TITLE: Poly-ornithine/laminin substrate for neural cell culture 
AUTHORS: Gist Croft, Regine Tipon

[DESCRIPTION]
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.

[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

[STEPS]
SECTION: Preparing Reagents
2. Filter sterilize and store at 4 degrees for 3 months
SECTION: Preparing Reagents
1. Dissolve unopened vial of Poly-Ornithine in Borate Buffer at 1mg/ml (P-Orn)
SECTION: Preparing Reagents
3. Pretreatment for coverslips:
8. Add Laminin in L15+ NaBicarbonate solution: final 10ug laminin/ml, same volume and incubate ≥ 12 hours in TC incubator
6. Wash wells 2-3x, 0-2 min each  with ≥coating volume of TC grade  H2O
SECTION: Coating
4. Add P-Ornithine solution  to cover well
SECTION: Preparing Reagents
3.1. Sterilize forceps in 100% EtOH
SECTION: Preparing Reagents
3.2. Dip 15mm coverslip generously in 100%EtOh, place in 24wp or 4wp wells
SECTION: Preparing Reagents
3.3. Wash wells 3x with TC H2O
SECTION: Preparing Reagents
3.4. Aspirate completely and Dry in hood
SECTION: Coating
4.1. ~0.5ml/24wp well or 8 well slide well
SECTION: Coating
4.2. Note: if using coverslip, make sure to tamp down coverslip with sterile tip
SECTION: Coating
4.3. Incubate 2-6 hrs to overnight in incubator
8.1. As long as laminin solution does not dry, substrate is good for several weeks in incubator, add water each week.
8.2. Alternatively, desalt wash 2-3x with H2O and dry (see Drying Protocol below).
7. When ready to seed cells, aspirate laminin (can save for reuse) and seed cell solution directly without drying substrate
9. Best to dry freshly prepared substrate, rather than one that has been incubating for some time
SECTION: Drying coated surfaces
10. Aspirate laminin
SECTION: Drying coated surfaces
12. Completely aspirate water, including residual droplets, without scratching surface
SECTION: Drying coated surfaces
11. Wash 3x with TC water
SECTION: Drying coated surfaces
13. Dry dishes open in the hood for 15-20 min.
SECTION: Drying coated surfaces
14. Parafilm and store at 4 degrees for 6 months
SECTION: Coating
5. Add P-Ornithine solution  to cover well
6.1. borate+polyamino acid solution is toxic, aspirate completely  during washes and use  greater than coating vollume for washes.