Polystyrene has always been a good choice for cultureware because of its optical clarity, its ability to be molded into many vessel types and the fact that it is fairly inexpensive. The major disadvantage is that it is hydrophobic and not a very hospitable surface for cell attachment without treatment. Over the years, polystyrene modifications were developed to promote better cell attachment. Modifications can be chemical or physical and each offers specific advantages. One common treatment is gas-plasma treatment, also called tissue culture treated. These modifications worked quite well for the majority of cell types when grown in serum supplemented media. Difficult to attach cell lines required coating of the plate with biological extracellular matrices. More recently there has been a large movement toward animal-free cell culture, which prompted the need for more advanced cell culture surfaces. Thus. a new generation of cell culture surfaces was born to address more specific cell needs and to allow for culturing of the most difficult cell lines in a reduced serum or serum-free environment.
These advanced animal-free cell culture surfaces can help in reducing or eliminating serum from culture, can promote better cell adhesion, allow for the culture of difficult cell types, provide better transfection efficiency, and can provide better anchorage when assay conditions are demanding. These new surfaces offer a variety of solutions to address several problems that are the result of culturing a new cell types and the move to animal-free culture.
Animal-Free Surfaces Available
Based on the cell type, plates often need to be coated with a biological extracellular matrix to allow cell adhesion. These biological coatings are animal products, which is problematic for anyone trying to move to an animal-free culture system. In addition, the use of animal products opens the door for contamination, safety concerns and lot-to-lot variability – all typical problems when animal-products are used. These next generation cell culture surfaces are completely animal-free because they are treated with synthetic peptide based coatings, which allow for good cell adhesion without the need for any animal derived coating. Animal-free surfaces provide more control of culture conditions and consistency across cultures. These products also come ready to use with no time consuming coating necessary and because they are not biological coatings, they are stable at room temperature and average a two-year shelf life.
Allows for the Reduction or Removal of Serum in Culture
As most cell culture has already either achieved animal-free production is or moving toward animal-free culture, removing serum and other animal products from media and cell culture surfaces is critical. With standard gas-plasma treated plates, removal or reduction of serum in culture is challenging because many cells need serum in media for attachment. This next generation of synthetic, animal-free cell culture surfaces provides improved attachment, which allows for the removal or reduction of serum in culture. This is particularly important for cell lines that are difficult to culture, have difficulty with attachment and/or proliferation challenges.
I wanted provide some examples of the companies and products in this space. This is not inclusive of all products available, but will give a good range of the types of next generation cell culture surfaces that are available.
BD Biosciences Discovery Labware has a line of next generation cell culture surface products in their BD PureCoat cell culture surfaces line. They offer two animal-free coatings, an amine-based surface that is positively charged and a carboxyl-based surface that is negatively charged. These coatings allow for enhanced attachment and proliferation of certain cell types that prefer either positive or negative charges. In addition they offer two novel peptide-based coatings that are designed to provide synthetic, animal-free alternatives to coatings with native extracellular matrices. Part of the BD PureCoat ECM Mimetic Cultureware line, these products are coated with animal-free synthetic extracellular matrix-peptides that are derived from either Fibronectin or Collagen Type 1 sequences.
The Fibronectin based surface contains the RGD amino acid sequence motif, that makes it a great surface for expanding mesenchymal stem cells (MSCs) or other alpha-5 integrin-positive cell types. The Collagen Type I based surface contains the GFOGER amino acid sequence motif and binds cells that attach via alpha-2 integrin, which makes this surface a good choice for the expansion of primary keratinocytes under serum or animal-free conditions. The fact that the Fibronectin and Collagen I Mimetic Surfaces support the expansion of MSCs (xeno-free) and primary keratinocytes (animal-free), respectively, makes these surfaces a better choice for pre-clinical or clinical research in stem and primary Cell Therapy applications.
As the stem cell industry continues to expand and therapeutics enter clinical trials and finally commercialization, scalable systems are key. While these surfaces are currently available in smaller scale vessels (up to 175 cm2 flasks), Discovery Labware is working on expanding to larger surface areas to meet the needs for commercial scale cell production.
Corning also offers a range of next generation surfaces. The Synthemax product line offers a synthetic peptide based substrate designed to improve the expansion and differentiation of stem cells in a serum free/reduced media. This is a self-coating substrate that can be used to coat plastic and glass surfaces. They also carry a specialized Osteo Assay Surface, which is an inorganic crystalline calcium phosphate coating designed to mimic the in vivo bone environment. This product is particularly valuable for cell growth and differentiation of precursor cells to osteoblasts and osteoclasts. This surface allows for the co-culture of bone and metastatic cells, drug screening and osteoblast/osteoclast cell function analysis.
Another specialized surface sold by Corning is the ultra low attachment coated polystyrene surface that has a covalently bound hydrogel layer that is hydrophilic and neutrally charged. This surface inhibits non-specific immobilization and inhibits cell attachment. Prevention of cell attachment is important in applications where stem cells need to be prevented from attachment-mediated differentiation. This surface can also be used to study tissue specific function of cancer cells and selectively culture tumor or virally transformed cells as unattached colonies.
Lastly Corning carries an improvement on the traditional gas-plasma treatment called CellBIND. CellBIND is a plasma treated surface that incorporates more oxygen into the surface to support low-serum applications or finicky cells.
Greiner has a next generation cell culture surface called Advanced TC. This surface is a synthetic modification to the polystyrene that is less polar and better mimics the in vivo environment and allows for culture of primary cells in serum free or serum reduced media. The Advanced TC surface has been tested on a variety of cell types and has been particularly successful with HEK 293 transfection in serum free culture. In addition Advanced TC provides better anchorage in situations where there are more demanding assay conditions with several washing steps necessary.
Thermo Scientific Nunc has a line of next generation cell culture surfaces that includes the Nunclon Vita Cell Surfaces Line. This is an energy treated polystyrene surface that is animal-free and specially designed for the culture of stem cells and other fastidious cells including human embryonic stem cells (hESC), human induced pluripotent stem cells, (HIPS) human embryonic kidney cells (HEK), and human mesenchymal stem cells (HMSC). Nunclon Vita eliminates the need for extracellular matrix coating or the use of feeder cells.
Another product in the Nunc line is the UpCell Surface. This surface has a covalently immobilized polymer poly (N-Isoproplacylamide), or PIPAAm, layer which is slightly hydrophobic at 37° C. This layer allows cells to attach and grow, however when the culture temperature is reduced to below 32° C the layer becomes very hydrophilic, binds water and swells resulting in the release of adherent cells. One benefit of the release of adherent cells using temperature is that there is no trypsin needed, which maintains cell surface proteins. Another plus is that there is no scraping used, so cell viability remains high and this product also enables the harvesting of cell sheets.
Nunc also has the Hydrocell Surface, which is a covalently immobilized super hydrophilic polymer that inhibits cell attachment and protein absorption. This surface can be used to prevent attachment-induced differentiation and provide higher cell secreted protein yields by allowing minimal protein absorption.
I hope that this has provided a sampling of new developments in cultureware. Have you tried any of the above surfaces? Do you have any others to recommend?