Sustained Delivery of Nerve Growth Factor Using a Porous Silicon Film in Rat Pheochromocytoma 12 Cells

Samenvatting

Source: Rosenberg, M., et al. Designing Porous Silicon Films as Carriers of Nerve Growth Factor. J. Vis. Exp. (2019).

This video demonstrates the use of a degradable nanostructured porous silicon (PSi) film for the sustained release of nerve growth factor (NGF) to support PC12 cell differentiation. NGF is adsorbed onto the film’s pores through electrostatic interactions and is gradually released into the media by diffusion and scaffold degradation. This controlled delivery promotes PC12 cell survival and facilitates neurite growth and branching over an extended period.

Protocol

1. Fabrication of Oxidized porous silicon (PSiO₂) Carriers

1. Cut a Silicon (Si) wafer (single side polished on the <100> face and heavily Boron-doped, p-type, 0.95 mΩ·cm) into 1.5 cm × 1.5 cm samples using a diamond-tipped pen.
2. Oxidize the Si samples in a tube furnace at 400 °C for 2 h in ambient air (heating rate: 25 °C/min, natural cooling).
3. Immerse the Si samples in a solution of aqueous hydrofluoric acid (HF) (48%), double-distilled water (ddH₂O), and ethanol (99.9%) (1:1:3 v/v/v) for 5 min; then rinse the samples with ethanol three times and dry under a nitrogen stream.
   NOTE: Prepare and store HF solution in plasticware only, as HF dissolves glass.
   CAUTION: HF is a highly corrosive liquid, and it should be handled with extreme care. In case of exposure, rinse thoroughly with water and treat the affected area with HF antidote gel; seek medical care immediately.
4. Mount the Si sample in a polytetrafluoroethylene etching cell, using a strip of aluminum foil as a back contact and a platinum coil as the counter electrode.
5. Etch a sacrificial layer in a 3:1 (v/v) solution of aqueous HF and ethanol (99.9%) for 30 s at a constant current density of 250 mA/cm²; then rinse the surface of the resulting PSi film with ethanol three times and dry under a nitrogen stream.
6. Dissolve the freshly etched porous layer in an aqueous sodium hydroxide (NaOH) solution (0.1 M) for 2 min. Then, rinse with ethanol three times and dry under a nitrogen stream.
7. Immerse the sample in a solution of aqueous HF (48%), ddH₂O, and ethanol (99.9%) (1:1:3 v/v) for 2 min. Then, rinse with ethanol three times and dry under a nitrogen stream.
8. Electrochemically etch the Si sample in a 3:1 (v/v) solution of aqueous HF and ethanol (99.9%) for 20 s at a constant current density of 250 mA/cm²; then rinse the surface of the resulting PSi film with ethanol three times and dry under a nitrogen stream.
9. Thermally oxidize the freshly-etched PSi samples in a tube furnace at 800 °C for 1 h in ambient air (heating rate: 25 °C/min, natural cooling) to form a porous SiO₂ (PSiO₂) scaffold.
10. Spin-coat the PSiO₂ samples with a positive thick photoresist at 4,000 rpm for 1 min; then bake the coated samples at 90 °C for 2 min (heating rate: 5 °C/min, natural cooling).
11. Dice the PSiO₂ samples into 8 mm × 8 mm samples using a dicing saw.
12. To remove the photoresist, soak the diced samples in acetone for 3 h; then thoroughly rinse with ethanol and dry under a nitrogen stream.

2. Loading PSiO₂ with nerve growth factor (NGF)

1. To prepare the NGF loading solution, dissolve 20 µg of murine β-NGF in 400 µL of 1:1 (v/v) solution of 0.01 M phosphate-buffered saline (PBS) and ddH₂O.
2. Add 52 µL of the loading solution on top of the PSiO₂ sample and incubate for 2 h at room temperature (RT) in a capped dish.
   NOTE: Maintain high humidity in the dish to prevent the solution from drying up during incubation.
3. Collect the solution on top of the sample for subsequent quantification of NGF content within the PSiO₂ carrier.
   NOTE: NGF loading into PSiO₂ should be performed immediately before the intended use; the protocol cannot be paused here due to the risk of drying and denaturation of the protein.

