primary outgrowth

Summary

The tissues were minced and washed, and the tissue blocks were inoculated on the surface of culture flasks or Picot dishes with a small amount of medium containing a high concentration of serum (40 % to 50 % ), and the surface tension kept the specimens in place until they spontaneously adhered to the surface, and subsequently the cells began to grow.

Operation method

Program 12.4 Primary Outgrowth

Principle

The tissues were minced and washed, and the tissue blocks were inoculated on the surface of culture flasks or Picot dishes with a small amount of medium containing a high concentration of serum (40 % to 50 % ), and the surface tension kept the specimens in place until they spontaneously adhered to the surface, and subsequently the cells began to grow.

Materials and Instruments

Growth media Petri dishes
Forceps Scalpel Pipettes Centrifuge tubes Containers

Move

1. Transfer the tissue to fresh, sterile BSS for rinsing.

2. Transfer to a second Petri dish (25 ^cm2 flask or 5-6 cm Picasso dish (tissue culture grade), volume size and amount of growth medium used depends on the amount of tissue taken, approximately 100 mg of tissue in 5 25 ^cm2 flasks) and excise any excess tissue such as fat or necrotic tissue before transferring to a third Petri dish.

3. Use a scalpel to cross-cut the tissue into small pieces of approximately 1 mm³.



4. Use a pipette (10-20 ml wide mouth tip) to transfer the tissue pieces into a 15 or 50 ml sterile centrifuge tube or a regular container (wet the pipette with BSS or culture medium first, otherwise the tissue pieces will tend to stick to the inner wall of the pipette).

5. Allow the block to settle.

6. Resuspend and rinse the block with DBSS (10 ml) and let it stand to remove the supernatant, repeating this step two or more times.

7. Transfer the blocks to culture flasks (remember to wet the pipettes first), inoculating each 25 ^cm2 flask with approximately 20-30 blocks of tissue.

8. Pipette off the excess liquid and add 1 ml of medium (growth medium (e.g. DMEM mixed with F12 in equal volume, plus 20 % fetal bovine serum)) per 25 ^cm2 of growth surface, and tilt the flasks gently to distribute the tissue blocks evenly on the growth surface.

9. Cover the bottle and place it in an incubator or a greenhouse at 37°C for 18~24 hours.

10. If the tissue mass has been adhered, the medium can be increased to 5 ml per 25 ^cm2 in the next 3-5 days and then changed once a week until the cells have grown stably.



11. Once outward growth has formed, the explants can be removed with forceps and re-transferred to a new culture vessel with a pre-moistened pipette (and return to step 7 for re-inoculation).



12. Replace the medium in the first vial until the cells have grown more than half of the growth area, at which point the cells are ready for passaging.

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