Neuroepithelial Differentiation Induced by ECM Molecules - Methods in Molecular Biology: Extracellular Matrix Protocols - page 248

In principle there are two approaches to this kind of in vitro assay, which
will be described below. The main application would be simply a comparison
of the response of dissociated retinal cells to different substrata, when dissoci-
ated cells are plated directly onto different ECM proteins. This has the disad-
vantage of lacking a real negative control. This problem can be circumvented
by the second approach, analyzing the effect of a particular ECM molecule or
ECM proteolytic fragment containing domains of interest compared with a
neutral substratum. In this case, the substrata that are the object of study are
added in a soluble form to the cells cultured onto fibronectin. These two
approaches will be further detailed below.

3.1. Coating with ECM Substrata
1. 10-mm round glass coverslips are pretreated with 65% HNO 3 for 1 h and are

rinsed several times with distilled water until reaching a neutral pH. Finally, they
are rinsed twice in ethanol and left to dry in sterile conditions (under a hood) at
room temperature.

2. Coverslips are then transferred to four-well Petri dishes using sterile forceps and

coated with polyornithine solution for at least 2 h at 37 ° C.

3. Remove the polyornithine solution by aspiration and rinse the coverslips three

times with distilled water. Coat with 10 g/mL (diluted in sterile Ca 2+ -, Mg 2+ -

free PBS) laminin-1, merosin, or fibronectin and incubate at 37

µ

° C for 2 h at least.

3.2. Differentiative Bioassay Using ECM Molecules as a Substrate
1.

Two neuroretinas from an E5 chick embryo are used as the source of biological
material. The procedure of dissection is as follows ( see Fig. 1 ). First, remove the
eyes using curved iris forceps and peel off the surrounding mesenchyme. Then,
transfer the eyes to a Petri dish covered with a thin layer of Ca 2+ -, Mg 2+ -free PBS
leaving the optic nerve exit facing up. Pick up the pigmented epithelium using
Dumont #5 tweezers and peel it off carefully. The dark color of this tissue facili-
tates its recognition. When the equator is reached, put the eye upside down and
remove the rest of the pigment epithelium by pulling up while the rest of the
eye is held with one of the tweezers. The neuroretina now can be isolated
easily by removing the lens and vitreous body (the transparent, gelatinous mass
inside the eye).

L Ca 2+ -, Mg 2+ -free PBS with 100

2. Mix 800 L freshly

prepared trypsin solution (10 mg/mL in Ca 2+ -, Mg 2+ -free PBS) and incubate the
two retinas in this buffer for 12 min at 37

µ µ L 30 mg/mL BSA and 50 µ

C with gentle shakings every 3 min.
After this time the retinas should look soft ( see Note 1 )

°

3. Immediately stop the enzymatic reaction by adding 100 µ L soybean trypsin

inhibitor (10 mg/mL).

4. Cells are then dissociated by passing the suspension gently through a fire-pol-

ished, enlarged bore of a Pasteur pipet five times ( see Note 2 ). The cell density is
then estimated using a hemocytometer.