Aug 14, 2024
Version 2
Poly-ornithine/laminin substrate for neural cell culture V.2
- 1New York Stem Cell Foundation
Protocol Citation: Gist Croft, Regine Tipon 2024. Poly-ornithine/laminin substrate for neural cell culture . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpz2mplzp/v2Version created by Gist Croft
Manuscript citation:
Takazawa T, Croft GF, Amoroso MW, Studer L, Wichterle H, Macdermott AB.
Maturation of spinal motor neurons derived from human embryonic stem
cells. PLoS One. 2012;7(7):e40154. doi: 10.1371/journal.pone.0040154.
Epub 2012 Jul 3. PMID: 22802953; PMCID: PMC3388990.
Ruzo A, Croft GF, Metzger JJ, Galgoczi S, Gerber LJ, Pellegrini C, Wang H
Jr, Fenner M, Tse S, Marks A, Nchako C, Brivanlou AH. Chromosomal
instability during neurogenesis in Huntington's disease. Development.
2018 Jan 29;145(2):dev156844. doi: 10.1242/dev.156844. PMID: 29378824.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 14, 2024
Last Modified: August 14, 2024
Protocol Integer ID: 105355
Keywords: ASAPCRN, astrocyte, neuron, cell culture, organoids, long term culture, adhesion, iPSC
Funders Acknowledgements:
Aligning Science Across Parkinsons
Grant ID: ASAP-000472
Abstract
This protocol is used to create adhesive and bioactive substrate for neural cell types, low to high density nuerons, astrocytes, or organoids. It is based on standard methods but includes several optimizations, use case recommendations, and alternatives, and advice.
Attachments
Guidelines
Under the substrate: plastic vs glass. Tissue culture treated plastic is much stickier (hydrophillic) and much softer (stiffness(kPa)) than glass and I prefer it for growth and imaging. Nunc delta surface is reliably stickier and more even than many alternatives. High-ornithine can compensate for the adhesion defecit of galls, but not completely and cannot soften the glass. If using glass, “german” borosilicate (decksglasser) glass grade is preferred and acid etching prior to coating is recommended. Neuvitro makes excellent No.1.5 german glass coverslips and they offer pre-etched, pre-coated coverslips.
Optical plastic cultureware is available from many suppliers. Needs to be No 1.5 glass equivalent and optically clear and tissue culture treated. I have had good experience with Greiner, 96wp black plates, Ibidi chamber slides and multiwell plates are excellent optically and TC treatment is great, and Mattek is good but mostly glass.
Commercial sources or precoated cultureware: We have found i house coated substrates are best but commercial providers are very useful as an alternative. Precoated lysine/laminin coverslips from NeuVitro are preferred; Becton Dickinson precoated slides and coverslips are usually good as well b home-coated
Poly-Ethyl-Imine in Borate buffer may be used in place of laminin at the same concentration
Drying: coated cultureware can be dried out for storage at 4 degrees and future use or for drop-seeding. Adhesion may be slightly less than freshly prepared (wet) substrate but this has not been methodically tested. Rule of thumb in lab is use fresh for very sensitive applications where consistency and maximal adhesion are crucial
Materials
Poly-L-ornithine hydrobromide,mol wt 30,000-70,000, Sigma-Aldrich P3655-50MG extremely hygroscopic, do not attempt to weigh, dissolve the whole container.
Borate Buffer: Boric Acid to 0.15M in ddH20, pH to 8.4 with NaOH, filter and store at room temperature
P-Orn Stock solution: store at 4 degrees for 3 months
Natural Mouse Laminin, Invitrogen, 23017015
L15+Bicarbonate: add 12.5ml sterile Sodium Bicarbonate (7.5g/L) to 500ml L15 with phenol red.
Poly-Ethyl-Imine in Borate buffer may be used in place of laminin at the same concentration
Safety warnings
Borate/PLL solution is toxic Aspirate well and wash before proceeding to laminin coating.
Preparing Reagents
Preparing Reagents
Dissolve unopened vial of Poly-Ornithine in Borate Buffer at 1mg/ml (P-Orn)
Filter sterilize and store at 4 degrees for 3 months
Pretreatment for coverslips:
Sterilize forceps in 100% EtOH
Dip 15mm coverslip generously in 100%EtOh, place in 24wp or 4wp wells
Wash wells 3x with TC H2O
Aspirate completely and Dry in hood
Coating
Coating
Add P-Ornithine solution to cover well
~0.5ml/24wp well or 8 well slide well
Note: if using coverslip, make sure to tamp down coverslip with sterile tip
Incubate 2-6 hrs to overnight in incubator
Add P-Ornithine solution to cover well
Wash wells 2-3x, 0-2 min each with ≥coating volume of TC grade H2O
borate+polyamino acid solution is toxic, aspirate completely during washes and use greater than coating vollume for washes.
When ready to seed cells, aspirate laminin (can save for reuse) and seed cell solution directly without drying substrate
Add Laminin in L15+ NaBicarbonate solution: final 10ug laminin/ml, same volume and incubate ≥ 12 hours in TC incubator
As long as laminin solution does not dry, substrate is good for several weeks in incubator, add water each week.
Alternatively, desalt wash 2-3x with H2O and dry (see Drying Protocol below).
Best to dry freshly prepared substrate, rather than one that has been incubating for some time
Drying coated surfaces
Drying coated surfaces
Aspirate laminin
Wash 3x with TC water
Completely aspirate water, including residual droplets, without scratching surface
Dry dishes open in the hood for 15-20 min.
Parafilm and store at 4 degrees for 6 months
Protocol references
Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L.
Human ES cell-derived neural rosettes reveal a functionally distinct
early neural stem cell stage. Genes Dev. 2008 Jan 15;22(2):152-65. doi:
10.1101/gad.1616208. Erratum in: Genes Dev. 2008 May 1;22(9):1257. PMID:
18198334; PMCID: PMC2192751.