TABLE 2.

Comparison of murine adult ENS primary culture protocols, with a detailed overview of mouse strains, age, dissection techniques, dissociation methods, coating agents, and culture medium specifications. Abbreviations: HBSS = Hank's balanced salt solution; FBS = fetal bovine serum.

Source	Culture type	Mouse strain and age	Dissection technique, intestine segment, and BSS	Cell dissociation	Coating substrates and plating	Medium and maintenance
Lowette et al. 2014 28	ENS culture	Adult C57BL/N6 Mouse (8–9 weeks)	Technique I Ileum Krebs‐HBSS	Digestion solution: Collagenase (14.67 mg/ml), protease (10 mg/ml), albumin (5% in PBS). Incubation time: 8 min at 37°C. Stopped with Krebs, 10% FBS at RT.	Poly‐D‐lysine‐laminin double coating: Poly‐D‐lysine hydrobromide (0.5 mg/ml in borate buffer). Laminin (20 µg/ml).	Complete medium: DMEM‐F12 (1:1), 10% FBS, 1% glutamine, 0.5% pen/strep. Serum‐free medium: DMEM‐F12, with NGF (0.05%), N2 (0.2%), G5 (0.2%). Medium replaced by serum‐free medium after 24h.
Zhang and Hu. 2013 25	ENS culture	Adult Mouse	Technique II Complete small intestine HBSS	Collagenase digestion medium: 1 mg/ml collagenase IV, 0.5 mM CaCl2, 10 mM HEPES in HBSS. Trypsin Digestion medium: 0.05% trypsin with 0.53 mM EDTA in HBSS. Digestion neutralizing medium: 500 U DNase I, 1 mg/ml BSA in DMEM/F12 medium.	Matrigel coating: BD GF‐reduced Matrigel: 60% laminin, 30% collagen IV, 8% entactin, diluted 1:2000 in ice‐cold DMEM/F12 medium. Density of seeding: 1 × 105/cm2; with enteric neuronal culture medium.	Enteric neuronal culture medium: DMEM/F12 medium (39.5 ml), chick embryo extract (7.5 ml), penicillin/streptomycin (100×, 0.5 ml), gentamicin (500×, 100 µl), amphotericin (100×, 0.5 ml), N2 (100×, 0.5 ml), B27 (50×, 1 ml), glutamine (100× 0.5 ml), FGF‐b (10 µg/ml, 50 µl), EGF (10 µg/ml, 100 µl), heparin (0.2%, 5 µl). Medium change every day.
Smith et al. 2013 27	ENS culture	Adult Mouse	Technique I Ileum Krebs solution	Collagen digestion solution: 13 mg collagenase type II, 3 mg BSA in 10 ml carbogen‐bubbled Krebs. Incubation time: 60 min at 37°C. Trypsin digestion solution (0.05% trypsin): 5 ml of 0.05% trypsin solution. Incubation time: 7 min at 37°C in shaking water bath. Rinse medium: F12 medium with 10% FBS and antibiotic/antimycotic: to 500 ml bottle F12 media add 50 ml FBS and 5 ml antibiotic/antimycotic 100× liquid.	Poly‐D‐lysine‐laminin double coating: 1 ml/25cm2 poly‐D‐lysine: 80 µl poly‐D‐lysine stock/CS. 5 µg/cm2 laminin: 200 µl stock/CS.	Neuronal culture medium: Neurobasal‐A medium, B27, 2 mM L‐glutamine, 1% FBS, 10 ng/ml GDNF and antibiotic/antimycotic 100× liquid. Change half of medium every 2 days.
Wahba et al. 2016 29	ENS culture	CD−1 mouse (4–6 month)	Technique Ib Complete small intestines Krebs‐Ringer Solution	Collagenase digestion solution: 1 ml per mouse: 1 mg/ml collagenase IV, 0.5 mM CaCl2, 10 mM HPES in HBSS. Incubation time: 15 min at 37°C in water bath with manual constant rotation once every 5 min. Trypsin digestion solution: 0.05% trypsin, 0.54 mM EDTA in HBSS, 1 ml/mouse. Incubation time: 10 min in 37°C water bath with manual rotation and inverting of tube every 5 min once.	5 different culture substrates tested: Poly‐D‐lysine (0.1 mg/ml, 50 µl, overnight at RT), Collagen (100 µg/ml, 50 µl, overnight at RT); Matrigel (MG) (1.8 mg/ml, 100 µl: 15 µl of MG in 85 µl culture media, 2h at 37°C); Poly‐D‐lysine /MG mix (Poly‐D‐lysine: 0.03 mg/ml 50 µl; MG: 0.5 mg/ml 15 µl in 85 µl media mix, 2h at 37°C); Collagen/MG mix: Collagen: 50 µl of 33µg/ml; MG: 0.5 mg/ml, 15 µl in 85 µl culture medium, 2h at 37°C). Wash once with pre‐warmed (37°C) HBSS.	Complete medium: DMEM/F12, 2% (v/v) FBS, 100 U/ml pen, 100 µg/ml strep, 1 × B27, N2, 7.2 mg/L uridine triphosphate, 50 mg/L gentamycin. Medium change every day.
