Organoids refer to 3D cell cultures cultivated in vitro using stem cells, progenitor cells, tumor cells, etc., which exhibit spatial structure and tissue-like organization. In 2021, organoid technology was designated as a key research and development focus in China's 14th Five-Year Plan. As disease models, organoids, compared to traditional 2D disease models, more closely mimic in vivo environments, making them crucial in various fields such as cell therapy, drug development, genetic engineering, immunology, and tissue regeneration.

The following content will guide you through the entire process of culturing mouse intestinal organoids and highlight relevant considerations to minimize pitfalls during the cultivation process.

 

Experimental Materials

Product Type	Product Name	Catalog Number	Specifications
Intestinal Tissue Digestion	Tissue Digestion Solution	C231131	30ml
Organoid Culturing Support	Arcegel Matrix for Organoid culture, Phenol Red-Free, LDEV-Free	C231009	5 mL
Culture Medium Components and Additives	DMEM/F-12 Reduced Serum Medium	C231114	500ml
	1×PBS for Cell Culture	C230135	500ml
	Recombinant Human RSPO1 Protein	C230254	100ug
	Recombinant Human Noggin Protein, His Tag	C230462	100ug
	Recombinant Human EGF Protein	C230328	100ug
	Recombinant Human Wnt -3a Protein	C230259	100ug
Organoid Collection	Cell recovery solution for Organoid	C231103	30mL
Organoid Cryopreservation	Cell freezing medium for Organoid	C231104	30mL

 

Cultivation Steps for Small Intestinal Organoids

1. Sample Preparation

 Euthanize mice by cervical dislocation and sterilize the surface with alcohol.

 Under aseptic conditions, extract the small intestine tissue from 3 to 15 cm near the stomach, carefully removing the mesentery and fat using forceps, and place it in DPBS solution containing 1% antibiotics/antimycotics at 4°C.

2. Sample Washing

 Rinse the intestine 2-3 times with a syringe, then carefully cut open the intestinal tube with surgical scissors, placing the luminal side facing up.

 Use a surgical blade to gently scrape off the villi from the luminal surface until the tissue becomes transparent. Wash the intestinal tissue in a new dish containing DPBS 2-3 times.

3. Sample Initial Processing

 Cut the cleaned small intestine tissue into 2 mm-wide pieces and transfer them to a new 50 ml centrifuge tube.

 Wash the tissue gently with DPBS 3-5 times to remove villus cells and floating adipose tissue.

4. Tissue Digestion

 Add 10-15 ml pre-cooled DPBS containing 3-5 mM EDTA to the cleaned small intestine tissue pieces and digest at 4°C for about 30 minutes, shaking the tube gently every 10 minutes.

 After digestion, discard the supernatant EDTA solution and wash the tissue gently 2-3 times with fresh DPBS to remove residual EDTA.

 Add 10-15 ml pre-cooled DPBS containing 0.1% BSA to the small intestine tissue pieces, repeatedly pipetting and suspending the tissue fragments to separate crypts from the basement layer.

 Stop pipetting when numerous crypt-like structures are observed, then filter the tissue suspension through a 70 μm filter and collect the tissue suspension that passes through the filter.

5. Mixture Formation

 Resuspend the crypt precipitate in Arcegel matrix gel, with each 10 μL matrix gel suspension containing 200-600 crypts.

 Place the mixed suspension on ice and proceed quickly to avoid gelation of the matrix gel.

 Dilute the matrix gel suspension with a dilution ratio of ≥50% to ensure the stability of the Arcegel matrix gel structure during cultivation.

 Plant the mixed suspension in the center of the bottom of a 24-well plate, about 30-50 μL per well, avoiding contact with the side walls of the plate.

6. Cultivation

 Place the plated culture in a 37°C CO₂ incubator for about 30 minutes until the Arcegel matrix gel is completely solidified.

 After the Arcegel matrix gel is fully solidified, slowly add pre-prepared small intestinal organoid culture medium along the walls, about 800 μL per well.

 Incubate the 24-well plate in a 37°C CO₂ incubator. Replace the medium with fresh culture medium every 3 days and monitor the growth of organoids. Typically, mouse small intestinal organoids form within 5-7 days.

 

Experimental Results

Figure Ex vivo culture results of mouse small intestinal organoids.

 

Passaging of small intestinal organoids

After culturing for 5-7 days or when the center of the small intestinal organoids turns black, passaging can be performed. Pre-incubate 24- or 12-well plates in the incubator for at least 30 minutes. Remove the old culture medium, add 1 mL cold DPBS per well, wait for 1 minute, lift the Matrigel gently, disperse it by pipetting with a 1 mL pipette tip, aspirate the suspension with a syringe, repeat this step once, transfer the suspension to a 5 mL centrifuge tube, centrifuge at 1200 rpm, 4°C for 5 minutes, discard the supernatant, and passage at a ratio of 1:3 per well.

Small intestinal organoid cryopreservation

Select vigorously growing organoids (generally 3-4 days after passaging) for cryopreservation, with two wells per cryovial. Remove the culture medium, add 1 mL cell freezing medium, disperse the Matrigel with a pipette tip, transfer the suspension to cryovials, place them in a freezing box, freeze at -80°C for 1 day, then transfer to liquid nitrogen for long-term storage.

Resuscitation of small intestinal organoids

Preheat a 24-well plate in the incubator for 30 minutes. Retrieve the cryovials from liquid nitrogen and place them in a 37°C water bath. Once the cryovial contents have thawed, aspirate with a 1 mL syringe, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1200 rpm, 4°C for 5 minutes, discard the supernatant, add 5 mL ice-cold DPBS to resuspend, repeat the centrifugation steps, and discard the supernatant. Plate and culture the organoids following the standard organoid culture protocol.

 

 FAQ

What digestion solution should be chosen for tissue digestion, and how long is the digestion period?

For digestion of tissues, one can choose between EDTA or collagenase. If digesting hollow organs, EDTA is suitable, such as for the small intestine and stomach. Collagenase comes in various types and can be used for a wide range of tissue types, often in combination with DNase. The duration of digestion varies significantly depending on the tissue type, ranging from several minutes to several hours.

What volume of matrix gel and culture medium is required for culturing organoids in different well plates?

The commonly used plate for culturing organoids is the 24-well plate, where 50 μl of matrix gel is added per well to form droplets, followed by the addition of 500 μl of culture medium to cover the droplets. For a 96-well plate, 10 μl of matrix gel is added per well to form droplets, followed by the addition of 200 μl of culture medium to cover the droplets. In a 6-well plate, multiple 50 μl droplets can be planted per well, and 2-3 ml of culture medium is added to cover the droplets.