1.   How stable is the product at room temperature w/o degradation?

2.   How far into the center do cells penetrate?

3.   Is there any data showing penetration and % cell population?

4.   What level of force can be used when refeeding?

5.   How long does spheroid formation take?

6.   What is the function of Alamar Blue?

7.   One slide shows "not suitable for adherent cells and primary cells"?

8.   Most people grow cells for 3 to 4 days and at certain density, they subculture cells.  How can I subculture in this matrix?

9.   Can AlgiMatrix™ be used for culturing mouse ES cells?

10. When staining cells or spheroids in the sponge, how problematic is background staining due to the AlgiMatrix™?

11. Are there references or protocols that describe how to culture and expand MSCs, HUVECS and HDMECS in the 3D AlgiMatrix™  sponge?

12. I would like more information on your AlgiMatrix™ 3D culture systems.  What are the optimal growth conditions?  Limitations? What plates provide the best growth?

13. What are the possible downstream analyses for this kind of 3D cell culture?

14. Matrigel is very well-known standard for metastasis studies / invasion studies with cancer cell lines or cancer primary cells.  Can AlgiMatrix™ be used for metastasis studies / invasion studies?

15. If AlgiMatrix™ is extract from brown seaweed how come it is of pharmaceutical raw material standard and also without lot-to-lot variations?

16. The significance of collagen is that it is just like any natural structures in body and that it makes it less "foreign" to human body for potential transplant applications so how about AlgiMatrix™?

17. Why do normal cells grow in this system?  I have done a lot of soft-agar assays and in that system, normal cells do not grow but cancer cells do.

18. One of our customers called in after seeing a picture in the Image Gallery on the 3D site which showed rat cardiac myocytes growing on RGD-decorated alginate.  Do you know how this process is done, or where we could find out more about it?

19. Has AlgiMatrix™ has been used to redifferentiate chondrocytes?

20. What is the depth of the AlgiMatrix™ sponge? If it enlarges after medium addition, I'd like to know both sizes (after and before).

Answers

1.    How stable is the product at room temperature w/o degradation?

Answer.    12 months unopened. Once opened, entire plate needs to be used.




2.    How far into the center do cells penetrate?

Answer.    When cells are hydrated, a certain amount of collapse observed.  Some segmentation seen.  More cells in upper layer and fewer in interior.  Proposed inoculation protocol including centrifugation, allows better penetration.




3.    Any data showing penetration and % cell population?

Answer.    Two kinds of data:

i.    Focusing on cells in culture show evidence of inoculation.
ii.    Paraffin imbedding and sectioning shows cells are inoculated throughout sponge.




4.    What level of force can be used when refeeding?

Answer.    Add media at an angle and gently add new media.  Be careful not to impel the matrix when refeeding.



5.    How long does spheroid formation take?

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



6.    What is the function of Alamar Blue?

Answer.    Alamar blue will be indicative of health and function of cells. Handy biological assay to monitor cell health.



7.    One slide shows "not suitable for adherent cells and primary cells?"

Answer.    A significant amount of data has not been done internally.  Dr. Cohen has performed research on primary cells and have evaluated for differentiation and formation of structures. Not seeing significant expansion or adherence of cells into matrix. AlgiMatrix™ does not allow attachment, however they differentiate nicely and representative of in vivo environment.



8.    Usually customers grow cells for 3 to 4 days and at certain density, they subculture cells.  How can they subculture in this matrix?

Answer.    Expand by dissolving matrix using calcium binding solution, Versene or sodium citrate.  Remaining spheroids can be dissociated with TrypLE, counted and expanded into other culture vessels or into new AlgiMatrix
sponges.  These steps are illustrated in our instruction manual that is supplied with each kit.



9.    Can AlgiMatrix™ be used for culturing mouse ES cells?

Answer.    Answer from Dr. Smadar Cohen: I believe that if the alginate scaffolds supported proliferation of human ES without feeder cells, so they can 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.



10.    When staining cells or spheroids in the sponge, how problematic is background staining due to the AlgiMatrix™?

Answer.    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.



11.    The ASCB-Alginate Sponge SC 1206 PowerPoint presentation regarding the AlgiMatrix product states that AlgiMatrix can be used to expand human mesenchymal stem cells (MSCs). Furthermore there is a list of other human cell types (HUVECS, HDMECS) that can be cultured in 3D.  Are there references or protocols that describe how to culture and expand MSCs, HUVECS and HDMECS in the 3D AlgiMatrix sponge?

Answer.    These references should be on the web site. Most of the articles are from Cohen's research at Ben Gurion University. The slide about expansion of human MSC was from her presentation. I saw good differentiation with human MSC but not much expansion. At ASCB I mentioned this to her and she did say that MSC would probably require surface modification with attachment factors for proper cell expansion.  Specifically for stem cells: the Methods Enzymol. 420: 303-315, 2006 and the Biotechnol. Bioeng. 88:313-320, 2004 articles are important for protocols.



12.    I would like more information on your AlgiMatrix 3D culture systems.  What are the optimal growth conditions? Limitations? What plates provide the best growth?  Our research team would like to use this system for umbilical cord stem cells. Any additional information you could offer would be appreciated.