Brun and Akbarali. 2018 26	ENS culture	Adult C57BL/6 J mice	Technique I Ileum Krebs solution	Neuronal digestion solution: 13 mg collagenase type II + 3 mg BSA in 10 ml RMPI medium 1640. Incubation time: 15 min at 37°C in water bath under shaking conditions. 0.05% Trypsin solution: 1 ml warmed 0.25% trypsin in 4 ml warmed RPMI medium 1640. Incubation time: 7 min at 37°C water bath shaking. Digestion stopped with Krebs solution.	Poly‐D‐lysine‐laminin double coating: Dilute poly‐D‐lysine stock (0.01% in ddH2O) and take 150 µl stock/cm2. Incubate at RT for 30 min. Dilute laminin stock to 5 µg/ml with ddH2O, take 100 µl of laminin solution/cm2. Incubate at RT for 1h.	Complete neuron medium: Neurobasal‐A medium/B27, 1 × L‐glutamine (2 mM), Pen/strep (100 U/ml and 100 µg/ml), sodium pyruvate (1 mM), 1%FBS, 10 ng/ml human recombinant GDNF. 150 µl of cell suspension in 1 ml complete medium. Medium change every 2 days. For electrophysiological studies: add 850 µl of cell/cm2 to the coated CS.
Verissimo et al. 2019 31	EGC culture	Adult mouse	Technique II Complete colon PBS with Pen/strep and fungizone	Enzymatic dissociation: Collagenase II (Gibco), DNase I (Sigma chemical). 60 min at 37°C. Mechanical dissociation and centrifugation 2×.	Poly‐L‐lysine‐laminin and Fibronectin single coating: Poly‐L‐lysine‐laminin substrate: 50 µg/ml in acid buffer (20 mM sodium acetate in 2 mM Calcium chloride), incubated with CS at least 12h at 37°C, 3× wash with PBS. Fibronectin substrate: 50 µg/ml in DMEM/F12, incubated with CS for 1h at 37°C, removed from wells and dried. Re‐plated after 3 days in culture to distribute cells homogenously.	Medium: DMEM/F12, 2 mM glutamine, 3 mM sodium bicarbonate (NaHCO3), 0.5 mg/ml pen/step, 10%FBS, 2% chick embryo extract.
Wang et al. 2018 30	EGC culture	Adult C57BL/6 mice (8 weeks)	Technique I, but isolation of mucosa, submucosa, and circular muscle Proximal small intestines DPBS w/o Ca2+ or Mg2+	Non‐enzymatic digestion: Sequential HEPES‐buffered EDTA incubations and gentle trituration. Isolation solution: EDTA/HEPES/DPBS dissociation solution (500 ml): sterile DPBS (w/o Ca2+ and Mg2+) to make final solution of 10 mM HEPES and 5 mM EDTA. Use 490 ml DPBS, 5 ml 1 m HEPES buffer, 5 ml 0.5 m EDTA stock. Cell recovery solution: commercially available.	Poly‐D‐lysine ‐laminin double coating: 1 mg/ml poly‐D‐lysine stock diluted 1:10 in sterile tissue culture grade water. Final concentration: 100 µg/ml; 2 ml per well in 6‐well plates; Incubate 1 h. Dilute laminin (0.5 mg/ml stock) 1:50 in DPBS. final concentration: 10 µg/ml; 1 ml per 6‐well plate. Incubate 2h at 37°C, then remove laminin wash gently 3× with sterile DPBS.	Glia cell resuspension media: 44.5 ml DMEM/F12, 5 ml FBS (10%), 500 µl Pen/Strep (100 U/ml Pen, 100 µg/ml Strep), 20 µl Gentamicin (20 µg/ml). Glial cell growth medium: 44 ml DMEM/F12, 5 ml FBS (10%), 500 µl Pen/strep (100 U/ml pen, 100 µg/ml strep), 20 µl Gentamicin (20 µg/ml), 50 µl GDNF (10 ng/ml), 500 µl L‐glutamine (2 mM). 200 µl cell suspension onto each CS. Complete medium change every day.