The science behind coating cell culture surfaces

Cell growth in vivo requires the extracellular matrix (ECM), the external scaffold of proteins that supports cell structure and function1. An important aspect of translating growth conditions for cell culture is replicating this adherent layer in vitro. Although some immortal cell lines can grow directly on tissue culture glass or plastic surfaces, many primary or iPSC-derived cell typeslines are unable to adhere to 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

Observations that cells generally adhere to proteins with a high content of positively charged amino acids paved the way for 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 proteases6,7. These artificial substrates have proven effective for adherence and proliferation of many different cell types5,8,9. Some cells like human iPSC and many iPSC-derived cells require additional growth substrate and are typically cultured with a layer of laminin over a layer of PLO, or with a layer of Matrigel10,11.

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 viability2,12. 

Non-peptide polymers

To address the problem of coating degradation, numerous non-peptide polymers have been synthesized that contain many positive amine groups and lack peptide bonds2,3,13–15. This includes polyethyleneimine, polypropyleneimene, polypyrrole, poly electrolyte multilayers (PEMs), and dendritic polyglycerol amine (dPGA). These polymers maintain the high density of positive charge that supports cell adhesion but are 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 polypeptides2,3,13–15.

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. Tsuyuki, E., Tsuyuki, H. & Stahmann, M. A. THE SYNTHESIS AND ENZYMATIC HYDROLYSIS OF POLY-d-LYSINE. Journal of Biological Chemistry 222, 479–485 (1956).
7. 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.
8. Letourneau, P. C. Possible roles for cell-to-substratum adhesion in neuronal morphogenesis. Developmental Biology 44, 77–91 (1975).
9. 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).
10. 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).
11. 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).
12. 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).
13. Lakard, S. et al. Culture of neural cells on polymers coated surfaces for biosensor applications. Biosensors and Bioelectronics 20, 1946–1954 (2005).
14. Vancha, A. R. et al. Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer. BMC Biotechnology 4, 23 (2004).
15. Landry, M. J., Rollet, F.-G., Kennedy, T. E. & Barrett, C. J. Layers and Multilayers of Self-Assembled Polymers: Tunable Engineered Extracellular Matrix Coatings for Neural Cell Growth. Langmuir 34, 8709–8730 (2018).