A Complete Guide to Standardized Organoid Passaging

Organoid technology has rapidly emerged as a powerful biological model for disease research, drug screening, and precision medicine. However, despite its promise, inconsistent passaging practices remain one of the biggest obstacles to reproducibility and long-term stability.

This guide provides a systematic, step-by-step framework for standardized organoid passaging, helping researchers achieve reliable expansion, high viability, and consistent experimental outcomes.

I. When to Passage Organoids: Defining the Right Time

1. Morphological Criteria

Organoids are generally ready for passaging when they reach a diameter of 200–500 μm

(human-derived organoids tend to be larger than mouse-derived ones).

Key indicators of healthy organoids include:

• Smooth, intact edges
• No central darkening, necrosis, or large vacuoles

Model-specific cues should also be considered:

• Esophageal organoids: Appearance of keratin pearls between days 9–14 marks the optimal passaging window
• Intestinal / pancreatic organoids: Branching or “cauliflower-like” structures indicate maturity (optional, not mandatory)
• Neural organoids: Functional validation is required, such as β-III tubulin staining or electrophysiological activity

2. Functional and Growth Indicators

Immediate passaging is recommended if:

Daily growth rate drops below 5%, or culture medium turns yellow within 48 hours

For secretory organoids, functional markers should be monitored:

• ≥ 20% positive cells (e.g., MUC2⁺ goblet cells in intestinal organoids) 

II. Standardized Passaging Workflow (24-Well Plate Example)

Step 1. Pre-Cooling of Reagents and Consumables

• Matrix gel: Thaw overnight on ice at 4 °C; avoid repeated freeze–thaw cycles

(>2 cycles may reduce gel strength by >30%)

• Consumables: Pipette tips, tubes, and PBS should be pre-cooled on ice for at least 30 minutes

to prevent premature gel polymerization

Step 2. Matrix Removal

• Aspirate culture medium
• Gently wash twice with ice-cold PBS along the well wall
• Place the plate on a horizontal shaker at 30 rpm for 30 seconds to remove residual gel fragments completely

Step 3. Dissociation

Mechanical dissociation (recommended for fragile or normal epithelial organoids):

• Gently pipette 5–10 times using a 200 μL tip
• Filter through a 100 μm strainer

Enzymatic dissociation (for dense or tumor organoids):

Add 200 μL ice-cold Accutase or TrypLE™ per well

• Incubate at room temperature for 5–7 minutes(extend slightly for dense tumors)
• Monitor under microscope; stop digestion when edges appear “fuzzy and bright”
• Immediately quench with an equal volume of ice-cold medium containing 10% FBS

Step 4. Washing and Centrifugation

• Collect suspension into a 15 mL tube
• Centrifuge at 200 × g, 4 °C, for 5 minutes
• Discard supernatant, resuspend in 1 mL ice-cold PBS, and repeat centrifugation to completely remove FBS

Step 5. Re-Embedding and Seeding

• Mix cell pellet with matrix gel at a 1:3 (v/v) ratio
• Final gel concentration should be ≥70%(concentrations <60% often cause 3D structure collapse)
• Deposit 20 μL droplets vertically in the center of a pre-warmed 24-well plate
• Polymerize at 37°C for 15 minutes
• Gently add 500 μL complete medium along the well wall

III. Post-Passaging Quality Control

1. Fragment Size Selection

Filter mechanically dissociated fragments through a 100 μm strainer

Discard fragments <50 μm(small fragments show only 10–15% organoid formation efficiency)

2. Do Not Change Medium for 48 Hours

• Retain autocrine survival factors
• Early medium replacement can reduce re-establishment efficiency by ~30%

3. Eliminate Air Bubbles

• Air bubbles inside gel result in 100% hypoxic necrosis
• If bubbles >200 μm appear, gently remove using a 10 μL pipette tip

IV. Recommended Passaging Ratios

Organoid Type	Ratio	Method	Recovery Rate
Normal intestine / stomach	1:4	Enzymatic + gentle pipetting	80–90%
Primary tumor (first passage)	1:2	Enzymatic	60–70%
Dense solid tumor (later passages)	1:6	Mechanical shearing	50–60%

VI. Troubleshooting Guide & FAQ

Issue / Symptom	Possible Cause	Recommended Solution
Organoids fail to bud or expand	Low seeding density; insufficient matrix concentration	Increase seeding density to ≥30 clusters per well; ensure final matrix concentration is ≥70%
Central darkening or necrosis	Excessive gel thickness; trapped air bubbles	Limit gel droplet thickness to ≤200 μm; carefully remove bubbles using a 10 μL pipette tip
Rapid medium acidification (yellowing within 24–48 h)	Over-digestion; excessive centrifugation force	Shorten digestion time; reduce centrifugation to ≤200 × g
Low organoid recovery after passaging	Fragment size too small; harsh dissociation	Discard fragments <50 μm; use gentle mechanical dissociation or reduce enzyme exposure
High variability between passages	Batch variation in matrix or growth factors	Use batch-qualified matrix and pre-formulated, consistent media
Premature gel polymerization during handling	Reagents or consumables not pre-cooled	Pre-cool matrix, PBS, tips, and tubes on ice for ≥30 minutes
Poor re-establishment after passaging	Medium changed too early	Do not change medium during the first 48 h post-passaging
Organoids collapse or lose 3D structure	Matrix concentration too low	Maintain matrix concentration at ≥70%
High cell death immediately after passaging	Excessive enzymatic digestion	Monitor under microscope and stop digestion when organoid edges become bright and fuzzy
Inconsistent growth across wells	Uneven gel droplet size or placement	Dispense 20 μL droplets vertically in the center of each well

VI. Yeasen’s Complete Organoid Culture Solutions

1. Ceturegel^TM Matrix

High biosafety: Free of LDEV, mycoplasma, bacteria, and fungi

Endotoxin ≤8 EU/mL—suitable for long-term culture and in vivo implantation

Batch-to-batch consistency: Concentration range 8–20 mg/mL; high-concentration option ≥18 mg/mL;

Single-batch production >30 L

Versatile 3D support: Rapid polymerization at room temperature; compatible with organoids, angiogenesis, tumor invasion, and in vivo tumorigenesis; Phenol-red-free option available for fluorescence imaging.

