Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

Summary

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Abstract

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest¹. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest^2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone^6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required¹⁰. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

Protocol

The authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Duisburg-Essen (Germany).

Part 1: Setting Up (not Shown on Video)

PREPARING MATERIALS AND SOLUTIONS (prior to taking the eggs out of the incubator)

• Eggs have to be incubated in a humidified incubator for 11 days at 37°C (100°F).
• 70% eth....

Discussion

This protocol is based on modifications of procedures that have been described in the past^10,11. In addition to former publications we here show the individual different steps of the process more expanded. This supports the experimenter with important details assuring correct proceedings and reliable outcomes. Further, as the dissection process is an essential part of CEE extraction we describe the procedure of taking the embryos out of the yolk sack in every detail. There are several important points to note .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
11 days incubated chicken eggs	Sorries-Trockels Vermehrungszucht GmbH
Alcoholic disinfection spray	Schülke Mayr GmbH
DMEMGlutamax	GIBCO, by Life Technologies
Syringe sterile (60 ml)	BD Biosciences
Tubes sterile (50; 15 ml)	Greiner Bio-One
Pipet tips sterile	Starlab GmbH
Hyaluronidase typeIV-S	Sigma-Aldrich
Milipore stericups sterile (0.45; 0.22 μl)	Fisher Scientific
Pipettes sterile (25; 10; 5 ml)	Greiner Bio-One
Centrifuge L5-50	Rotor type Ti-45