Matrices for Cell Culture – How to develop healthy attachment

Introduction

According to the dictionary, there are many definitions for the word matrix.  From my point of view the following three pertain to cell culture; 1)  a substance in which something is embedded or enclosed; 2) a situation or set of circumstances that allows or encourages the origin, development, or growth of something and; 3) the substance that exists between cells and from which tissue., for example, cartilage and bone, develops. Many of the common cell types we use are robust and grow on cell culture treated plastic. However, there are many cell types that have difficulty attaching and/or spreading on cell culture treated plastic.  In other cases, cells may attach but not differentiate unless the attachment surface correct. To learn more about matrices or if you are using them and have questions, this Ask The Expert topic is your chance to learn more about attachment surfaces and the rationale for using them.

Question 1

Can you recommend any matrices that don’t require enzymes during passaging. I am using iPSCs.

This is a good question. You know most of the matrices out there do require some sort of enzymatic digestion if your goal it to obtain single cells. Some matrices are such that the matrix can be dissolved to liberate the cells for assays or passaging. One example is Life Technologies AlgiMatrix 3D where a tri-sodium citrate solution is used to solubilize the matrix allowing cell isolation. Another approach is to use something like Versene which is a Ca/Mg free buffer with 0.43 mM EDTA. Versene chelates the Ca in media and buffer, and its use causes the attachment proteins do disassociate somewhat and can help some cell types loose attachment without using an enzyme. Primorigen Biosciences producers of StemAdhere XF allows for the growth of stem cells and iPSCs and non-enzymatic release of cells with a cell release buffer. Finally I found an interesting paper that may be pointing to the future for stem cell culturing. A Nature Communications 2013, Vol 4:1335 publication entitled, "A Thermoresponsive and Chemically Defined Hydrogel for Long-Term Culture of Human Embryonic Stem Cells" by Rong Zhang et al. These gels permit gentle reagent-free passaging using using temperature as a means to dissociate the matrix and allow passaging without enzymes.

Question 2

Respected sir , I am currently working on the stem cell research for the assessment of nano particles in Caprine Wharton’s jelly derived mesenchymal stem cells . Last month I take the sample from local abbatior and separate the whartons jelly and pour it in different patriplates after proper procedure and kept the plates in C02 Incubator after 15-20 days I observed the plate under microscope and cells were grown and were healthy and contamination free but there were not any attachment of cells. Sir please advise me and guide me what will be the problem. Thanking you Sir

Hello Sir. So, I need to tell the readers a bit of background to your question before I answer it. Caprine are related to animals of the subfamily Caprinae" (goat-antelope) and Whaton's jellyi(from Wkipedia) is a gelatinous substance within the umbilical cord also present in vitreous humor of the eyeball, largely made up of mucopolysaccharides (hyaluronic acid and chondroitin sulfate). It also contains some fibroblasts and macrophages. Now, from your message I noticed you mentioned plating cells on " different patriplates". If this is a misspelling and you meant petri-dishes then there is your problem. Petri-dishes are plastic dishes for growing bacteria. Animal cells grow on cell culture treated charged plastic and the petri-dishes are hydrophobic. When animal cells are grown on petri-dishes they do not attach and will not attach. Use cell culture treated plastic dishes and use the Alpha modification of MEM supplemented with 20% FBS and you should be fine. I would be interested if the answers your question.

Question 3

Respected Sir, I want to know that, above mentioned Matrices for Cell attachment are autoclavable? What concentration will be effective per square centimeters of surface area?

This is a difficult question to answer since it depends on the matrix you are using. Generally for things like collagen, laminin, fibronectin, poly-L-lysine or something like that, we use 0.1 mg/ml using for example, 2 mls for a 35 mm cell culture dish. I use sterile water to reconstitute and dilute, then I add the volume to the dish and let it sit in the hood for longer than about 2 hours. Then I rinse with sterile water several times and let dry. I then store the dried dishes at room temp in the hood. For other matrices I recommend following the manufactures instructions. As far as autoclaving goes...generally matrices come sterile and all the buffers and media would also be sterile so there should be no need to autoclave.

Question 4

Do you know of any available clinical grade quality matrices? Or maybe specialty plates that allow for less or no plate coating for use with iPSCs.

Primorigen Biosciences has something called StemAdhere which they say is a defined matrix for hPSC and I imagine iPSCs. Another is Thermo Scientific Nunclon Vita Stem Cell Culture Surface. This format allows for the direct attachment and growth of cells without the need of feeder layers or matrix coatings. I hope this helps and good luck

Question 5

How do matrices affect differentiation? Is there a way to control this?

Yes they do. One of the major reasons people use matrices is to maintain or promote differentiation. Years ago attachment was thought of as simply that...now we know attachment proteins in a cell contacting the proper matrix is a survival signal and a differentiation signal. Many of the different attachment proteins in a cell act as signal transducers that alter gene expression pathways in a cell. This is one reason why you read so much about 3D culture. Most cells do not live in 2D but in 3D and allowing them to grow in a more natural way causes cells to do things in 3D that they can not do in 2D. I often do experiments with a pheochromocytoma cell line that will differentiate into neuronal like cells if plated on a positive charged dish.

Question 6

What are the differences, advantages/disadvantages to 3-D matrices?

Lets start with the advantages. When we grow cells the way most people do, by placing them in a dish and allowing them to attach we are growing cells in 2D. As you are aware few cells in the body grow in 2D, they actually grow in 3D. The idea behind 3D culture is to mimic the natural environment of the cell most closely making cell cultures more authentic in both structure and function. Cells growing in 2D have unlimited access to nutrients from the media above and the ability to remove waste products directly into the media. We also now know that cell-cell attachment and cell-matrix attachment are signal transducers and affect gene expression. As an example, Dr. Sangeeta Bhatia at M.I.T. has developed a system where rat hepatocyte cells are placed to mimic the natural organization of liver cells and has built a complete miniaturized ex vivo liver for drug metabolism studies (The Scientist May 1, 2013). Her approach is slightly different than 3D culture but it does point to the cell organization-funtion relationship. Others have shown how intestinal cells grown in 2D do not uptake blood pressure drugs because the bottom of the cells is where uptake occurs. In 2D cultures the bottom portion of the cell is not structurally relevant for uptake. In 3D culture uptake occurs.

On the downside, people have found some disadvantages to 3D culture but these disadvantages are being overcome by innovation. Some matrices are made from animal origin components which may make implementation for clinical work difficult. Some matrices made from tissue such as basement membrane extracts, can contain unknown or unwanted components like growth factors or viruses. Other matrices allow for attachment but not efficient removal of the cells making assay development difficult. To overcome these problems Gibco's AlgiMatrix 3D culture System are completely animal origin free. As the name implies, AlgiMatrix 3D is made from algae and once the cells are grown, the 3D matrix can be dissolved using a dissolving buffer, liberating the cells for assays or whatever is needed. Other approaches to minimize these potential problems have resulted in matrix-free 3D culture. N3DBIO's Bio-Assembler System actually levitates cells causing them to cluster together as groups making them have 3D contact without using a physical matrix. N3DBIO accomplishes this by providing a solution containing nanomagnetic particles that are incubated with the cells in culture. During a several hour incubation the magnetic particles are taken up by the cells. Once a supplied magnet is suspended above the wells the cells levitate together and grow as a cluster of cells in 3D.

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