Brain Organid Culture Kit,Absin_specification/price/image

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Product Specification

Usage

1. Organizational pre-processing:
1. Experimental materials:
Primary buffer B (pre-cooled at 4 ℃), tissue preservation solution E, sampling tube, tissue transport box and ice pack should be prepared in advance.
2. Acquisition and transportation of organizations:
Tissue sampling and transportation is the first and most easily neglected link in the successful construction of organoids. If the tissue is not preserved properly in the early stage, it will lead to problems such as poor cell activity, pollution, and few normal cells, which will reduce the success rate of organoid construction.
The tissue should be put into tissue preservation solution E within 30 minutes of isolation, and the primary buffer solution B should be washed for 3-5 times. The blood on the tissue surface should be washed clean, and put into tissue preservation solution E. The sampling tube should be stored and transported at 4 ℃ at low temperature (within 72 hours).
2. Primary culture of organoids (taking 24-well plates as an example)
1. Experimental materials:
Primary buffer B (4 ℃), primary tissue digestion juice C (37 ℃), Matrigel (melted in 4 ℃ refrigerator 24 h in advance), brain organoid culture medium A (room temperature or 37 ℃), tweezers (10 ㎝), pointed ophthalmic surgical scissors/blade, disposable 60 mm culture dish, 1.5 mL/15 mL/50 mL centrifuge tube, 100um cell strainer, 3 mL pasteurization pipette/1000 uL pipette gun, 24-well cell culture plate, metal ice box.
2. Brain organoid construction:
2.1 Handling of the organization
The sampled animal brain tissue is recommended to be stored and transported at 2-8 ℃, and quickly transported to a clean laboratory for the experimental process of animal brain organoid construction, organizing photos and registering detailed information.
(1) Cleaning of tissue
After the sampling tube is disinfected, the tissue is removed from the ultra-clean table, placed into a culture dish, added primary buffer B, and cleaned by pipetting with a 3 mL pasteurization pipette or a 1000 uL pipette gun, and the cleaning operation is repeated three times or more.
(2) Dissociation and digestion of tissues
Use ophthalmic scissors or surgical blade to remove tissue impurities, transfer tweezers to 1.5 mL EP tube, further mechanically dissociate the tissue into tissue blocks with a volume of about 1 ~ 3 mm3 with ophthalmic scissors, transfer them to 15 mL EP tube, add 6 mL of primary tissue digestive juice and shake digestion at 37 ℃ for 20-25 min. During the digestion process, the tissue digestion was observed under a microscope every 10 minutes, and a small amount of digestive juice was taken to observe under a microscope. After more cell clusters or single cells below 70 um were observed, the next operation was carried out. The degree of tissue digestion is shown in Figure 1.
If the tissue volume is too small or the biopsy tissue is digested with 1 mL of primary tissue digest juice C in a 1.5 mL EP tube.

Figure 1 Tissue digestion into more cell clusters or more single cells

(3) Filtering of tissues
After digestion, the tissue was filtered through a cell sieve with a pore size of 100 um, the filtrate was collected, 3 times the volume of primary buffer B was added to rinse to terminate the digestion, and the supernatant was discarded after enrichment and centrifugation at 300 g for 5 minutes; If the cell pellet contains red blood cells, add 1-2 mL of red blood cell lysate for 1-2 min, dilute to 10 mL, enrich 300 g and centrifuge for 5 min, and then discard the supernatant. Brain organoids were cultured directly when there were too little or no red blood cells precipitated.
(4) Brain organoid culture
Observe the volume of cell pellets collected by centrifugation, add 25 times the volume of Matrigel to resuspend, form a 3D culture space structure, and avoid bubbles during resuspension. The volume of cell pellet is shown in Figure 2. 300 uL, 200 uL, 150 uL, and 100 uL Matrigel are added according to the volume of cell pellet as shown in Figure 2.

Figure 2 Cell pellet volume

The 24-well cell culture plate was dispensed at 30 uL/well, and the Matrigel was maintained at 0-4 ℃ throughout the process. The cell culture plate was placed in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulated, 750 uL of brain organoid medium A (preheated at 37 ℃ in advance) was added to each well for culture.
3. Organoid subculture (taking 24-well plate as an example)
1. Experimental materials:
Passage buffer G (4 ℃), organoid passage digestion juice D (room temperature or 37 ℃), Matrigel (melted in a 4 ℃ refrigerator 24 h in advance), brain organoid medium A (room temperature or 37 ℃), 1.5 mL/15 mL centrifuge tube, 24-well cell culture plate, ice box.
2. Organoid passage:
Select suitable organoids for passage, usually for about 10 days of growth. More than 20 organoids, or organoids with a size of 100-200um, can be seen under a microscope of 10X.
Aspirate the medium, add an equal volume of passage buffer G to each well, gently blow the Matrigel with a pipette gun, collect it in a 15 mL centrifuge tube, transfer it to a centrifuge tube every 6-8 wells, and stand at 4 °C for 10-15 minutes.
2.1 Organoid digestion
According to the growth of organoids, it is decided whether digestive passage is needed. After centrifugation, if there is little sediment at the bottom of the tube, no cells are seen, and the matrigel is not stratified, it can be resuspended again, the centrifugal force can be increased, and centrifuged again.
When the number of organoids is insufficient or the volume is small, the supernatant is discarded by centrifugation at 300 g for 5 min.
When the number of organoids is large or the volume is large, centrifuge at 300 g for 5 minutes to discard the supernatant, and digestive juice digestion or mechanical digestion can be selected.
Digestive juice digestion: Add 1-2 mL of organoid passage digestive juice D, blow away the cell pellet, digest at 37 ℃ for 120 seconds, pipetting every 60s, and observe under a microscope until the digestion reaches the state (Figure 3A-B) It can be stopped. Add 3 times the volume of organoid digest fluid in passage buffer G to terminate the digestion, centrifuge at 300 g for 5 min, and discard the supernatant.

