B009-Organoid Cryopreservation Solution

Organoid Cryopreservation Solution

Product Description

This cryopreservation solution is an organoid cryopreservation solution that is generally applicable to the low-temperature cryopreservation of various organoid cells. Its chemical components are clearly defined, and it contains no protein components. It can eliminate the risks of potential pathogens or immune responses caused by serum or proteins, making it safer to use. Moreover, it can maintain the recovery viability of multiple types of cells above 90%.

Storage

Store at 2-8℃ for 12 months.

Instructions for Use

(1) Observe the state of organoids under a microscope. When the organoid density reaches 500 per well or the diameter of a single organoid is within 100 - 200 μm, perform cryopreservation to maintain the good state and rapid proliferation rate of the organoids.

(2) Before the experiment, prepare an ice box to thaw the basement membrane matrix. Place the rinsing solution, organoid washing solution, cell recovery solution, enzymatic digestion solution II, organoid complete culture medium, and additive I in the ice box for later use. Meanwhile, preheat the culture plates.

(3) Inside a biological safety cabinet, aspirate the old culture medium from the culture plate to be subcultured, add pre-cooled organoid washing solution, wash once, change the pipette tip, aspirate the organoid recovery solution and add it into the wells (500 μL per well for a 24-well plate), and then incubate at room temperature for 5 minutes.

(4) Use the 1000 μL pipette tips rinsed with the rinsing solution to disperse or scrape off the basement membrane matrix. Collect the scraped materials into a 15 mL centrifuge tube, add 5 mL of organoid washing solution, pipette the organoids 5 - 10 times, place on ice for 10 minutes, centrifuge at 200 g at 2-8 °C for 5 minutes, and discard the supernatant.

(5) Select the mechanical method or enzymatic digestion method according to the culture period or morphology of the organoids:
a) Mechanical method (recommended for diffuse and cystic types): Add 1 mL of organoid culture medium (containing additive I) to resuspend the organoids, gently pipette 5 - 50 times, aspirate 20 μL of the cell suspension onto a glass slide, and observe under the microscope. When most of the cells are single cells and small cell clusters, it is okay.
b) Enzymatic digestion method (recommended for solid and mixed structures): Add 500 μL of enzymatic digestion solution II to resuspend the organoids, transfer the organoid suspension to a 24-well plate for enzymatic digestion, allow it to stand and digest in a 37 °C incubator for about 5 - 15 minutes, pipette 5 - 10 times every 5 minutes. When observing under the microscope that most of the cells are single cells and small cell clusters, stop the enzymatic digestion. After pipetting evenly, transfer the cell suspension to a new 15 mL centrifuge tube, add 9.5 mL of pre-cooled organoid washing solution to the centrifuge tube to stop the digestion. After centrifugation, remove the supernatant as much as possible, use 1 mL of organoid culture medium (containing additive I) to resuspend the organoids, and place the centrifuge tube on a constant-temperature metal heating block for counting.

(6) Centrifuge at 200 g at 2-8 °C for 5 minutes to collect the cell pellet, remove the supernatant as much as possible, and then add the organoid cryopreservation solution for cryopreservation.

Note: The recommended cryopreservation density of organoids is 105~5×105 cells per vial. Before putting them into a liquid nitrogen storage tank, gradient cooling is required. After the programmed cooling in an -80 °C ultra-low temperature freezer is completed, put them into the liquid nitrogen tank for cryopreservation as soon as possible.

Precautions

This product is strictly reserved for scientific research and shall not be applied to clinical diagnosis, treatment, food, or pharmaceuticals. Additionally, it is prohibited from being stored in residential premises.