| Usage |
I. Organizational Handling
It is recommended that the animal brain tissue after sampling be 2 – 8 ℃ Store and transport under these conditions, and quickly transfer to a clean laboratory for animal brain organoid construction experiments. Organize photography and record detailed information. After sterilizing the sampling tube, the tissue was removed from the ultra-clean bench, placed in a petri dish, and primary buffer was added. 3 mL pasteurized straw or 1000 μL The pipette gun is blown and cleaned, and the cleaning operation is repeated three or more times.
II. Tissue Dissociation and Digestion Tissue impurities are removed with ophthalmic scissors or a surgical blade, and the forceps are transferred to 1.5 mL EP In the tube, ophthalmic scissors are used to further mechanically dissociate the tissue into a volume of approximately 1~3 mm3 The tissue block was transferred to 15 mL EP In the tube, add 6 mL primary tissue digestive fluid 37 ℃ oscillation digestion 20-25 min 。 Each digestive process 10 min The tissue digestion was observed under a microscope. A small amount of digestive juice was taken and observed under the microscope, and a larger amount was observed. 70 μm The following cell clusters or individual cells are followed by the next step. The degree of tissue digestion is shown in Figure 1. If the amount of tissue is too small or the biopsy tissue is used 1 mL primary tissue digestive fluid C In 1.5 mL EP Tubular digestion. 
Figure 1 Tissue digestion into more cell clusters or more single cells
III. Tissue Filtration
Digestion completes tissue passage 100 μm The cell sieve with a pore size is filtered, the filtrate is collected, and added... 3 Double the volume of primary buffer B Rinsing terminates digestion. 300 g enrichment centrifugation 5 min Abandoning the supernatural; If the cell pellet contains red blood cells, add 1-2 mL erythrocyte lysate 1-2 min Then, dilute to 10 mL ， 300 g enrichment centrifugation 5 min They abandoned their superiors. Brain organoid culture was performed directly when there was too little sedimentation or no red blood cells.
IV. Brain organoid culture
Observe the volume of cell pellets collected by centrifugation, and add 25 Double the volume of matrix glue is resuspended, forming 3D Cultivate the spatial structure to avoid bubbles during the resuspension process. The cell pellet volume is shown in Figure 2. Add the cell pellet volume according to the figure. 300 μL 、 200 μL 、 150 μL 、 100 μL Matrix glue.
Figure 2 cell pellet volume
24 well cell culture plate according to 30 μL/ When dispensing the adhesive through the holes, the matrix adhesive is 0-4 ℃ Operate under these conditions. cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 750 μL Brain organoid culture medium (in advance 37 ℃ (Preheating) for cultivation.
V. Organoid passage
Select appropriate organoids for passage, generally for growth. 10 About a day, microscope 10X You can see more below 20 Organoids, or size 100-200μm Organoids.
Aspirate off the culture medium, add an equal volume of passage buffer to each well, and gently blow away the matrix gel with a pipette, collecting it. 15 mL In the centrifuge tube, each 6-8 The hole is transferred to a centrifuge tube, 4 ℃ standing 10-15 min 。
VI. Organoid digestion
The need for digestive passaging is determined based on the growth of the organoid. If there is little precipitation at the bottom of the tube after centrifugation, no cells are seen, and the matrix glue is not delaminated, it can be resuspended again to increase the centrifugal force and centrifuge again.
When the number of organoids is insufficient or the volume is small, 300 g centrifuge 5 min Abandon the Shangqing.
When the number of organoids is high or the volume is large, 300 g centrifuge 5 min Discard the supernatant, and you can choose digestive juice digestion or mechanical digestion.
Digestive juice digestion: Add 1-2 mL After the organoid is passed through the digestive juice, the cell precipitate is blown away, 37 ℃ digestion 120 Seconds, every other 60s Blow once and observe under a microscope until digested (Figure 3). A-B It can be stopped when in the ) state. Add 3 A passage buffer of twice the volume of organoid digestive fluid terminates digestion, 300 g centrifuge 5 min They abandoned the Shangqing
VII. Subgenerational organoid culture
Observe the volume of organoid pellets collected by centrifugation; if there is very little pellet, it can be reserved. 1 Dispense the supernatant with twice the precipitation volume; a large amount of precipitated supernatant can be absorbed. Add... 25 The organoids were resuspended in an amount of matrix gel twice the organoid pellet volume. The volume of matrix rubber can be referenced. “ Organoid primary culture operation Figure 2 ” 。
24 well cell culture plate according to 25 μL-30 μL/ When dispensing the adhesive through the holes, the matrix adhesive is 0-4 ℃ Operate under these conditions. cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 750 μL Brain organoid culture medium (in advance 37 ℃ (Preheating) for cultivation.
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| Description |
Brain organoid culture medium, which can cultivate mammalian brain organoids such as mice, humans, and sheep (fetal brain is recommended for materials), is not sourced from IPSC, can be passaged and amplified, has a short construction cycle, and is produced according to GMP standards.
Product Information:
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Product Composition |
Specification |
Storage conditions and periods |
| 1 |
brain organoid culture medium |
100 mL |
4℃, 3 months or -20℃, 1 year |
Reagent arrival processing:
Brain organoid culture medium can be stored at 4℃ for 3 months, and stored at 4℃ after receipt. It is recommended to use it within 1 month. If not used for a long time, it is recommended to store it at -20℃ to avoid repeated freezing and thawing more than twice.
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