Answer.    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 types. Both primary and established cells will differentiate in a 3-dimensional format over 1-2 weeks of culture.  Presently we offer AlgiMatrix in a 96 well tray only, but we are interested in learning if other tray sizes or formats would be desired. On the Invitrogen web site are listed articles on stem cells that provide culture methods information. 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".



13.    What are the possible downstream analyses for this kind of 3D cell culture? For instance if someone would like to do immunofluorescence / immunohistochemistry staining, how can this be done with a 3D structure? Can the normal fluorescence / light microscope handle the 3D structure staining? If yes, any application notes?

Answer.    Standard IF testing can be done on 3D structures. The staining can be done with either intact spheroids within the AlgiMatrix or you can dissolve the AlgiMatrix 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 and stain as dissociated cells. Our flyer insert with publication references is good for applicationnotes.



14.    Matrigel is very well-known standard for metastasis studies / invasion studies with cancer cell lines or cancer primary cells. So if AlgiMatrixbe used for metastasis studies / invasion studies? How does the property of "not so compatible to adherent cell culture" hamper its potential applications in metastasis studies?

Answer.    Invasion studies measure the ability of cells to penetrate through a matrix, simulating what would be going on in vivo. Matrigel is good for this since it contains collagen and laminin among other molecules which also occur in vivo (human) and a parallel could be drawn between situations that would impede invasion in Matrigel and in vivo. However, AlgiMatrix 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.  (We are working on other formulations of scaffolds to remedy this).



15.    If AlgiMatrix is extract from brown seaweed how come it is of pharmaceutical raw material standard, and also without lot-to-lot variations?

Answer.    Good question. Our supplier has been producing pharmaceutical grade alginate and hyaluroinic acid for years. Although 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, very standard product can be obtained.




16.    The significance of collagen is that it is just like any natural structures in the body and then it makes it less "foreign" to the human body for potential transplant applications, so how about AlgiMatrix?

Answer.    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 become rejected. In general two directions seem to be desired by researchers: using either totally chemically-defined, non-natural scaffolds or highly purified chemicals that are the same as those inside the body (collagen, etc.).



17.    Why do normal cells grow in this system?  I have done a lot of soft-agar assays. In that system, normal cells do not grow but cancer cells do. This is a hallmark of cancer cells.   If normal and cancer cells form spheroids in this matrix, how do you distinguish normal from cancer cells in a traditional sense?

Answer.    Alginate, from which AlgiMatrix 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 but you are right that expansion will be limited since most normal cells need to adhere to increase in number. Again as you are aware, cancer cells are able to expand without attachment and do so in AlgiMatrix. (One exception to this is embryonic stem cells which can expand as embryoid bodies in AlgiMatrix).  However, one major difference: soft-agar is a hydrogel and AlgiMatrix is a sponge with relatively larger pore sizes and cells that do grow will be able to do so easier and faster in AlgiMatrix than when encased in a hydrogel (soft-agar). So a traditional way to tell cancer from normal 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.



18.    One of our customers called in after seeing a picture in the Image Gallery on the 3D site which showed rat cardiac myocytes growing on RGD-decorated alginate.  Do you know how this process is done, or where we could find out more about it?

Answer.    The RGD process covalently links a 3 amino acid peptide sequence (arginine, glycine, aspartic acid, which is in various ECM molecules) onto alginate to support cell adherence. The covalent attachment is usually via carbodiimide, a linker between amine groups on the peptide and the carboxyl groups on the alginate (which is a polysaccharide). This and other means of getting cells to adhere to alginate are actively being researched by us (Invitrogen) now so that most primary cells in addition to the cardiac myocytes in the photo will be able to attach and expand in culture. Your answer to the 3T6 fibroblast question is a good one. Those cells would form spheroids in AlgiMatrix, but would probably not expand significantly since fibroblasts need attachment for this. However, if the customer is using 3T6's as a feeder layer to support differentiation of another cell population, a co-culture in AlgiMatrix may result in in vivo-like tissue constructs as both cells should segregate and even form cell layers in the spheroid as in vivo, something that does not happen in 2D.



19.    Do you know if your AlgiMatrix has been used to redifferentiate chondrocytes? I usually grow chondrocyte cultures in sodium alginate beads.

Answer.     I'm not sure what is meant by "redifferentiate chondrocytes"? Does this mean differentiate MSC into chondrocytes? (We have done this in AlgiMatrix). Does it mean redifferentiating (actually "de-differentiate" is the term mostly used) chondrocytes back into earlier cell types (e.g. MSC)? This has not been done in AlgiMatrix by anyone to my knowledge.



20.    How much is the depth of the AlgiMatrix sponge? If it enlarges after medium addition, I'd like to know both sizes (after and before).

Answer.    The height of the sponge is ~3-4mm dry. When the sponge is hydrated its volume is essentially the same-it doesn't swell and increase in size, in fact it becomes a slight bit smaller as it essentially goes back into a hydrogel consistency and 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).