Keep the animal warm using a 37°C heating-pad. Adjust anesthetic gas concentration to
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