TABLE 4.

Comparison of human adult ENS primary culture protocols, with a detailed overview of donor material, dissection techniques, dissociation methods, coating agents, and culture medium specifications. Abbreviations: FCS = fetal calf serum; BSA = bovine serum albumin; HBSS = Hank's balanced salt solution; FBS = fetal bovine serum.

Source	Culture type	Donors	Dissection technique, intestine segment, and BSS	Cell dissociation	Coating substrates and plating	Medium and maintenance
Cirillo et al. 2011 72 and Turco et al. 2014 71	EGC culture	Colorectal cancer patients	Technique II Small intestine Krebs	Digestion solution: Protease (1 mg/ml), collagenase (1.25 mg/ml). Incubation time: 30 min at 37°C.	No coating.	Medium: DMEM‐F12, 10% heat‐inactivated FCS, 1% antibiotic‐antimycotic solution. Grow for 3–4 weeks and use Thy−1.1 antibody‐coated magnetic beads while passaging to eliminate fibroblasts and smooth muscle cells. Perform purification twice.
Soret et al. 2013 74	EGC culture	Patients with colorectal cancer, polyps, Hirschprung, fistula, sigmoiditis, volvulus, or pancreatic adenoma	Technique II Jejunum, ileum, colon (normal segments of resection margin) Krebs	Digestion solution: 250 µl protease (type I from bovine pancreas; stock solution: 5 g/l), 250 µl collagenase (Clostridium histolyticum; stock solution:20 g/L), and 400 µl BSA (stock solution: 50 g/L) in 5 ml of complete medium. Dissociated in a GentelMACS dissociator procedure A−01: 25s at cycles of 400 rpm clockwise and 300 rpm counter clockwise, once before and once after incubation in digestion solution. Incubation time: 45 min at 37°C on a rocker.	Gelatin coating: gelatin type A from Porcine skin 90–110; 0.5% in PBS.	Complete medium: DMEM/F12 supplemented with 10% heat‐inactivated FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 1.1 µg/ml amphotericin B, 20 µg/ml gentamicin, 6 mM glutamine, 2.1 g/l NaHCO3. After 48h half the medium is replaced with complete DMEM medium: DMEM 4.5 g/L glucose, 10% FCS, 2 mM glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin.
Liñán‐Rico et al. 2016 73	EGC culture	Patients with polyps undergoing colectomy or patients undergoing Roux‐en‐Y‐ bypass surgery	Technique II Sigmoid colon or jejunum Krebs	Digestion solution: Protease (1 mg/ml), collagenase (1 mg/ml) in HBSS. Incubation time: 60 min at 37°C. Spin down and resuspend in HBSS (once) and then in DMEM‐F12 0.1% BSA and DNase (50 µg/ml). Collect ganglia with micropipette.	Laminin/Poly‐D‐Lysine coating: 20 mg/ml on 50 mm bottom glass #0 culture dishes.	Medium: DMEM‐F12 (1:1), 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (0.25 µg/ml). Grow for 3–4 weeks and use magnetic microbeads linked to D7‐Fib while passaging to eliminate fibroblasts. Perform purification twice.
Grubišić et al. 2020 70	EGC culture	Patients with Crohn's disease	Technique II Ileum Krebs	Digestion solution: Liberase (0.125 mg/ml), Amphotericin B (0.5µg/ml) in DMEM‐F12. Incubation time: 60 min at 37°C. Spin down and resuspend in DMEM‐F12 0.1% BSA and DNase (50 µg/ml). Collect ganglia with micropipette.	No coating.	Medium: DMEM‐F12 (1:1), 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (0.25 µg/ml). Grow for 3–4 weeks and use magnetic microbeads linked to D7‐Fib while passaging to eliminate fibroblasts. Perform purification twice.