Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Summary

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Abstract

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.

For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used^1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay^5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye^7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM^10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice¹³ as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM¹⁴.

In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications.

Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Protocol

All equipment and reagents have to be purchased sterile or needs to be heat or steam sterilized or sterilized with 70% ETOH.

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: Incubation of eggs

1. Fertilized eggs can be stored at 13°C for up to one week.
2. Eggs a....

Discussion

Innovative or just another chick culture protocol?

With former shell-less culture protocols^1-4 the total survival rates of the chick embryos were low, e.g. ca. 30%¹. The refined ex ovo culture protocol described in this video article, by contrast, allows for survival rates over 50%. Compared to classical in ovo cultures ex ovo culture of chick embryos significantly facilitates the accessibility of the CAM and chick embryo and enables their expe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fertilized Eggs	Animal	Sorries-Trockels Vermehrungszucht GmbH
Mice	Animal	Charles River Laboratories
Incubator Type 3000 with rotating egg tray	Tools	Siemens AG	9503	Maintain at 37°C with relative humidity set above 60%
Egg incubator BSS 160	Tools	Grumbach, Germany	8101	Maintain at 37-387deg;C with relative humidity set above 60%
PBS	Reagent	Sigma-Aldrich		PBS should be cold (> 4°C) and sterile
Dulbecco’s modified eagle’s medium	Reagent	Sigma-Aldrich	D 6429	DMEM culture medium
Leibovitz’s L-15 Medium	Reagent	Invitrogen	31415-029	here: collection medium for tumor samples
Tumor cell -specific medium	Reagent	Sigma-Aldrich		each research group should use the medium they culture their tumors cells in or common culture media, e.g. Leibovitz’s Medium or Dulbecco’s modified eagle’s medium supplemented with 15% fetal bovine serum (FBS), 4mM L-glutamine and 50 μM insulin
70% EtOH	Reagent	Sigma-Aldrich
Magnetic stir bar, triangled 80 mm	Tool	VWR international	442-0391	A triangled magnetic stir bar serves well to crack the eggs shell.
Forceps DUMONT #5	Tool	Fine Science Tools	11252-30	bevelled very fine shanks (0.05 mm x 0.02 mm tip)
Forceps DUMONT #7	Tool	Fine Science Tools	11271-30	curved shanks (0.07 mm x 0.10 mm tip)
Spring scissors, straight, 8cm	Tool	Fine Science Tools	15000-00	fine, small straight blades
Fine Iris scissors, straight	Tool	Fine Science Tools	14094-11	Use to cut our the CAM
Standard scissors, straight, sharp/blunt	Tool	Fine Science Tools	14007-14	Use for decapitation or cervical dislocation
microliter syringe 1702 TLLX, 25 μl	Tool	Hamilton Co	80222	Use for injection into the vitreous
Hamilton 33-G needle (15 mm)	Tool	Fine Science Tools	18073-15	Use for injection into the vitreous
Sterile scalpels	Tool			Use for dissecting tumor samples
Small drain spoon	Tool	Geuder	15758	Use to transfer of small chick embryos
Wax board with fixing pins	Tool	Home made		Use to fix of animals for dissection
Petri dishes 60 x 15 mm	Tool	Greiner Bio-One	628102
Petri dishes 92 x 16 mm	Tool	Sarstedt Ltd	82.1472
Sterile Petri dish 100 x 20 mm	Tool	Greiner Bio-One	664160	extra deep, with spacers for ventilation
Beaker	Tool			300-600 ml
FALCON tubes	Tool	Falcon BD		15 ml and 50 ml
Eppendorf tubes	Tool	Eppendorf		1.5 ml and 2 ml
1ml pipette tips	Tool			Use to cut plastic rings for application of substances / cells
Peripheral venous catheter (PVL)	Tool	Viggo®		Use to cut silicon rings from the tube for application of substances / cells
Pipettes and tips	Tool	Eppendorf
Autoclaved folded Filters 595 1/2, 110 mm diameter	Tool	Schleicher Schuell	311643	Carrier, which caused least irritation of the CAM; used in the paper
PTFE-Pledget, non resorbing, 6,3 x 152.4 x 1.6 mm	Tool	santec-medical	REF 277, LOT 832/511-1	Carrier; caused false positive results
Hxdroxyethylcellulose Tylose H 100000	Reagent			Carrier mixed with Kollidon 17 PF; caused false positive results
Kollidon 17 PF	Reagent	BASF	S30200	mixed with Hydroxyethylcellulose; caused false positive results
Collagen Biomatrix TissuDura	Tool	Baxter Internationl Inc.	REF B2246000999999	Carrier; caused false positive results
Round glass cover slips, 11 mm	Tool	Assistent Germany	1001/0011	Carrier; causes false positive results
Bovine Collagen Sponge	Tool	Wyeth Animal Health		Carrier; caused false positive results
Resodont Absorbable Collagen Membrane	Tool	Resorba Woundcare Germany	LOT 280303	Carrier; caused false positive results
Non-Woven Swabs	Tool	Fink Walter GmbH	REF 328132	Carrier; caused false positive results