Bovine Hepatic Organoid Culture Medium Kit

1.Pre-screening of tissue specimens: Cell viability of isolated tissue must reach ≥25% to proceed with organoid derivation.

2.Digestion time optimization: Digestion duration for tumor tissues ranges from 20–120 min, which should be adjusted according to tissue hardness and fibrosis degree.

3.Storage and transportation of ex vivo tissues: Total ex vivo storage time of isolated tissues should be controlled within 72 h to guarantee cellular activity.

Literature has reported 2D and 3D culture using cryopreserved tissues, yet the overall establishment success rate remains low. Organoid culture demands high viability of starting stem cells, and cryopreservation causes irreversible damage to stem cell populations. Therefore, fresh ex vivo tissue specimens are strongly recommended over frozen tissues for organoid construction.

Large quantities of red blood cells are commonly present in liver, lung and other tumor tissue samples. Most erythrocytes can be eliminated via repeated tissue washing; commercial red blood cell lysis buffer is an optional alternative. Red blood cell removal is not compulsory and can be decided based on specimen conditions.

Animal-derived specimens carry higher contamination risks than human samples, especially gastrointestinal and urogenital tissues. The culture system can be supplemented with 3–5% gentamicin, penicillin-streptomycin (P/S), and laboratory antifungal agents when necessary to build a multi-layer anti-contamination system.

Tissue-derived organoids:

• Ethical considerations apply to human tissue sources
• Short culture cycle
• Lineage profile highly consistent with original tissue, closely recapitulating human physiological and pathological features; ideal for clinical research and drug screening

iPSC-induced organoids:

• Derived from directed differentiation of pluripotent stem cells
• Longer construction cycle
• High cell purity
• Limited passaging capacity in traditional protocols; optimized systems can generate hepatobiliary organoids within approximately 3 weeks

Passage capacity depends on the stemness status of organoids; organoids with robust stem cell activity can be stably passaged over 20 times. Dedicated serum-free organoid cryopreservation medium is mandatory; general cell freezing medium is not suitable for organoid cryopreservation.

Dissociated single primary cells activate replicative senescence pathways, leading to restricted proliferation. In contrast, organoid cultures retain self-renewing stem/progenitor cell populations, and specialized organoid culture medium maintains stem cell properties while suppressing excessive differentiation, enabling long-term serial passaging.