Category	Product Name	Cat. No.	Size	Note
Basic concentration (8–12 mg/mL)	Ceturegel™ Matrix LDEV-Free	40183ES08/10	5 mL / 10 mL	Suitable for 2D/3D culture, invasion and migration assays, and in vivo tumor formation studies.
	Ceturegel™ Matrix Phenol Red-Free, LDEV-Free	40184ES08/10	5 mL / 10 mL
High concentration (18–20 mg/mL)	Ceturegel™ Matrix High Concentration, LDEV-Free	40187ES08/10	5 mL / 10 mL	Viscous and gels quickly; ideal for in vivo tumor formation with hard-to-grow cell lines.
	Ceturegel™ Matrix High Concentration, GFR, LDEV-Free	40189ES08/10	5 mL / 10 mL
	Ceturegel™ Matrix High Concentration, Phenol Red-Free, LDEV-Free	40188ES08/10	5 mL / 10 mL
Low Growth Factor	Ceturegel™ Matrix GFR, LDEV-Free	40185ES08/10	5 mL / 10 mL	Minimizes growth factor interference in signaling pathway studies.
	Ceturegel™ Matrix GFR, Phenol Red-Free, LDEV-Free	40186ES08/10	5 mL / 10 mL
Stem Cell	Ceturegel™ Matrix hESC-Qualified, LDEV-Free	40190ES08/10	5 mL / 10 mL	Mainly used for hESC/iPSC stem cell culture.
Organoid-Specific	Ceturegel™ Matrix for Organoid Culture, Phenol Red-Free, LDEV-Free	40192ES08/10	5 mL / 10 mL	Upgraded organoid matrix gel for normal and tumor tissues with improved culture performance.

 2. Cebrary^TM Ready-to-Use Culture Media

Open and use: Pre-mixed at optimized ratios—no repeated titration required

High performance: Efficient crypt isolation and robust intestinal organoid formation

Serum-free & stable: Batch CV <10%, improving experimental success rates by ~30%

Human Tumor Organoid Culture Medium
Colorectal Cancer Organoid Growth Medium	Gastric Cancer Organoid Growth Medium	Liver Cancer Organoid Growth Medium	Breast Cancer Organoid Growth Medium
Lung Cancer Organoid Growth Medium	Esophageal Cancer Organoid Growth Medium	Ovarian Cancer Organoid Growth Medium	Pancreatic Cancer Organoid Growth Medium
Prostate Cancer Organoid Growth Medium	Kidney Cancer Organoid Growth Medium	Bladder Cancer Organoid Growth Medium	Endometrial Cancer Organoid Growth Medium
Head and Neck Squamous Cancer Organoid Growth Medium
Normal Tissue Organoid Culture
Human Colorectal Organoid Growth Medium	Human Colorectal Organoid Growth Medium	Human Colorectal Organoid Growth Medium	Human Colorectal Organoid Growth Medium
Human Small Intestine Organoid Expansion Medium	Human Small Intestine Organoid Expansion Medium	Human Small Intestine Organoid Expansion Medium	Human Small Intestine Organoid Differentiation Medium

 3. DIY Modular Components

Flexible selection of growth factors (EGF, R-spondin1, Noggin). Small molecules (Y-27632, CHIR-99021) added as needed. Buy only what your project requires—no waste, no redundancy

Cat.NO.	Specification	Product Name	Category
41420ES76	500 mL	DMEM/F-12 Reduced Serum Medium	Culture Medium Additive
60117ES60	100 mL	HEPES Solution (1M), Cell Culture Grade
60713ES03	1 mL	N-2 Serum-Free Supplement, 100X (Non-Animal Source)
60712ES03	1 mL	NSC-27 Serum-Free Supplement, Vitamin A Free (Non-Animal Source), equivalent to B-27
92262ES08	5 g	Recombinant Mouse Noggin Protein	Protein / Cytokine
92277ES08	5 µg	Recombinant Mouse RSPO1 Protein
92703ES60	100 µg	Recombinant Mouse EGF Protein
92294ES10	10 µg	Recombinant Mouse Wnt-3a Protein
52604ES08	5 mg	Recombinant Human Wnt-3a Protein (Liquid)
53002ES03	1 mg	A83-01	Small Molecule Compound
50303ES05	2 g	NAC (N-Acetyl-L-Cysteine), Antioxidant
60701ES60	100 mL	L-Alanyl-L-Glutamine (GlutaMAX)
53005ES08	5 mg	SB-202190
60281ES50	50 mg	Primocin
51402ES03	1 g	Nicotinamide
92294ES10	5 mg	Y-27632 Dihydrochloride

4. Essential Reagents for Passaging and Cryopreservation

Cat.NO.	Reagent Name
41424ES	Tissue Preservation Solution
41423ES	Tissue Digestion Solution
41451ES	Specialized Wash Solution
41421ES	Organoid Recovery Solution
41422ES	Organoid Cryopreservation Solution

 Whether you aim for rapid startup, long-term stability, or cost-effective scaling, Yeasen provides a complete toolkit to make organoid culture accessible, reliable, and reproducible.

Spend less time troubleshooting—and more time answering real biological questions.