Intestinal organoids | • Chopping into ~5 mm pieces[81]• Cutting tissues into 5 mm pieces; mechanically separating crypts in HBSS–EDTA[82]• Mincing tissue to ~ 1 mm pieces with scalpels[83]• Digesting with LiberaseTM DH and hyaluronidase[84]• Mincing into small pieces and enzymatically digesting with collagenase IV[85]• Finely mincing of human colorectal tissues and digesting with collagenase Type 1[86]• Chopping rectal cancer samples to 1 mm pieces in PBS-DTT buffer; resuspending vigorously in ADF medium; settling for crypt inspection by microscopy, repeating until no crypts detected[87] | +++ |
Liver organoids | • Mincing the tissue into pieces of roughly 0.5 mm3 using fine scissors, pipetting the sample up and down to remove red blood cells and fat, then digesting with collagenase and dispase II for mouse and collagenase D for human[88]• Mincing into pieces (~ 0.25–1 cm3) and incubating at 37 °C with the digestion solution[89] • Collagenase-accutase digestion after mechanical dissociation[60, 89–91]• Isolating hepatocytes from mice or human adult liver via two-step collagenase digestion; mechanically fragmenting and re-seeding organoids 14 days post-seeding[91] | + |
Pancreatic organoids | • Mincing into small pieces, then digesting with collagenase II and further digested with TrypLE[92]• Mincing into pieces, washing with 10 mL AdvDF+++, and digesting with collagenase II[93, 94]• Mincing into small portions using a scalpel, then digesting with digest medium including collagenase XI, DNase, and Y27632[95]• Mincing the tissue into pieces of roughly 0.5 mm3 using fine scissors; Pipetting the sample up and down to remove red blood cells and fat. Digesting with medium containing collagenase and dispase II for mouse and collagenase D for human[88] | + |
Kidney organoids | • Chopping into small pieces with surgical blade, shaking for 25 min, pipetting ~ 25 times to dissociate the tissues into single cells[96]• Mincing into ~ 1-mm3 pieces, following digestion with AdDF+++ containing collagenase and Y-27632[97]• Isolating tubular fragments from human cortical kidney or mouse kidney tissue through collagenase digestion[98]• Mincing into small pieces and digesting with collagenase II with ROCK inhibitor Y-27632 dihydrochloride, following enzymatic digestion in TrypLE Express. Subsequently, centrifuged pellets were pipetted up and down to further dissociate tissue fragments[99] | ++ |
Lung organoids | • Pipetting up and down to passage lung organoids. If single cells are not required, or enzymatic digestion is not suitable for downstream applications, mechanical dissociation is recommended[100]• Dissecting pleura and large airways; processing into single-cell suspension with dispase, collagenase I, and DNase I[101]• Digesting human tumor specimen with DNase I and collagenase/dispase in DMEM/F12 medium[102]• Mincing tumor samples with scissors; digesting with collagenase II and Y-27632, preceded by TrypLE Express and Y-27632[103]• Mincing non-small-cell lung cancer biopsies with scissors; digesting in collagenase type II in Advanced DMEM/F12 with gentle shaking[104] | + |
Brain organoids | • Either mildly disaggregating into small cell clusters by incubation in StemPro™ Accutase™ or cutting into 0.5–1 mm pieces using tweezers and scalpels[105].• Dissociating patient tissue samples by either finely mincing or enzymatic digesting into single-cell suspensions[106, 107]• Cutting into 1 mm in diameter pieces using fine dissection scissors, and later cutting into 0.5 mm in diameter pieces for propagation[29]• Cutting glioma specimens into 1–2 mm³ pieces with dissection scissors and suspending in Short-Term Glioma Organoid Medium[108] | +++ |
Gastric organoids | • Mechanically dissociating mouse stomach with micro-dissecting scissors and fine forceps; cutting into pieces < 5 mm²; shaking in DPBS (without Ca2+ and Mg2+, with EDTA); followed by pipetting up and down[109]• Cutting human samples into ~ 5 mm pieces; incubating with chelating solution; pipetting up and down to extract glands[16, 110]• Mincing epithelial tissue with surgical razors; digesting fragments with collagenase type I[111]• Mincing gastric cancer specimens following enzymatic digestion with dispase II and collagenase XI[112, 113], or with EDTA and TrypLE[13], or Liberase TH and TrypLE Express[114], or collagenase and hyaluronidase[115] | ++ |
Breast/Female genital organoids | • Mechanically shearing Stage IV high-grade serous cancer (HGSC) specimens[28]• Mincing mouse mammary glands into 1 mm pieces; digesting with collagenase and hyaluronidase[116]• Cutting human ovary cancer tissues into 3–5 mm3 pieces; digesting remaining large pieces in Advanced DMEM/F12 with RHO/ROCK pathway inhibitor and collagenase[117]• Mechanically dissociating human breast cancer tissue samples with scalpels or razor blades; obtaining single-cell suspensions or cell clumps through enzyme digestion using collagenase, DNase, dispase, hyaluronidase, trypsin (TrypLE), or enzyme mixes like liberase[61] | ++ |
Male genital organoids | • Finely cutting prostate cancer samples with scissors to produce small tissue fragments, termed “aggregates”[118]• Mincing mouse or human prostate lobes into small pieces (~ 1–5 mm³) with a scalpel, followed by digestion in collagenase II with Y-27632[8]• Mechanically dissociating prostate cancer samples with scissors or generating a cell suspension through further digestion with Collagenase II[118–120]• Cutting human testis cancer specimens into approximately 1 mm³ pieces and culturing in hanging drops[121] | ++ |