Key Points for Culturing bEnd.3 Cells:

1. The most common issues encountered with bEnd.3 cells are changes in cell morphology and difficulty in digestion.

2. Cell Morphology:
   bEnd.3 cells inherently grow slowly. At low density, they exhibit irregular morphology, while at high density, they appear as regular, fibrous structures. Therefore, if abnormal cell morphology is observed during cultivation, it may be due to low cell density. It is recommended to passage the cells when they reach a high confluency.

3. Digestion Issues:
   The longer bEnd.3 cells are cultured, the harder they are to digest.

   • For cells passaged after 4 days of culture, digestion typically takes 3-5 minutes.

   • For cells passaged after 7 days of culture, digestion typically takes 5-10 minutes.
     The exact digestion time should be determined by observing cell status under a microscope.

   If digestion difficulties arise, there is no need to worry. The issue can be resolved through stepwise digestion, as follows (using a T25 flask as an example):

   (1) Aspirate the original culture medium.
   (2) Add approximately 2 mL of PBS, gently swirl the flask to rinse the cells, and then aspirate and discard the PBS.
   (3) Add about 1 mL of trypsin, gently swirl the flask to ensure all cells are coated.
   (4) Place the flask in the incubator for digestion. Digest for 3-5 minutes (adjust slightly based on cell conditions). When cells begin to detach under the microscope, add 2 mL of PBS and swirl the flask to suspend the cells. Avoid pipetting the remaining adherent cells.
   (5) Aspirate the PBS and cell suspension from the flask and transfer them to a sterile centrifuge tube. Add 3 mL of serum-containing medium to the tube to stop digestion and disperse the cells into a single-cell suspension as much as possible.
   (6) Add 0.5 mL of trypsin to the original flask, swirl to coat the cells, and return the flask to the incubator for further digestion until the cell clumps contract and round up, detaching when the flask is swirled.
   (7) Stop digestion with 3 mL of serum-containing medium, gently pipette to detach all remaining cells, and disperse them into single cells.
   (8) Combine the cell suspension with the cells collected from the first digestion into one centrifuge tube.
   (9) Centrifuge to pellet the cells (1100 rpm for 4 minutes), then aspirate and discard the supernatant.
   (10) Resuspend the cell pellet in fresh medium, mix gently, and seed into new flasks as needed. Top up with medium and loosen the cap or use a vented cap for cultivation.
   (11) Check the CO₂ level, temperature, and water tray in the incubator.

4. Cultivation Notes:
   (1) Cell morphology varies during cultivation. At low density, cells may appear radial, seeming to cover the flask surface while actually being sparse. Passage when cells are densely packed.
   (2) Avoid frequent medium changes. Replace or half-change the medium every 2-3 days.
   (3) About 10% of viable single cells may float during cultivation. Collect floating cells when adherent cell density is low; discard them during medium changes when adherent cell density is high.
   (4) Seeding density affects cell growth. Too low a seeding density may result in slow proliferation and excessive floating cells. Control the passaging ratio carefully.
   (5) Cells are sensitive to temperature and pH. Warm the medium to room temperature for at least 4 hours before passaging or medium changes. Avoid using medium with altered pH (yellowish if acidic, purplish-red if alkaline).
   (6) If digestion takes excessively long during passaging, use stepwise digestion. Never forcibly pipette cells off, as mechanical stress can damage them.