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This page contains protocols for using AlgiMatrix™ Firming Buffer with 24-well and 96-well AlgiMatrix™ 3D Culture System plates. The following products are discussed in this protocol, each sold separately and are also available under "Ordering Information":

Products Catalog numbers
AlgiMatrix™ 3D Culture System 24-well plate 12684-023 (1 plate) 12684-048 (4 plates)
AlgiMatrix™ 3D Culture System 96-well plate 12684-015 (1 plate) 12684-031 (5 plates)
AlgiMatrix™ Firming Buffer A10915-01 (50 ml)


The AlgiMatrix™ 3D Culture System is an animal origin-free bioscaffold (sponge) that facilitates three-dimensional (3D) cell culture. AlgiMatrix™ sponges are provided in a plate-well format. AlgiMatrix™ Firming Buffer is an isotonic, neutral-pH buffered solution designed to increase the firmness of the bioscaffold. Firmer sponges are less susceptible to pore expansion and contraction by cellular and environmental forces, and can also be easily picked up and manipulated by sterile forceps. This allows sponges to be transferred to different wells, or inverted to seed or observe cells on both sides of the sponge.

Guidelines for Using the Firming Buffer

For a firmer sponge, add AlgiMatrix™ Firming Buffer to cells and culture medium prior to adding the cells to the sponge. Alternatively, for cell culture applications that require lower calcium levels, you may rehydrate the sponge with Firming Buffer before adding cells and medium. See the following step-by-step protocols.

Recommended Concentration of Firming Buffer

For general use, we recommend a Firming Buffer concentration of 10% in solution. For a firmer AlgiMatrix™ sponge, the Firming Buffer concentration can be increased as high as 50%. Lower Firming Buffer concentrations will lead to softer sponges with better optical clarity.

Recommended Volume of Firming Buffer

The volume of cells plus Firming Buffer needed per AlgiMatrix™ sponge varies depending on the size of the plate

  • 96-well plates: For each sponge, prepare 100 μl of cells in culture medium plus Firming Buffer. For example, for a 10% concentration of Firming Buffer, add 10 μl of Firming Buffer to 90 μl of cells in medium.
  • 24-well plates: For each sponge, prepare 500 μl of cells in culture medium plus Firming Buffer. For example, for a 10% concentration of Firming Buffer, add 50 μl of Firming Buffer to 450 μl of cells in medium.

Standard Protocol for 24-well and 96-well AlgiMatrix™ Plates

To use AlgiMatrix™ Firming Buffer:

  1. Resuspend cells in standard cell culture medium, then add 10% AlgiMatrix™ Firming Buffer to this suspension (e.g., add 10 μl Firming Buffer to 90 μl cells plus medium). The optimal final cell concentration will vary by cell type, but in general 1 × 106 cells/ml is a reliable target. While a 10% Firming Buffer concentration provides an optimal balance between sponge transparency and firmness, certain applications may benefit from optimization of this formulation (see the guidelines above).

  2. Remove the AlgiMatrix™ 3D Culture System plate from its package and discard the desiccant.

  3. 96-well plates: Inoculate 100 μl of the cell suspension with Firming Buffer onto the top of each dry sponge with a pipette. 24-well plates: Inoculate 500 μl of the cell suspension with Firming Buffer onto the top of each dry sponge with a pipette. 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 the pipette tip.

  4. Approximately 5 minutes after rehydration, the sponge should appear foamy with a dry or soggy surface.

  5. 96-well plates: Add 100 μl of culture medium without Firming Buffer to the top of each sponge with a pipette. 24-well plates: Add 500 μl of culture medium without Firming Buffer to the top of each sponge with a pipette.

  6. Incubate plate(s) in an incubator (36–38°C in a humidified atmosphere of 4–6% CO2 in air). If using multiple plates, do not stack plates. Note: 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.

  7. Change the medium based on cell proliferation or when the medium begins to change color. 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 aspirating the sponge (see image to the right). If the sponge becomes stuck in the aspirator, stop suction right away and release the sponge, then gently try again. If a sponge has been partially aspirated, do not use that well for quantitative assays.

  8. After several days, remove the plate from the incubator and exam under light microscopy (low magnification) for the presence of spheroid formation.


Alternative Protocol for Cell Culture Applications that Require Lower Calcium Levels

AlgiMatrix™ Firming Buffer contains elevated calcium levels. If lower calcium environments are required for specific cell culture applications, modify the first steps of the culturing protocol as follows:

  1. Add cell culture medium with Firming Buffer to the AlgiMatrix™ sponge without cells.

  2. Rehydrate for ~5 minutes.

  3. Optional: If you are using a high concentration of Firming Buffer in the cell seeding medium (e.g., 50%), rinse the sponge once with culture medium without Firming Buffer.

  4. Inoculate cells in standard culture medium (without Firming Buffer) to the sponge, then proceed with incubation.

Learn more

A11200      Rev. date:   13-Jan-2009