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Benefits of dPGA as a cell culture substrate coating

Benefits of dPGA as a cell culture substrate coating

What is dPGA?

A lot of biological conditions must be replicated to grow healthy dissociated cells in culture. One important factor for cell growth in vivo is the extracellular matrix (ECM), the external scaffold of proteins that supports cell structure and function1. Although some immortal cell lines can even grow directly on tissue culture glass or plastic surfaces, many primary or iPSC-derived cell lines are unable to adhere to these materials and require a growth substrate that mimics properties of the ECM1–3. 

Many ECM components have been purified and isolated for use in cell culture, however this process is expensive and difficult1. Cells generally adhere to proteins with a high content of positively charged amino acids, so synthetic polypeptides were createdstarted being used as inexpensive alternative coating substrates4. Poly-lysine and poly-ornithine (PLO) are homopolymeric long chains of the positively charged amino acids lysine and ornithine, respectively4. Poly-lysine has two enantiomers, poly-L-lysine (PLL), and poly-D-lysine (PDL), the latter of which is preferentially used for primary neuronal cultures due to its higher resistance to enzymatic degradation5,6. Due to the long-term nature ofSome cells like human iPSCand many iPSC-derived cells differentiation, these cells require additional growth substrate and are typically cultured with a layer of laminin over a layer of PLO, or with an additional layer of Matrigel7,8. 

Synthetic polypeptides contain peptide bonds, so these coating substrates arecan be eventually degraded by proteases released from the cultured cells2,3,5. This is where dendritic polyglycerol amines (dPGAs) come in. Unlike poly-lysine and poly-ornithine, dPGA does not have peptide bonds, making it resistant to proteolysis2. This family of polymers has a highly branched structure with many amine functional groups, and presents a high density of positive charges at neutral pH, thereby replicating the positive amino acid nature of synthetic polypeptide substrates9,10. Unlike poly-lysine and poly-ornithine, dPGA does not have peptide bonds, making it resistant to proteolysis2. ItsTh unique architecture and its is resistance to degradation makes dPGA an optimal growth substrate for many cell lines and cell culture experiments. 

Which cell typeslines can you grow on dPGAdoes dPGA benefit?

Pretty much any adherent cells can be grown on dPGA, from cell lines, to primary cells to iPSC-derived cells, but we found that dPGA brings several benefits for neuronal cell cultures:

Primary rat cortical and hippocampal neurons: when grown on dPGA coated plates, primary cortical neurons have normal physiology and electrical properties. When compared to cultures grown on PDL-coated plates, dPGA neuron cultures are 50% denser at 7 DIV, and almost 300% denser at 90 DIV2. 

iPSC-derived cortical neurons: when dPGA is used to replace the foundation PLO layer beneath a laminin matrix layer, iPSC-derived cortical neurons are healthier, as determined by phase contrast microscopy, and have a higher neuronal density2. 

iPSC-derived dopaminergic neurons: when dPGA is used in place of PLO, these cultures have a higher yield of dopaminergic neurons and show reduced cell clustering2. 

iPSC-derived hippocampal neurons: when dPGA is used in place of PLO, hippocampal neurons show a significant reduction in clustering2. 

iPSC-derived motor neurons: when cultured on dPGA + Matrigel, in comparison to Matrigel alone, iPSC derived motor neurons show similar differentiation, significantly reduced clumping, and a 5-fold increase in adherence. These cultures can be maintained on dPGA + Matrigel for 2-months without clustering11. 

iPSC-derived sensory neurons: unpublished data shows that healthy iPSC-derived sensory neuron cultures can also be maintained using dPGA as a growth substrate. 

 

How can dPGA improve your cell cultures?

Increased culture longevity

The ability to maintain long-term neuronal cell cultures is especially important for in vitro modelling of neuronal dysfunction and degeneration in neurodegenerative diseases such as Alzheimer’s, Parkinsons, Huntington’s, and amyotrophic lateral sclerosis(ALS)12,13. Since dPGA is more resistant to protease degradation, it is better able to support long-term cultures, as is observed in primary cortical neurons at 90 DIV and iPSC-derived motor neurons at 2-months post-plating2,11. 

