Ovarian Organoid Kit CAT : OCK006

“One-stop” Organoid Construction 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%.

Kit Specifications

Table 1.1 Reagents Contained in Kit

Table 1.2 Consumables Included in Kit 

Storage Conditions

Instructions for Use

Primary Construction of Organoids

Tissue Cleaning: Upon completion of tissue cleansing with pre-chilled tissue cleaning 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 centrifuge 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. Subsequently, add 5 mL of digestion solution Ⅰ to resuspend the tissue and transfer it to a new 6 cm culture dish. Place the culture dish in a 37 °C incubator for enzymatic digestion. Remove it every 10 minutes, pipette gently, and observe under the microscope to assess the extent of digestion. Terminate the digestion process when no conspicuous tissue fragments are observable and a substantial number of single cells and small cell clusters emerge in the microscopic field.

Termination of Digestion: Transfer the tissue digestion suspension to a 15 mL centrifuge tube and supplement it with pre-chilled tissue cleaning solution to a final volume of 10 mL to halt the digestion. After centrifugation, discard the supernatant and add 5 mL of pre-chilled tissue cleaning solution to repeat the cell washing procedure 2 to 3 times.

Cell Collection: Add pre-chilled tissue cleaning solution to resuspend the cell pellet. Employ the natural sedimentation method to harvest the completely digested cell clusters and filter them through a 100 μm cell strainer to eliminate extracellular matrix contaminants. Transfer the filtrate to a 15 mL centrifuge tube and discard the supernatant after centrifugation.

Red Blood Cell Lysis: Add 500 to 1000 μL of red blood cell lysis buffer to the cell pellet and pipette gently 5 to 10 times to ensure homogeneous mixing. Incubate on ice for 3 minutes. Then, add 5 times the volume of pre-chilled organoid cleaning solution to terminate the red blood cell lysis. Discard the supernatant after centrifugation and collect the cell pellet.

Cell Counting: Add 1 mL of organoid culture medium (containing additive Ⅰ) to the cell pellet. After thorough pipetting to achieve uniform mixing, aspirate 10 μL of the cell suspension for viable cell detection and enumeration.

Cell Seeding in Matrigel: Taking a 24 well plate as an illustrative example, inoculate one 50 μL gel droplet per well with a cell density ranging from 10,000 to 50,000 cells per 50 μL gel droplet. Incorporate an equal volume of Matrigel into the cell suspension, pipette gently and thoroughly to ensure homogeneity, and promptly inoculate it into the central position of the pre-warmed 24-well plate. Incubate in a 37 °C incubator for 5 minutes for pre-solidification. Subsequently, invert the 24-well plate gently and continue incubation in the 37 °C incubator for approximately 10 minutes until the Matrigel is completely solidified.

Maintenance Culture: Once the gel droplets have solidified, slowly introduce 500 μL of organoid culture medium (containing additive Ⅰ) along the well wall. Replace the organoid culture medium (without additive Ⅰ) 48 hours after initiating the organoid culture. During the maintenance culture, replace the culture medium every 2 days. Conduct passage of the organoids within 7 to 15 days of organoid culturing.

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

Precautions

Make every effort to avoid repeated freezing and thawing. Appropriate aliquots can be prepared prior to utilization.

This product is exclusively intended for scientific research applications and must not be employed for clinical diagnosis or treatment. It is not to be used for food or pharmaceutical purposes and should not be stored in ordinary residential premises.

For the safety and well-being of laboratory personnel, it is recommended to don laboratory coats, masks, and disposable gloves during the experimental process.