A Practical Guide for Beginners: Full Workflow for Construction of Mouse Small Intestinal Organoids

Organoids are three-dimensional cellular structures cultured in vitro using stem cells, progenitor cells or tumor cells. They recapitulate the spatial architecture and functional features of their tissue of origin. Compared with conventional two-dimensional cell culture models, organoids better mimic the in vivo physiological microenvironment, rendering them irreplaceable tools for disease modeling, drug screening, genetic engineering, immunology and tissue regeneration.

Among diverse organoid models, mouse small intestinal organoids represent a classic benchmark. In 2009, Hans Clevers’ team reported a landmark study published in Nature. Using Lgr5⁺ intestinal stem cells, they successfully generated “mini-guts” with crypt and villus structures within an in vitro three-dimensional culture system. This milestone work marked the official dawn of modern organoid research and established the core paradigm for subsequent organoid construction (e.g., 3D Matrigel culture and defined growth factor cocktails).

For this reason, mouse small intestinal organoids serve as the origin and cornerstone of the organoid field. Mastery of their construction unlocks the core principles of organoid technology. Good tools are prerequisite to successful work. Selection of a well-validated and stable kit is critical for successful organoid generation. Absin’s Organotial Mouse Normal Small Intestinal Organoid Culture Kit (Cat. No.: abs9514) provides a complete solution tailored for in vitro culture of primary mouse normal small intestinal organoids.

1. Overview of Absin Organotial Mouse Normal Small Intestinal Organoid Culture Kit

This dedicated kit for mouse normal small intestinal organoids contains core components listed in the table below. Combined with Absin organoid-grade Matrigel abs9495 (low growth factor, phenol-red free), organoid culture can be performed. All reagents in the kit are sterile. The culture medium requires no extra growth factor supplementation or optimization, and no secondary preparation is needed; the kit is ready-to-use upon opening.

Component Name	Specification
Mouse Normal Small Intestinal Organoid Medium A	100 mL
Primary Culture Buffer B	250 mL
Mouse Normal Small Intestinal Primary Tissue Digestion Solution C	30 mL
Organoid Passaging Digestion Solution D	30 mL
Tissue Preservation Solution E	100 mL
Organoid Cryopreservation Medium F	20 mL
Organoid Passaging Culture Buffer G	250 mL

2. Full Workflow for Construction of Mouse Small Intestinal Organoids

2.1 Pre-experiment Preparation

2.1.1 Instruments and Equipment

CO₂ incubator, biosafety cabinet, inverted microscope, refrigerated benchtop centrifuge, water bath (abs72023) or water bath shaker, -20 / -80 °C refrigerator, pipette set, ophthalmic scissors, ophthalmic forceps.

2.1.2 Reagents and Consumables

Mouse Normal Small Intestinal Organoid Culture Kit (abs9514), Matrigel (low growth factor, phenol-red free) (abs9495), 60 mm cell culture dish (abs7005), 100 μm cell strainer (abs7233), 15 mL centrifuge tube (abs7102), 1.5 mL EP tubes (abs7119), 24-well cell culture plate (abs7035), metal cooling block (abs7289), ophthalmic scissors, ophthalmic forceps.

2.2 Tissue Harvesting and Processing

2.2.1 Sample Preparation

Euthanize mice by cervical dislocation and disinfect the body surface with 75% ethanol. Under sterile conditions, open the abdominal cavity and isolate the small intestinal segment located 3–15 cm distal to the stomach. Carefully remove mesentery and adipose tissue with forceps.

2.2.2 Tissue Preservation and Transportation

Immediately place the freshly harvested normal mouse small intestinal tissue into pre-cooled (2–8 °C) Tissue Preservation Solution E in a sampling vial. Rapidly transport the sample to a clean laboratory for tissue processing and stem cell isolation. Record tissue information (size, color, texture, etc.) with photographic documentation.

2.2.3 Tissue Washing and Processing

Add pre-cooled (4 °C) Primary Culture Buffer B into a 60 mm cell culture dish (abs7005). Disinfect the sampling vial and transfer tissue into the dish. Rinse tissue with Primary Culture Buffer B and remove residual fat, mesentery and blood vessels on the intestinal surface. Flush the intestinal lumen with buffer using pipette tips to remove luminal contents. Longitudinally incise the intestine to fully expose the mucosal surface. Fix one end of the intestinal tissue with forceps and gently scrape off intestinal villi using a surgical blade until the tissue becomes translucent, followed by repeated rinsing.

2.2.4 Tissue Mincing

Mince the cleaned small intestinal tissue into fragments of approximately 1–3 mm³ using ophthalmic scissors or a surgical scalpel, then transfer tissue fragments into a 15 mL centrifuge tube.

2.3 Tissue Digestion and Crypt Isolation

2.3.1 Enzymatic Digestion

Add Primary Tissue Digestion Solution C at a volume ratio of 5:1 (digestion solution : tissue volume). If tissue volume is difficult to estimate, 5 mL digestion solution is generally sufficient. Perform digestion at 4 °C for 15–30 min and monitor digestion progress continuously. Every 10 min, withdraw an aliquot for microscopic observation. Once abundant crypts are observed, quench digestion by adding Primary Culture Buffer B at a volume ratio of 3:1 (buffer : digestion solution).

