AlgiMatrix 3D Cell Culture Scaffold

*Gift courtesy of Smadar Cohen and Tsiona Elkayam Ben-Gurion, University of Negev

1. How stable is the product at room temperature? 
2. How far into the center of the sponge do cells penetrate? 
3. Is there any data showing penetration and percent cell population? 
4. How should media be added during feedings? 
5. How long does spheroid formation take? 
6. What is the function of alamarBlue reagent? 
7. Is the AlgiMatrix system suitable for adherent cells and primary cells?
8. How can I subculture my cells 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 material?
11. I would like more information on your AlgiMatrix 3D culture systems. What are the optimal growth conditions?  Limitations? What plates provide the best growth?
12. What are the possible downstream analyses for this kind of 3D cell culture? 
13. Matrigel is very well-known standard for metastasis studies/invasion studies with cancer cell lines or cancer primary cells. Can AlgiMatrix sponge be used for these types of studies?
14. If AlgiMatrix material is extracted from brown seaweed, how can it be of pharmaceutical raw material standard and also without lot-to-lot variations?
15. Collagen is considered to closely resemble natural structures in the body, making it less "foreign" with respect to potential transplant applications. How does AlgiMatrix material compare in this regard?
16. In soft agar assays, cancer cells grow whereas normal cells do not, making soft agar assays a method to distinguish cancerous from non-cancerous cells. In the Matrigel system both normal cells and cancer cells grow, so how do you distinguish between the two in this system?
17. In the 3D image collection within the Image Gallery, there are images of 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?
18. Has AlgiMatrix sponge been used to re-differentiate chondrocytes?
19. Can you tell me the height of AlgiMatrix sponge when dry and when hydrated?

1. How stable is the product at room temperature?
   The plate is stable for 12 months at room temperature as long as it is unopened. Once opened, the entire plate needs to be used.
   
   
2. How far into the center of the sponge do cells penetrate?
   When cells are hydrated, a certain amount of collapse observed. Some segmentation is observed, with more cells in the upper layer and fewer in the interior. Following the recommended inoculation protocol, including centrifugation, allows better penetration.
   
   
3. Do you have any data regarding penetration and percent cell population?
   We have two kinds of data:
   i.    Focusing on cells in culture show evidence of inoculation.
   ii.    Paraffin embedding and sectioning shows cells are inoculated throughout sponge.
   
   
4. How should media be added during feedings?
   Media should be added gently and at an angle. Be careful not to impel the matrix when feeding.
   
   
5. How long does spheroid formation take?
   After inoculation, spheroid formation takes about 5–7 days. Air bubbles will be observed at the time of inoculation. These will dissipate within several days as the cells consume the oxygen.
   
   
6. What is the function of alamarBlue reagent?
   alamarBlue reagent is a mix-and-read cell viability and vitality reagent. The assay assay is based on the ability of metabolically active cells to convert the reagent into a fluorescent and colorimetric indicator. Damaged and non-viable cells have lower innate metabolic activity, and generate a proportionally lower signal. The dye itself has low toxicity, and is generally safe for cells. alamarBlue reagent may be added to AlgiMatrix spheroids to assess cell viability and proliferation.
   
   
7. Is the AlgiMatrix system suitable for adherent cells and primary cells?
   We don't have a significant amount of in-house data on those scenarios. AlgiMatrix sponge does not allow attachment or significant expansion of primary cells, but anecdotal reports suggest that cells grown in AlgiMatrix do differentiate.
   
   
8. How can I subculture my cells in this matrix?
   Expand by dissolving matrix using calcium binding solution, Versene solution or sodium citrate. Remaining spheroids can be dissociated with TrypLE reagent, 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 sponge be used for culturing mouse ES cells?
   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 sponge?
    There is virtually no background staining with alginate. The same surface characteristics that prevent cells from adhering also make it very difficult for molecules such as fluorescein-labeled antibodies to adhere, which makes background staining very unlikely.
    
    
11. I would like more information on your AlgiMatrix 3D culture systems. What are the optimal growth conditions? Limitations? What plates provide the best growth?
    This scaffold is great for forming 3D spheroids that allow maximum cell–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 3D systems 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 feeding of cultures, as the medium turns "yellow".
    
    
12. What are the possible downstream analyses for this kind of 3D cell culture?
    Standard IF testing can be performed on 3D structures. The staining can be performed with either intact spheroids within the AlgiMatrix sponge, or you can dissolve the AlgiMatrix sponge using Versene solution or 55 mM trisodium citrate dihydrate and stain the spheroid alone. You can further dissociate the spheroid into individual cells using TrypLE reagent and stain as dissociated cells.
    
    
13. Matrigel is very well-known standard for metastasis studies/invasion studies with cancer cell lines or cancer primary cells. Can AlgiMatrix sponge be used for be used for these types of studies?
    Invasion studies measure the ability of cells to penetrate through a matrix, simulating the in vivo situation. Matrigel is good for this since it contains collagen and laminin among other molecules that occur in vivo (human), and a parallel may be drawn between situations that would impede invasion in Matrigel and in vivo. However, AlgiMatrix sponge 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 3D systems.
    
    
14. If AlgiMatrix material is extracted from brown seaweed, how can it be of pharmaceutical raw material standard and also without lot-to-lot variations?
    Good question. Our supplier has been producing pharmaceutical-grade alginate and hyaluronic acid for years. Although a natural product, 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.
    
    
15. Collagen is considered to closely resemble natural structures in the body, making it less "foreign" with respect to potential transplant applications. How does AlgiMatrix compare in this regard?
    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 pancreatic B cells 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.).
    
    
16. In soft agar assays, cancer cells grow whereas normal cells do not, making soft agar assays a method to distinguish cancerous from non-cancerous cells. In the Matrigel system both normal cells and cancer cells grow, so how do you distinguish between the two in this system?
    Alginate, from which AlgiMatrix material 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 sponge, 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 sponge. (One exception to this is embryonic stem cells which can expand as embryoid bodies in AlgiMatrix sponge). However, one major difference: soft-agar is a hydrogel and AlgiMatrix material is a sponge with relatively large pore sizes, and cells that do grow will be able to do so easier and faster in AlgiMatrix sponge than when encased in a hydrogel (soft agar). So one way you can distinguish cancerous 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.
    
    
17. In the 3D image collection within the Image Gallery, there are images of 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?
    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 so that most primary cells in addition to the cardiac myocytes in the photos will be able to attach and expand in culture. 3T6 fibroblasts would form spheroids in AlgiMatrix sponge, but would probably not expand significantly since fibroblasts need attachment for this. However, if you are using 3T6s as a feeder layer to support differentiation of another cell population, a co-culture in AlgiMatrix sponge 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.
    
    
18. Has AlgiMatrix sponge been used to redifferentiate chondrocytes?
    If by "redifferentiate" you mean differentiate MSCs into chondrocytes, then yes, we have done this using AlgiMatrix sponge. If you mean de-differentiating chondrocytes back into earlier cell types (e.g., MSCs), then no. This has not been done in AlgiMatrix sponge by anyone to our knowledge.
    
    
19. Can you tell me the height of AlgiMatrix sponge when dry and when hydrated?
    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 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).