A B

Figure Diagram of passage digestion degree of three types of organs

2.2 Passage organoid culture
Observe the volume of organoid precipitate collected by centrifugation. If there is little precipitate, reserve 1 time the precipitate volume of supernatant for dispensing. If a lot of precipitate supernatant can be aspirated. Add 25 times the matrigel volume of organoid precipitate to resuspend organoids. For the volume of matrigel, please refer to "Organoid Primary Culture Operation Figure 2".
24-well cell culture plates were dispensed according to 25 uL-30 uL/well,The Matrigel is maintained at 0-4 ℃ throughout the process。 The cell culture plate was placed in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulated, 750 uL of brain organoid medium A (preheated at 37 ℃ in advance) was added to each well for culture.
four, organoid cryopreservation (taking 24-well plate as an example)
1. Experimental materials:
Passage buffer G (4 ℃), organoid cryopreservation solution F (4 ℃), 15 mL centrifuge tube, cell cryopreservation tube, program cooling box, pipette gun.
2. Organoid cryopreservation:
Organoids that are not in use temporarily should be frozen and stored in a low temperature environment.
Aspirate the medium, add an equal volume of passage buffer G to each well, gently blow the Matrigel with a pipette gun, collect it in a 15 mL centrifuge tube, transfer it to a centrifuge tube every 6-8 wells, and stand at 4 °C for 10-15 minutes. Centrifuge at 300 g for 5 min to discard the supernatant and add 2 mL organoids every three wellsCryopreservation solution F, mix well with gentle pipetting, and transfer to cell cryopreservation tubes, 1 mL per tube.
Make the mark information, put it in the program cooling box, move it to a-80 ℃ refrigerator, and store it in a liquid nitrogen tank after 48 hours. Or put it in a 4 ℃ refrigerator for 40 minutes, put it in a-20 ℃ refrigerator for 2 hours, move it to a-80 ℃ refrigerator, and store it in a liquid nitrogen tank after 48 hours.
5. Organoid resuscitation culture (taking 24-well plate as an example)
1. Experimental materials:
Passage buffer G, brain organoid medium A, Matrigel (melted in a 4 ℃ refrigerator 24 h in advance), 24-well cell culture plate, ice box, 15 mL centrifuge tube, water bath pan, 3 mL pasteurization pipette/pipette gun.
2. Organoid resuscitation culture:
Take out the frozen organoids from the low temperature environment and quickly put them in a 37 ℃ water bath to thaw. During the water bath thawing process, the cryopreservation tube should be gently shaken to ensure that the cryopreservation solution is completely thawed in a short time. The thawed organoids were quickly transferred to a 15 mL centrifuge tube, gently pipetted 6-8 times with a pipette gun, and centrifuged at 300 g for 5 minutes to discard the supernatant; Add an appropriate amount of passage buffer G to resuspend, transfer it into a 1.5 mL centrifuge tube and centrifuge 300 g for 5 minutes, and discard the supernatant.
Add 100 uL of Matrigel to resuspend per cryovial, dispense 24-well cell culture plates at 25 uL-30 uL/well, and maintain the Matrigel at 0-4 ℃ throughout the process. The cell culture plate was placed in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulated, 750 uL of brain organoid medium A (preheated at 37 ℃ in advance) was added to each well for culture.
6. Use of matrigel
Matrigel was thawed overnight at ambient conditions of 2-8 °C. When using Matrigel, keep it in an ice box to prevent premature setting. The matrigel formed a gel at 37 °C within 20 minutes.
1. Matrigel features:
(1) 4 ℃ can still maintain good fluidity for 14 consecutive days
(2) Put it in a 37 ℃ incubator for 10-15 min to solidify
(3) It is not easy to break during the culture process, and the glue is removed to clean the non-stick culture plate
7. Brain organoid identification map

Description

This kit contains the whole process reagents of brain organoids, which can cultivate mammalian brain organoids such as mice and sheep (fetal brain is recommended). It is not from IPSC source, can be passaged and amplified, has a short construction period, and is produced according to GMP standards.

Product composition:

	Product composition	Specifications	Storage conditions and cycles
1	Brain organoid medium A	100 mL	4 ℃, 3 months or-20 ℃, 1 year
2	Primary Buffer B	2 * 250 mL	4/-20 ℃, 2 years
3	Primary tissue digestive juice C	50 mL	4/-20 ℃, 1 year
4	Organoid passage digestive juice D	50 mL	4/-20 ℃, 1 year
5	Tissue preservation solution E	100 mL	4/-20 ℃, 1 year
6	Organoid Cryopreservation Solution F	20 mL	4/-20 ℃, 2 years
7	Passage Buffer G	2 * 250 mL	4/-20 ℃, 2 years

Remarks: All of the above ingredients are available separately.

Reagent Arrival Handling:

1. Brain organoid culture medium A can be stored at 4 °C for 3 months. It is stored at 4 °C after receiving the goods. It is recommended to use it within 1 month. It is recommended to store it at-20 °C for a long time to avoid repeated freezing and thawing more than 2 times。
2. Tissue preservation solution E and primary tissue digestion solution C contain nutrients to maintain cell activity. In order to maintain the activity of reagent nutrients, it is not recommended to store them at-20 °C for a long time to avoid repeated freezing and thawing more than 2 times。

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.