2.3.2 Filtration

Filter the digested mixture through a 100 μm cell strainer (abs7233). Examine an aliquot of filtrate under the microscope. Collect filtrate into a 15 mL centrifuge tube and centrifuge at 300 × g for 5 min at 4 °C to pellet crypts, then discard the supernatant.

2.4 Primary Culture of Organoids

2.4.1 Preparations

Thaw Matrigel overnight at 4 °C in a metal cooling block. Pre-chill pipette tips and centrifuge tubes at -20 °C for at least 30 min. Thawed Matrigel can be stored at 4 °C and should be used within 2 weeks.

Metal cooling block (abs7289)

2.4.2 Seeding Density

Recommended volume ratio of Matrigel : crypt pellet = 25:1. If pellet volume is hard to estimate, 300 μL Matrigel is generally adequate. For 24-well plates (abs7035), seed 25 μL Matrigel-crypt mixture per well.

2.4.3 Matrix Loading and Plating

Add Matrigel (abs9495) to the crypt pellet and mix gently (avoid vigorous pipetting to prevent bubble formation). Perform plating on ice or a pre-chilled metal cooling block. After practice, complete Matrigel addition, mixing and plating within 30 s to maintain appropriate Matrigel fluidity.

Plating density

2.4.4 Medium Addition

Place the seeded plate in a 37 °C incubator for 40–60 min to allow Matrigel polymerization. Subsequently add 500–750 μL Organoid Medium A for culture. Within 10–14 days, most organoids reach 200–500 μm in diameter and are ready for passaging.

Mouse small intestinal organoids

2.5 Organoid Passaging

Select organoids for passaging when cultured for approximately one week. Suitable candidates show >20 organoids under 10× objective or are 100–200 μm in diameter.

2.5.1 Organoid Harvesting

Aspirate culture medium. Add 1–2 mL pre-chilled (4 °C) Organoid Passaging Culture Buffer G per well to gently dissolve Matrigel. Collect organoids into 15 mL centrifuge tubes (pool 5 wells of a 24-well plate per tube). Top up to 14 mL with Passaging Culture Buffer G. Incubate at 4 °C for 40 min or at -20 °C for 5 min to soften Matrigel. Adjust freezing time empirically; Matrigel is sufficiently softened when no gel clumps remain after tube shaking.

2.5.2 Organoid Dissociation

Determine dissociation strategy according to organoid growth status.

For low abundance or small-sized organoids: centrifuge at 300 × g for 5 min at 4 °C and discard supernatant.

For abundant or large organoids: centrifuge at 300 × g for 5 min at 4 °C and discard supernatant; either enzymatic digestion or mechanical dissociation can be applied.

Enzymatic digestion: Add 2–3 mL Organoid Passaging Digestion Solution D and incubate in the biosafety cabinet for 2–3 min. Pipette gently 1–2 times during incubation. The target product is cell clusters rather than single cells; single cells exhibit extremely low survival. Check an aliquot under microscope; stop digestion when abundant cell clusters appear.

2.5.3 Re-plating

Quench digestion by adding Passaging Culture Buffer G at a volume ratio of 5:1 (buffer : digestion solution). Centrifuge at 300 × g for 5 min at 4 °C and discard supernatant.

Passaging ratio: organoids are typically split at 1:2. For example, harvest from 5 wells of a 24-well plate for re-seeding into 10 wells, requiring 250 μL Matrigel (25 μL per well).

Matrigel loading, plating and medium addition follow the same procedure as primary culture.

2.6 Organoid Cryopreservation

2.6.1 Cryopreservation Principles

Organoids are cryopreserved without prior digestion (digestion drastically reduces post-thaw viability). Harvest organoids at exponential growth phase. Organoids at Day 3–4 post-passaging with diameter of 100–200 μm are optimal for cryopreservation; viability declines once organoids reach routine passaging time-point.

2.6.2 Cryopreservation Density

For 24-well plates, recommended density: 2 wells per mL Cryopreservation Medium F. Resuspend organoids gently in Cryopreservation Medium F and cryopreserve immediately. Prolonged exposure to DMSO impairs organoid viability.

2.6.3 Freezing Protocol

Programmed freezing: Place cryovials in a controlled-rate freezing container and store at -80 °C overnight, then transfer to liquid nitrogen the next day.

Manual freezing: Hold at 4 °C for 30 min → transfer to -20 °C for 1 h → store at -80 °C overnight, then transfer to liquid nitrogen the next day.

2.7 Organoid Thawing

2.7.1 Pre-thaw Preparation

Preheat water bath to 37 °C; perform routine disinfection of cell culture laboratory; prepare centrifuge tubes, pipettes and culture plates.

2.7.2 Thawing Process

Immerse cryovial rapidly in pre-warmed water bath and agitate continuously to accelerate thawing, leaving only tiny ice crystals (approximately 1–2 min).

Transfer organoid suspension from cryovial into a 15 mL centrifuge tube. Add Passaging Culture Buffer G at 10× volume of cryopreservation medium, mix gently, centrifuge at 300 × g for 5 min at 4 °C and discard supernatant.

2.7.3 Post-thaw Culture

Recommended seeding ratio 1:1 (thaw the same number of wells as originally frozen). Subsequent Matrigel embedding, plating and medium exchange follow the primary culture protocol.

Appendix: Serial micrographs of mouse small intestinal organoid growth

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