It is often difficult to utilize larger biopsy specimens and all the valuable material provided by them. In these cases, the tissue may be preserved by freezing. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition
Operation method
Programme 24.2 Experiments with frozen biopsy tissue
Principle
Tumors were excised, and tumor blocks were immersed in DMSO and then frozen in liquid nitrogen in portions.
Materials and Instruments
Biopsy Specimen Move I. Materials 6. utensils (scalpel, dissecting forceps, petri dish, etc., same as primary culture) 5. Thaw the cryotubes by placing them in warm water at 37°C (with appropriate precautions, see Option 20.2). For more product details, please visit Aladdin Scientific website.
DBBS collection solution Dimethyl sulfoxide
Plastic freezing tubes Dissecting knives Dissecting forceps Petri dishes
aseptic
1. Biopsy specimens
2. 1.2 ml plastic cryopreservation tubes (Nagle Nunc)
3. DBBS
4. Collection fluid
5. Dimethyl sulfoxide (semi-sterile if placed in a sterile container)
Second, the operation steps
1. After removing necrotic tissue, fat and fibrous tissue, cut the tumor into small pieces of about 1~2 mm. As in primary culture, put the tumor pieces into DBBS for washing.
2. Place 4 or 5 tumor blocks in each cryotube.
3. Add 1 ml of growth medium containing 10% DMSO to the frozen tubes with tumor blocks and leave at room temperature for 30 min.
4. Freeze the tube at 1°C/min (see protocol 20.1) and transfer the tube to a liquid nitrogen tank. To avoid the risk of explosion, do not immerse the tubes in liquid nitrogen.
6. Open the cryotube after wiping it thoroughly with ethanol to allow the tumor mass to settle and then aspirate half of the culture.
7. Slowly replenish with fresh culture medium without DMSO, mix with gentle shaking and leave for 5 min.
8. Gradually replace all of the culture medium with DMSO-free culture medium, and then transfer the tumor mass into Petri dishes and proceed as for conventional primary culture, but with more than twice as many cells per culture flask.