B008-Organoid Recovery Solution

Organoid Recovery Solution

Product Description

The organoid recovery solution can recover the cells cultured on the basement membrane matrix. The whole process is gentle and rapid, while maintaining cell viability at the same time.

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 subculture to maintain the good state and rapid proliferation rate of the organoids.

(2) Prepare the reagents and consumables required for the experiment in advance.

(3) Inside a biological safety cabinet, aspirate the old culture medium from the culture plate to be subcultured, add pre-cooled organoid washing solution (B004), wash once, change the pipette tip, aspirate the organoid recovery solution (B008) 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 rinsing solution (B006) to rinse the used 1000 μL pipette tips, and then use them 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: S001) 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 (D002) 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 basement membrane matrix or organoid cryopreservation solution for subsequent amplification culture or cryopreservation.

Note:

(1) The subculture ratio of organoids needs to be adjusted according to the growth state of organoids before subculture. For organoids in a good growth state, the recommended subculture ratio is 1:2 - 1:3.

(2) Regarding the planting density of subcultured organoids, taking a 24-well plate as an example, plant one droplet of matrix per well, with a cell density of 5000 - 10000 cells per 50 μL droplet for normal cells and a density of 300 - 500 cells per 50 μL droplet for larger cell clusters.

(3) 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.