AlgiMatrix™ 6-Well, 3D Culture System, Flat-Bottom Microplate, 4 x 6 well plates - FAQs

AlgiMatrix Matrix is stable for 12 months unopened. Once opened, the entire plate needs to be used.

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Geltrex Matrix is a basement membrane extract that contains collagen IV, laminin, and other matrix proteins and growth factors that are required for cell adhesion. AlgiMatrix Matrix contains only alginate which does not facilitate cell adhesion. There is nothing in the alginate sponges for the cells to interact with.

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Other than the Invitrogen alamarBlue Cell Viability Reagent, AlgiMatrix Matrix is also compatible with Invitrogen PrestoBlue Cell Viability Reagent and Invitrogen CyQuant Direct Cell Proliferation Assay. The protocol is almost the same as the 2D cell culture, but it needs the blank AlgiMatrix Matrix as a blank control.

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The firming buffer works by adding calcium to the sponge, making it stiffer. The dissolving buffer works by removing calcium from the sponge, making it softer.

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3D cell culture with AlgiMatrix Matrix approaches a more physiological state in terms of cell growth and behavior, but along with that comes the higher variability typically associated with more complex systems. You may need to do more replicates or more controls.

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Some primary cells (with the exception of stem cells) need to adhere in order to expand. As cells won't adhere to AlgiMatrix Matrix, these cells can still be cultured, but will tend to differentiate rather than expand. We recommend that you plate at a high cell density for primary cells.

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The height of the sponge is ~3-4 mm dry. When the sponge is hydrated, its volume is essentially the same - it doesn't swell and increase in size, in fact it becomes slightly bit smaller as it essentially goes back into a hydrogel consistency, especially if the inoculated sponges are centrifuged briefly (as suggested to force cells more into the interior and also to spread the sponge uniformly over the bottom of the well).

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Alginate, from which AlgiMatrix Matrix is made, presents a very hydrophilic surface, and lipid-containing cell membranes and even molecules do not adhere. Normal cells will aggregate to form spheroids and differentiate into structures in AlgiMatrix Matrix, but expansion will be limited since most normal cells need to adhere to increase in number. Cancer cells are able to expand without attachment and do so in AlgiMatrix Matrix (one exception to this is embryonic stem cells, which can expand as embryoid bodies in AlgiMatrix Matrix).

However, here is one major difference between soft-agar and AlgiMatrix Matrix: soft-agar is a hydrogel and AlgiMatrix Matrix is a sponge with relatively large pore sizes, and cells that do grow will be able to do so easier and faster in AlgiMatrix Matrix than when encased in a hydrogel (soft-agar). So a traditional way to tell cancer cells from normal cells is to observe the culture for expansion; cancer cells (example HepG2 hepatocarcinoma) will form expanding spheroids while normal cells will form spheroids that do not increase in size or number.

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The recent more highly purified forms of alginate do not generate a sensitivity reaction as is the case with less pure versions. Alginate has been used for some years to encapsulate, for instance, B-cells of the pancreas for in vivo transplantation (diabetes research) and it does not get rejected.

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Our supplier has been producing pharmaceutical-grade alginate and hyaluroinic acid for years. Although these are natural products, by specifying among other things (in the case of alginate) harvest location, water temperature, which part of the seaweed to harvest, use of stringent purification SOPs, and rigorous QC criteria, a very standard product can be obtained.

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Invasion studies measure the ability of cells to penetrate through a matrix, simulating a metastatic cells activity in vivo. AlgiMatrix Matrix contains only alginate (brown seaweed) with no in vivo-like molecules in the undecorated structure, so invasion may not be a very relevant use for AlgiMatrix Matrix.

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Standard immunofluorescence testing can be performed on 3D structures. The staining can be done with either intact spheroids within the AlgiMatrix Matrix or you can dissolve the AlgiMatrix Matrix using Versene or 55 mM tri-sodium citrate dihydrate and stain the spheroid alone. You can further dissociate the spheroid into individual cells using TrypLE reagent and stain as dissociated cells.

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For umbilical cord stem cells, you might want to try a relatively high inoculation density of 100,000-300,000 cells per sponge with daily or every-other-day re-feeding of cultures, as the medium turns “yellow”.

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This scaffold is great for forming 3D spheroids that allow maximum cell to cell interaction. Established cell lines expand by increasing size of spheroids up to ~100 cells or so depending upon the cell type. Both primary and established cells will differentiate in a 3-dimensional format over 1-2 weeks of culture.

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Please see the following references specifically for stem cells:

- Methods Enzymol 420:303 (2006)
- Biotechnol Bioeng 88:313 (2004)

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There is virtually no background staining with alginate. The same surface characteristic that prevents cells from adhering also makes it very difficult for molecules such as fluoresceinated antibodies to adhere, thus essentially no background staining is observed.

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While we have not tested this in-house, the common belief is that if the alginate scaffolds support proliferation of human ES without feeder cells, they should also support mouse ES. Both mouse and human form embryoid bodies, and seeding within the macro-porous alginate scaffolds have been found very advantageous for these structures, in terms of proliferation and differentiation.

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Yes, start by dissolving the matrix using calcium binding solution, Versene, or sodium citrate. The remaining spheroids can be dissociated with TrypLE reagent, counted, and expanded into other culture vessels or into new AlgiMatrix Matrix sponges. These steps are illustrated in the instruction manual that is supplied with each kit.

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Spheroid formation requires about 5 to 7 days once inoculated. Air bubbles will be observed at the time of inoculation. This will dissipate within several days by the cells consuming the oxygen.

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Add medium gently at an angle. Be careful not to impel the matrix when re-feeding.

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We have shown the following data:

- Focusing on cells in culture shows evidence of inoculation.
- Paraffin embedding and sectioning shows that cells are inoculated throughout the sponge.

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Gibco Geltrex Matrix and collagen (rat tail and bovine) can be used as either a coating solution or a 3D gel matrix. CELLstart Substrate was developed to be used as a xeno-free coating matrix for only ESC applications (as a substitute for Gibco Geltrex Matrix or Matrigel Matrix, which are of animal origin). AlgiMatrix Matrix is a 3D scaffold-type matrix that does not support cell attachment but does provide a good environment for growing spheroids that can be easily harvested for downstream applications.

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