Mouse Kidney Organoid Construction Mini Kit CAT : MOCKs011

“One-stop” Organoid Construction Mini Kit

Product Description

The “One-stop” Organoid Construction Kit developed by Jiyan Biotech is an all-in-one solution for organoid construction, integrating tissue sampling, organoid culturing, subculturing, cryopreservation, and resuscitation. It is characterized by its user-friendly nature and ready-to-use design, achieving a remarkably high success rate of over 90% in organoid construction. There are over 50 varieties of human/mouse/monkey organoid construction kits available to meet diverse requirements. Additionally, on-site technical support is provided, and we offer a free reconstruction service in case the organoid construction fails.

This product equips customers with all the necessary consumables and reagents throughout the entire experimental process, minimizing the time consumption on non-experimental procedures. According to the operation manual of this product, provided that the sampling prerequisites are fulfilled and the uncontrollable factors such as sample-induced contamination are eliminated, the success rate of organoid construction can exceed 90%.

Mouse Kidney Organoid Construction Mini Kit

Mini Kit Specifications

Table 1.1 Reagents Contained in the Mini Kit

Table 1.2 Consumables Included in the Mini Kit

Storage Conditions

The Mouse Kidney Organoid Culture Medium should be stored below -20°C and has a shelf life of 6 months. It can be stored at 2-8°C for two weeks.

The rest of the reagents should be stored according to their specific conditions.

Instructions for Use

Primary Construction of Organoids

Tissue Washing: Upon completion of tissue rinsing solution with pre-chilled Tissue Rinsing Solution, transfer the tissue into a 6 cm culture dish and meticulously remove the adipose, connective tissue, and necrotic regions in the vicinity of the tissue.

Tissue Fragmentation: Transfer the tissue into a 1.5 mL microcentrifuge tube and mince the tissue with sterile scissors approximately 100 to 200 times until the tissue fragments attain a diameter of around 0.5 to 1 mm.

Tissue Digestion: Centrifuge the tissue suspension and discard the supernatant. Resuspend the pellet in 5 mL of Dissociation Solution I and transfer it to a new 6 cm culture dish. Place the dish in a 37°C incubator for enzymatic digestion. Every 10 minutes, remove the dish, pipette gently, and observe under a microscope to monitor the digestion progress. Terminate the digestion when no visible tissue fragments remain and a large number of single cells and small cell clusters are present in the field of view.

Termination of Digestion: Transfer the digestion suspension to a 15 mL centrifuge tube. Quench the digestion by adding pre-chilled Tissue Rinsing Solution to a final volume of 10 mL. Centrifuge the mixture and discard the supernatant. Wash the cells by repeating this process (resuspending in 5 mL of pre-chilled Tissue Rinsing Solution, centrifuging, and discarding the supernatant) 2-3 times.

Cell Collection: Resuspend the cell pellet in pre-chilled Tissue Rinsing Solution. Collect the well-digested cell clusters using the natural sedimentation method and filter the suspension through a 100 μm cell strainer to remove extracellular matrix contaminants. Transfer the filtrate to a 15 mL centrifuge tube, centrifuge, and discard the supernatant.

Red Blood Cell Lysis: Add 500–1000 μL of Red Blood Cell Lysis Buffer to the cell pellet. Pipette 5-10 times to mix thoroughly and incubate on ice for 3 minutes. Then, add a 5x volume of pre-chilled Organoid Wash Solution to stop the lysis. Centrifuge and discard the supernatant to collect the cell pellet.

Cell Counting: Add 1 mL of Organoid Medium (containing Supplement I) to the cell pellet. Pipette to mix well, then take 10 μL of the cell suspension for viable cell counting.

Cell Seeding in Matrigel: Using a 24-well plate as an example, seed one 50 μL droplet per well at a density of 10,000–50,000 cells per droplet. Mix the cell suspension with an equal volume of extracellular matrix (e.g., Matrigel) by gentle pipetting. Quickly inoculate the mixture into the center of each pre-warmed well. Incubate the plate right-side up at 37°C for 5 minutes for pre-solidification. Then, gently invert the plate and continue incubation at 37°C for approximately 10 minutes to allow complete solidification.

Maintenance Culture: After the matrix droplets have solidified, carefully add 500 μL of Organoid Medium (containing Supplement I) slowly along the wall of each well. Replace the medium with Organoid Medium (without Supplement I) 48 hours after culture initiation. For maintenance, change the medium every 2 days. Passage the organoids within 7-15 days. 

For more detailed steps and other experimental procedures, please refer to the "Instruction Manual for Organoid Construction Kit".

Precautions

Avoid repeated freeze-thaw cycles. Aliquoting reagents prior to use is recommended.

This product is for research use only. It is not intended for clinical diagnosis or treatment, nor for use in food or pharmaceuticals. Do not store in ordinary residential areas.

For the safety and health of laboratory personnel, wear lab coats, masks, and disposable gloves during experiments.