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1. Resuspend the cells in standard cell culture medium, and then add 10% (v/v) AlgiMatrix™ Firming Buffer to this suspension (i.e., 1 part Firming Buffer to 9 parts cells plus medium). The optimal final cell concentration will vary by cell type, but in general 1 × 10 6 cells/mL is a reliable target. While 10% (v/v) Firming Buffer provides an optimal balance between sponge transparency and firmness, certain applications may benefit from optimizing this concentration.
2. Remove the AlgiMatrix™ 3D Culture System plate from its package and discard the desiccant.
3. Depending on your plate type, inoculate the following volume of the suspension from Step 1 onto the top of each dry sponge with a pipette:
6-well Plate 24-well Plate 96-well Plate
2 mL 400–500 μL 80–120 μL
4. Optional: Dynamically seed the sponges by immediately centrifuging the plates at 100 × g for 4 minutes. Certain cell types may be embedded more thoroughly within the sponge with dynamic seeding.
Note: The sponge will become wet and translucent. Gas bubbles may appear in the matrix; they will decrease or disappear with time. If large bubbles appear inside or under the sponge, release by gently pushing the sponge against the plate bottom with pipette tip.
5. Approximately 5 minutes after rehydration, the sponge should appear foamy with a dry or soggy surface. Depending on your plate and cell type, add the following additional volumes of culture medium without Firming Buffer to the top of each sponge (volumes are intended as a general starting point; volumes may vary by cell type):
2–3 mL 400–500 μL 80–120 μL
6. Incubate the plate(s) in an incubator (36–38°C in a humidified atmosphere of 4–6% CO 2 in air). Do not stack multiple plates.
7. Change the medium based on cell proliferation or when the medium begins to change color. To observe the cells inside or on top of the AlgiMatrix™ sponge, decrease the medium volume or relocate the sponge to a dry well. Sponges can be inverted for better observation of cells at the top of the sponges.
Note: Do not allow the aspirating pipette tip to contact the sponge when removing spent medium. Keep the tip on an angle against the wall of the well and block the tip with another pipette tip to avoid sucking up the sponge (see image). If the sponge becomes stuck in the aspirator, stop suction right away and release the sponge, then gently try again. If the sponge has been partially aspirated, do not use that well for quantitative assays. Refer to Tips at the top of the page for additional suggestions regarding medium exchange. Picture 1
8. After several days, remove the plate from the incubator and examine under light microscopy (low magnification) for the presence of spheroid formation.
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