spheroid culture (biology)
spheroid culture (biology)
Digest monolayers of cells or disperse the original tissue with trypsin and inoculate the cells onto a substrate coated with agar. The aggregates are transferred to 24-well culture plates and analyzed.
Operation method
spheroid culture (biology)
Principle
Digest monolayers of cells or disperse the original tissue with trypsin and inoculate the cells onto a substrate coated with agar. The aggregates are transferred to 24-well culture plates and analyzed.
Materials and Instruments
Noble's Agar 0.25% Trypsin Move I. Coating 25 cm2 culture flasks with globular agar Caveat When applying agar, all culture flasks, plates, and petri dishes should be sterile, but do not need to be tissue culture grade. For more product details, please visit Aladdin Scientific website.
Growth medium Ultrapure water
Culture flasks 24-well culture plates Petri dishes
1. Add 1 g of Noble's agar to a 100 ml borosilicate glass flask with a loose screw cap containing 20 ml of UPW.
2. Heat the agar in a water bath at 100°C for 10 min, or until the agar is completely dissolved.
3. Immediately add the contents of the flasks to 60 ml of preheated growth medium at 37°C and then add 5 ml to each culture flask. volume to each bottle. Ensure that there are no air bubbles in the agar.
4. Leave the agar at room temperature for about 5 min to prepare 1.25% agar-coated culture bottles.
II. Coating Multi-well Plates with Agar
1. Add 0.5 g of agar to 10 ml of UPW as heated in Step 2 of coating 25 cm2 culture flasks, then add 40 ml of UPW.
2. Prepare 1% agar-coated culture wells by adding 0.5 ml of the final solution to each well of a 24-well plate. Precise and careful addition is important to ensure easy microscopic focusing of individual wells during subsequent sphere observation.
Sphere Formation
1. For established cell lines, trypsinize confluent monolayers of cells or disperse interstitial tumor cells to produce single cell suspensions.
2. If desired, trypsin can be neutralized with serum-containing culture medium.
3. Cells are counted using either an electronic cell counter or a blood cell counting plate.
4. To each agar-pre-coated 25 cm2 culture flask add 5 ml of growth medium containing 5x105 cells in growth medium and then incubate the cells. If the cells have the ability to form spheres, small clusters of aggregates (about 100~300 um in diameter) can be formed spontaneously within 3~5 d. The cells should be incubated for a period of time.
In order to facilitate future growth, the spheroids should be transferred to new 25 cm2 culture flasks or 24-well culture plates.
Transfer to 25 cm2 culture flasks
1. Transfer the culture from the original culture flasks into a conical centrifuge tube or a regular container.
2. After allowing the spheres to settle, remove the supernatant and individual cells.
3. Resuspend the spheres with fresh culture medium and transfer the suspension into new agar pre-coated culture flasks. The spheroplasts can continue to grow in these culture flasks by dividing the cells in the outer layer.
V. Transfer to 24-well plates
1. Transfer the culture from each 25 cm2 culture flask into a 6 cm Petri dish.
2. To each agar pre-coated well of a 24-well plate, add 0.5 ml of culture solution.
3. Under low magnification (40x), select spheroids of a certain size one by one. Using a Pasteur pipette and Pipump or other pipettes with appropriate fine adjustments, pipette the selected spheres of similar diameter into each well of the agar pre-coated 24-well plate one by one.
4. Place the 24-well plate into a CO2 incubator.
5. Change the culture medium 1 or 2 times a week, 0.5 ml per change. or add 0.5 ml of culture medium (without removing any culture medium) 1 or 2 times a week for 2 to 4 weeks. Or add 0.5 ml of culture solution 1 or 2 times a week (without removing any culture solution), and the culture solution reaches 2 ml/well after 2~4 weeks.