Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Summary

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Abstract

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Introduction

The embryonic chicken (Gallus gallus) is an excellent model organism commonly used in the field of biology. Its incubation period is roughly 21 days and many eggs can be incubated simultaneously, making experimentation quick and efficient. Perhaps most importantly, the embryo is also easily manipulated, enabling the extensive study of key developmental processes and of the genes and proteins that drive these processes.

The embryonic chicken eye is a complex organ that develops via the interaction of a number of different tissues similar to many other body systems. This method enables the study of the development of these t....

Protocol

NOTE: For embryo stages, utilize the Hamburger and Hamilton¹² (HH) staging table.

1. Embryo Incubation

1. Incubate fertilized chicken eggs in a sterile, temperature-controlled incubator at 37 °C ± 1 °C and ~40% humidity.
2. Turn the eggs 1x per day and allow them to incubate to HH34 (8 days post-fertilization).

2. Preparation and Sterilization of the Materials

1. For 12 embryos, autoclave 2 L of distilled water, 1 L of 0.85% chick saline, 12 glass pipettes, 1 box of paper tissue, 24 2.5 cm x 2.5 cm squares of semi-porous filter paper, and 24 2....

Results

Using this method, an embryonic chicken eye can be cultured from day 8 of its development (HH34) in vitro for 4 days. Four days of in ovo development corresponds to HH38.

This culturing method supports the development of feather buds surrounding the eye and on the eyelids (Figure 1B). These feather buds are not present in ovo at HH34 prior to the culturing (

Discussion

This protocol makes use of established tissue culturing techniques to achieve the growth of a chicken eye from embryonic day 8 (HH34) in vitro for 4 days. This grid-culturing method was originally described by Trowell¹⁵. We optimized a protocol from Pinto and Hall's 1991 study¹⁶ utilizing a semi-porous membrane with the grid to study inductive signals between separated tissue layers of the embryonic chicken eye¹⁵. Using this method, Roac.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm cell culture petri dishes	Corning	353001	easy grip tissue culture dish, polystyrene, non-pyrogenic
100 mm cell culture petri dishes	Corning	353003	tissue culture dish, polystyrene, non-pyrogenic
paper tissue	Kimtech	34155	Kimtech Science Brand Task Wipers, 280 per carton
wire mesh	n/a	n/a	stainless steel wire mesh (grid size 0.7 mm)
disposable glass pipettes	VWR	14673-010	Borosilicate glass disposable 5 3/4"
nutrient medium	Gibco	12591-038	Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)
penicillin-streptomycin	Sigma-Aldrich	P4458	10000 units/mL penicillin streptomycin solution stabilised
filter paper	Whatman	1454 090	semi-porous filter paper 90mm
fertilized chicken eggs	Dalhousie University Agricultural College	n/a	can be obtained from local farms
sodium chloride (NaCl)	EMD	SX0420-3	sodium chloride crystals, reagent grade
1 L glass bottle	VWR	89000-240	1 L pyrex autoclavable glass bottle
ethanol	Fisher Scientific	BP82011	70% molecular biology grade
tupperware containers	n/a	n/a	store-bought and sterilized with EtOH
disposable razor blades	VWR	55411-050	single edge industrial razor blades (surgical carbon steel)
plastic spoons	n/a	n/a	store-bought and sterilized with EtOH
dust mask	3M	n/a	3M 8500 Comfort Mask
paraformaldehyde	Sigma-Aldrich	P6148	paraformaldehyde, reagent grade, crystalline
neutral-buffered formalin	Fisher Scientific	72210	10% neutral buffered formalin
phosphate buffered saline (PBS)	n/a	n/a	10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)
15 ml falcon tubes	VWR	21008-216	presterilized centrifuge tubes
forceps	FST	n/a	fine forceps
chick saline	n/a	n/a	0.85% NaCl
tinfoil	n/a	n/a	store-bought
paper towel	n/a	n/a	store-bought