The science behind coating cell culture surfaces

The science behind coating cell culture surfaces

Cell growth in vivo requires the extracellular matrix (ECM), an external scaffold of proteins that supports cell structure and function1. An important aspect of translating growth conditions for cell culture is replicating this supportive layer in vitro. Although some immortal cell lines can grow directly on tissue culture glass or plastic surfaces, many primary or iPSC-derived cell lines are unable to adhere or survive on these materials and require a growth substrate that mimics properties of the ECM1–3. The technology of cell culture coating has evolved substantially over the years. 

Purified ECM components

An early solution to replicating the ECM in vitro was to extract individual components of the ECM itself1,4. Extracted ECM components included collagen, fibronectin, and laminin. The first studies to incorporate purified ECM proteins into cell culture observed significant improvements in culture health and lifespan1. However, this process of extraction is difficult, expensive, and prone to variation between batches1,2.  

Synthetic polypeptides

As it turns out, cells do not require an exact replication of the ECM to grow in vitro, but rather a growth surface that contains a high density of positive charge5. This can be achieved by coating culture surfaces with polymers composed of basic amino acids. Positively charged surfaces are believed to promote cell adhesion through interaction with heparan sulfate proteoglycans (HSPGs) on the cell surface6,7. Negatively charged HSPGs have strong electrostatic interactions with basic amino acids and are important for cell adhesion via interactions with the ECM in vivo. 

These observations paved the way for the use of synthetic polypeptides as inexpensive alternative coating substrates5. The most commonly used in cell culture are poly-lysine and poly-ornithine (PLO). These molecules are long chains of the positively charged amino acids lysine and ornithine, respectively5. Poly-lysine has two enantiomers, poly-L-lysine (PLL), and poly-D-lysine (PDL). The d enantiomer is preferentially used by many due to its higher resistance to enzymatic degradation by secreted proteases8,9. These artificial substrates have proven effective for adherence and proliferation of many different cell types5,10,11. However, some applications, such as the culture of iPSC-derived cells still require additional growth substrate.   These are typically cultured with a layer of laminin over a layer of PLO, or with a layer of Matrigel12,13.

Optimal conditions for coating surfaces

These are typically cultured with a layer of laminin over a layer of PLO, or with a layer of Matrigel12,13. Because ofDue to the high content of positive charge in these synthetic polypeptides like poly-lysine and poly-ornithine, they are best adsorbed onto negatively charged materials (such as silica your typical laboratory glass))14. Similarly, TC treated plastic plates and dishes are made of polystyrene that has been treated, typically with plasma, to exhibit more negative charges on its surface to facilitate the adsorption of synthetic polypeptides as well as the nonspecific adsorption of serum proteins contained in the media (https://doi.org/10.1089/ten.teb.2018.0056). 

 Additionally, adsorption isof PLL and PLO is best performed in a basic solution, which reduces electrostatic repulsion within the polymer, allowing it to adopt an α-helix conformation14,15. 

Both purified ECM components and synthetic polypeptides are effective substrates for cell growth and adhesion in vitro, however, they are composed of peptide bonds and are therefore prone to degradation by proteases secreted by cells2. Particularly in long-term cultures, degradation of the coating substrate can cause cells to aggregate in clumps, disrupting culture stability and viability2,16. 

Non-peptide polymers

Both purified ECM components and synthetic polypeptides are effective substrates for cell growth and adhesion in vitro, however, they are composed of peptide bonds and are therefore prone to degradation by proteases secreted by cells2. Particularly in long-term cultures, degradation of the coating substrate can cause cells to aggregate in clumps, disrupting culture stability and viability2,16. 

To address the problem of coating degradation we have developed a new synthetic polymeric coating based on dendritic polyglycerol amine (dPGA). These polymers maintain the high density of positive charge that supports cell adhesion but lack peptide bonds and are therefore resistant to degradation from cellular proteases. When tested in various neuronal culture systems, these non-peptide polymers support cellular attachment and proliferation with as much or improved effectiveness as synthetic polypeptides2,3,16.

References

1. Kleinman, H. K., Luckenbill-Edds, L., Cannon, F. W. & Sephel, G. C. Use of extracellular matrix components for cell culture. Analytical Biochemistry 166, 1–13 (1987).
2. Clément, J.-P. et al. Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture. ASN Neuro 14, 17590914211073276 (2022).
3. Schmidt, S., Lilienkampf, A. & Bradley, M. New substrates for stem cell control. Philosophical Transactions of the Royal Society B: Biological Sciences 373, (2018).
4. Kleinman, H. K., Klebe, R. J. & Martin, G. R. Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol 88, 473–485 (1981).
5. McKeehan, W. & Ham, R. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J Cell Biol 71, 727–734 (1976).
6. Yamada, Y., Onda, T., Hamada, K., Kikkawa, Y. & Nomizu, M. Octa-arginine and Octa-lysine Promote Cell Adhesion through Heparan Sulfate Proteoglycans and Integrins. Biological and Pharmaceutical Bulletin 45, 207–212 (2022).
7. Siow, W. X. et al. Interaction of poly-l-lysine coating and heparan sulfate proteoglycan on magnetic nanoparticle uptake by tumor cells. Int J Nanomedicine 13, 1693–1706 (2018).
8. Tsuyuki, E., Tsuyuki, H. & Stahmann, M. A. THE SYNTHESIS AND ENZYMATIC HYDROLYSIS OF POLY-d-LYSINE. Journal of Biological Chemistry 222, 479–485 (1956).
9. Li, J. & Yeung, E. Real-Time Single-Molecule Kinetics of Trypsin Proteolysis. https://pubs.acs.org/doi/epdf/10.1021/ac801365c (2008) doi:10.1021/ac801365c.
10. Letourneau, P. C. Possible roles for cell-to-substratum adhesion in neuronal morphogenesis. Developmental Biology 44, 77–91 (1975).
11. Yavin, E. & Yavin, Z. ATTACHMENT AND CULTURE OF DISSOCIATED CELLS FROM RAT EMBRYO CEREBRAL HEMISPHERES ON POLYLYSINE-COATED SURFACE. J Cell Biol 62, 540–546 (1974).
12. Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275–280 (2009).
13. Shi, Y., Kirwan, P. & Livesey, F. J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc 7, 1836–1846 (2012).
14. Choi, J.-H. et al. Influence of pH and Surface Chemistry on Poly(l-lysine) Adsorption onto Solid Supports Investigated by Quartz Crystal Microbalance with Dissipation Monitoring. J. Phys. Chem. B 119, 10554–10565 (2015).
15. Stil, A. et al. A simple method for poly-D-lysine coating to enhance adhesion and maturation of primary cortical neuron cultures in vitro. Front Cell Neurosci 17, 1212097 (2023).
16. Thiry, L., Clément, J.-P., Haag, R., Kennedy, T. E. & Stifani, S. Optimization of Long-Term Human iPSC-Derived Spinal Motor Neuron Culture Using a Dendritic Polyglycerol Amine-Based Substrate. ASN Neuro 14, 17590914211073381 (2022).