Healthier culture

Primary cortical neurons grown on dPGA show higher cell density starting after 3 days in culture2 and reduced cell death. Analysis of scRNAseq data from iPSC-derived motor neurons cultured with dPGA + Matrigel shows a significant reduction in the number of damaged cells and cells undergoing cell death, compared to cultures grown on Matrigel alone11.

Promotion of iPSC-derived cell line differentiation

When dPGA is used in place of PLO as a culture substrate, iPSC-derived primary cortical neurons show an increase in the neuron to glia ratio, suggesting a bias towards neuronal differentiation2. Similarly, iPSC-derived dopaminergic neurons show a higher ratio of tyrosine hydroxylase (TH) positive dopaminergic to TH-negative neurons, suggesting that dPGA also supports this targeted differentiation2. Theseis evidenceevidence suggests indicates that dPGA favours early differentiation of neural progenitors into the intended neuronal type with certain differentiation protocols. 

 

Clustering reduction

When cell culture growth substrates are degraded, cells tend to clump together and adhere to each other11. Use of dPGA as a growth substrate has been shown to reduce cell clumping in iPSC-derived dopaminergic, hippocampal, and motor neurons2,11. The ability to isolate single cells in culture is important for many types of assays including electrophysiological measures2, 11, microscopic analysis from morphological analysis to live-cell imaging, as well as metrics to test culture composition such as single-cell assays like RNA sequencing (scRNAseq) 11. Analysis of scRNAseq data from iPSC-derived motor neurons cultured with dPGA + Matrigel shows a significant reduction in the number of damaged cells and cells undergoing cell death, compared to cultures grown on Matrigel alone11. 

Improved multi-electrode array (MEA) coating

Reduced cell clumping also has important implications for the use of multi-electrode arrays (MEAs) with cell culture. MEAs allow for electrophysiological characterization of large populations of neurons by detecting extracellular voltages, however cell aggregation results in a low number of electrodes in near enough vicinity for recording11. When tested with iPSC-derived motor neurons, cells grown with dPGA remained evenly distributed on MEA plates, while those grown with Matrigel alone formed large cell clusters 11. As a result, all 16 electrodes in the dPGA + Matrigel plate remained in the vicinity of cells over the 3-week recording period, which was reflected in an increase in number and frequency of spikes recorded11. 

Promotion of iPSC-derived cell line differentiation

When dPGA is used in place of PLO as a culture substrate, iPSC-derived primary cortical neurons show an increase in the neuron to glia ratio, suggesting a bias towards neuronal differentiation2. Similarly, iPSC-derived dopaminergic neurons show a higher ratio of tyrosine hydroxylase (TH) positive dopaminergic to TH-negative neurons, suggesting that dPGA also supports this targeted differentiation2. This evidence indicates that dPGA favours early differentiation of neural progenitors into the intended neuron type with certain differentiation protocols. 

 

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. McKeehan, W. & Ham, R. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J Cell Biol 71, 727–734 (1976).
  5. Tsuyuki, E., Tsuyuki, H. & Stahmann, M. A. THE SYNTHESIS AND ENZYMATIC HYDROLYSIS OF POLY-d-LYSINE. Journal of Biological Chemistry 222, 479–485 (1956).
  6. 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.
  7. 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).
  8. 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).
  9. Frey, H. & Haag, R. Dendritic polyglycerol: a new versatile biocompatible material. Reviews in Molecular Biotechnology 90, 257–267 (2002).
  10. Hellmund, M. et al. Systematic adjustment of charge densities and size of polyglycerol amines reduces cytotoxic effects and enhances cellular uptake. Biomater. Sci. 3, 1459–1465 (2015).
  11. 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).
  12. Lesuisse, C. & Martin, L. J. Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death. Journal of Neurobiology 51, 9–23 (2002).
  13. D’Souza, G. X. et al. The application of in vitro-derived human neurons in neurodegenerative disease modeling. Journal of Neuroscience Research 99, 124–140 (2021).