Neural-like cell growth on biological surface coatings

Author - Laura Middeldorp
Supervisors - Laura Poole-Warren, Penny Martens

Introduction and Aims

This study aims to investigate adhesion of a neural-like cell line to different biological surface coatings on, and find the minimum effective amount of growth factor for the cells to grow axon-like processes. Ultimately the coatings could be applied to retinal electrodes to provide better cell contact, and the minimum dose used to distinguish between growth effects of electrical stimulation and the growth factor itself.

Materials and Methods

Culture materials and methods

• Glass and tissue culture grade plastic (TCP) wells
• Collagen type 1 (CN) (6mg/cm2), Laminin (LN) (2mg/cm2), Fibronectin (FN) (6mg/cm2), surface coating
• Dulbecco’s modified eagle media (DMEM), with 1% horse serum media, containing nerve growth factor (NGF)
• Rat pheochromocytoma neuron-like cell line (PC12 cells), density 2 x 104 cells/cm2
• Cultures incubated at 37oC, 5% CO2, , media changed every 2 to 3 days

Experiment 1 - NGF Dose response on CN coated TCP
Cells were plated on CN coated TCP, at [NGF]=0,4, 6,8,10, 20 or 40ng/ml.

Experiment 2 - Cell response on different substrates
Cells were plated on CN, FN or LN coated TCP or glass, with [NGF]=0,5,10, or 15ng/ml; or plated on wells half coated CN, FN or LN, with [NGF]=10ng/ml

Analysis methods

• Photographs were taken of each well at 10x magnification at 24, 48, 72 and 96 hours post plating
• Number of cells present, number of attached cells, and lengths of extensions were recorded for each photograph, using the ImageJ and NeuronJ cell analysis programs (NIH imageJ).

Results and Discussion

Experiment 1

• Increase in average process length and in percent of cells with processes with increasing [NGF] (see figure 1), with the minimum effective [NGF] around 6-8ng/ml.
• A slight decrease in average length was seen at 96 hours. This could be attributed to the media change at 72 hours, at which point the existing media may have depleted in NGF, and replacing media would have increased the NGF concentration, promoting new extension growth.

Figure 1. [NGF] vs percent of cells with processes, CN coated TCP, 72 hours post plating

Figure 2. [NGF] vs average extension length, both CN coated TCP, 72 hours post plating

• Increase in average process length as [NGF] increased (see figure 2), indicating minimum effective dose could be in the range of 8-20ng/ml.

Experiment 2

• Increase in average process length over time, until slight decrease at 96 hours, for all 3 coatings and both surfaces
• Similar length increases across all 3 coatings and both surfaces for given [NGF] (see figure 3), indicating that PC12 cells may use same surface receptors for LN, FN and CN attachment, as in agreement with Tomaselli et al, 1987².

Figure 3. Average process length vs coating and surface, [NGF]=10ng/ml, 72 hours post plating

• Almost no adherence of cells to glass, some to TCP, and significant adherence to all coatings, indicating that TCP provides a suitable surface for adhesive media derived proteins to settle, encouraging cell adhesion. Glass does not, and any media proteins would settle on the coating, providing extra support for process outgrowth.

Figure 4. Half FN coated, half uncoated glass, 72 hours post plating. Arrows indicate edge of coating

Conclusions and Future Studies

• Minimum effective [NGF] for process outgrowth was in the range of 8-20ng/ml. This could be further refined by repeating the current experiments for a much larger sample size, and for a longer time frame.
• CN, LN and FN all appeared to promote cell attachment and growth to a similar extent, investigation into combining different coatings could reveal the mechanisms behind this.
• Possible depletion of [NGF] in the media as shown in both experiments may slow process growth, testing NGF depletion rate may help optimise cell growth.
• Another useful growth indicator could be the rate of process growth, which could be measured by time lapse photography.

These studies provide assay conditions and measurement techniques for assessing neural cell interactions with novel bioactive coatings.

References

1 Liu X, et al, 1999. IEEE Trans Rehab Eng. 7(3):315-325

2 Tomaselli KJ, Damsky CH, Reichardt LF, 1987. J Cell Biol. 105: 2347-2358.

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