3. Cell Viability and Growth in the Presence of NGF-Loaded PSiO₂ Carriers

1. Rat pheochromocytoma (PC12) cell culture
   1. Prepare the basic growth medium by adding 10% horse serum (HS), 5% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin-streptomycin, and 0.2% amphotericin to Roselle Park Medical Institute (RPMI) medium.
   2. Prepare a differentiation medium by adding 1% HS, 1% L-glutamine, 1% penicillin-streptomycin, and 0.2% amphotericin to the RPMI medium.
   3. Grow cell suspension (10⁶ cells) in a 75 cm² culture flask with 10 mL of basic growth medium for 8 days; every 2 days, add 10 mL of basic growth medium to the flask.
   4. To generate a differentiated PC12 cell culture, transfer the cell suspension to a centrifuge tube; centrifuge cells for 8 min at 200 x g and RT. Discard the supernatant.
   5. Suspend the cells in 5 mL of fresh basic growth medium and re-centrifuge the cells for 5 min at 200 x g and RT; discard the supernatant and resuspend the cell pellet in 3 mL of basic growth medium.
   6. To separate cell clusters, aspirate the cells ten times using a 23 G syringe.
   7. Count the cells using a hemocytometer cell counter and seed 10⁴ cells/cm² working area on collagen type I-coated plates in the presence of differentiation medium.
   8. After 24 h, add fresh murine β-NGF (50 ng/mL) or NGF-loaded PSiO₂ carrier per plate.
      NOTE: Higher NGF concentrations (>50 ng/mL) possess the exact effect as the specified concentration.
   9. Renew the differentiation medium every 2 days.
   10. To evaluate cell viability, add 10% (v/v) of the viability indicator solution (resazurin-based) at representative time points and incubate for 5 h at 37 °C; measure the absorbance at 490 nm using a spectrophotometer.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Acetone	Gadot	830101375
Amphotericin	Biological Industries	03-028-1B
Aqueous HF (48%)	Merck	101513
AZ4533 photoresist	Metal Chem, Inc.	AZ4533
BSA fraction v	MP biomedicals	216006950
BSA solution (10%)	Biological Industries	03-010-1B
Collagen type l	Corning Inc.	354236
Collagenase	Enco	LS004176
Collagen-coated plastic coverslips	NUNC Thermanox	1059846
D-(+)-glucose	Sigma-Aldrich Chemicals	G8170
Dispase-II	Sigma-Aldrich Chemicals	4942078001
Ethanol absolute (99.9%)	Merck	818760
FBS	Biological Industries	04-121-1A
Guanidine-HCl	Sigma-Aldrich Chemicals	G7294
Ham's F-12 nutrient mixture	Thermo Scientific	11765054
HBSS	Thermo Scientific	14185-045
HEPES (1M)	Thermo Scientific	15630-056
HS	Biological Industries	04-124-1A
L-15 medium	Sigma-Aldrich Chemicals	L5520
Laminin	Thermo Scientific	23017015
L-glutamine	Biological Industries	03-020-1A
Murine β-NGF	Peprotech	450-34-20
Normal donkey serum (NDS)	Sigma-Aldrich Chemicals	G9023
Papain	Sigma-Aldrich Chemicals	p-4762
PBS (pH 7.4)			Prepared by dissolving 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, and 2.7 mM KCl in double-distilled water (ddH₂O, 18 MΩ·cm).
PBS X10	Biological Industries	02-020-1A
PC12 cell line	ATCC	CRL-1721
Penicillin–streptomycin	Biological Industries	03-032-1B
Poly-L-lysine	Sigma-Aldrich Chemicals	P4832
PrestoBlue reagent	Thermo Scientific	A13261
RPMI medium	Biological Industries	01-100-1A
Si wafer	Siltronix Corp.		Highly-B-doped, p-type, 0.00095 Ω-cm resistivity, <100> oriented
Sodium azide	Sigma-Aldrich Chemicals	S2002
Sodium hydroxide (NaOH)	Sigma-Aldrich Chemicals	S8045
Tannic acid	Sigma-Aldrich Chemicals	403040
Triton X-100	Chem-Impex International Inc.	1279