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 tissues, particularly at advanced stages of development. For example, the multi-layered retina may be of particular interest to those studying the development of the nervous system. Alternative methods that enable the study of other eye tissues such as the cornea, the vitreal body, the lens, the sclera, and the eyelids are of benefit to researchers. The chicken embryonic eye also contains a series of flat bones, the scleral ossicles, which can be used as a model for the study of intramembranous bone induction and ossification in vertebrates¹.

Currently, there are a number of methods used to study embryonic development. Microinjections of inhibitory antibodies or other inhibitory molecules^2,3, surgically implanted microbeads soaked in inhibitor⁴, and electroporation⁵ are all methods that can be used to downregulate genes or proteins of interest in an embryo. Similar methods are used to upregulate proteins. These methods are not without their limitations. For example, when using chemicals to alter the embryonic development, the lethal effects on the embryo must be evaluated, and this limits the use of the aforementioned methods to localized sites of application at doses low enough to ensure the survivability of the embryo.

In vitro tissue culturing has been used in a wide range of organisms to study development and can be used to bypass some of the aforementioned limitations. For example, the femora⁶, feather buds^7,8, and limbs⁹ of the chicken have all been studied using tissue culturing methods, as have the testes of the mouse¹⁰ and the roots and stems of plants¹¹. These methods grant scientists a high degree of control over the tissue development, such as the ability to fluctuate the temperature and alter the nutrient availability. The isolation of the tissue from the whole embryo also makes it far less susceptible to the lethal effects of chemicals, thus enabling manipulation studies on a global scale at higher concentrations. Another notable advantage of in vitro culturing is the preservation of the tissue's cellular environment; the arrangement of tissues remains relatively unchanged, making it possible to study the interactions between different tissue types⁹. Thus, in vitro culturing opens doors to additional experimental approaches not available in in vivo or in ovo models.

Currently, studying the development of the embryonic chicken eye using chemicals is particularly challenging. A number of extraembryonic membranes cover the embryo, making it difficult to apply microbeads or chemicals; the embryo is also very active within the egg as it gets older, further complicating an already difficult method. This protocol enables easy access to the eye and its surrounding tissues, eliminating these barriers, while also providing new opportunities to examine the developmental processes within the eye. This protocol was established to study the induction of the scleral ossicles within the embryonic eye.

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 2.5 cm x 2.5 cm squares of steel wire mesh with the edges curved downwards. To sterilize the materials, autoclave them at 121 °C for at least 15 min.
   NOTE: The autoclaving of the materials can be done in advance.
2. Sterilize the incubator with 70% ethanol by wiping the sides, shelves, and door. Place 2 plastic containers containing autoclaved distilled water inside the incubator to maintain humidity. Prewarm the culture incubator to 37 °C ± 1 °C and ~40% humidity. Ensure the incubator is completely dark; if the door is transparent, cover it with tinfoil.
3. Prewarm 100 mL of nutrient medium and 1 mL of penicillin by placing them in the culture incubator.
4. Cover the workspace with a paper towel and sterilize the bench by spraying it with 70% ethanol. Sterilize 2 pairs of forceps, dissection scissors, a razor blade, and a plastic spoon with 70% ethanol in a similar manner.
5. Place 12 sterile 100 mm Petri dishes and 24 sterile 35 mm Petri dishes on the bench for use. Ensure the dishes remain in a sterile bag until use.

3. Embryo Preparation

NOTE: From this point onwards, wear a protective dust face mask to avoid contaminating the cultures. A bacterial or fungal infection will ruin the culture and there is a risk of it spreading quickly to all cultures in the incubator.

1. Crack open the egg and transfer the embryo to a 100 mm Petri dish containing 0.85% chick saline. Stage the embryo according to the anatomical features described in the Hamburger and Hamilton staging guidelines¹² and confirm the embryo is at HH34.
2. Cut the neck and then bisect the head of the embryo at the midline using a sterilized razor blade. Using sterile forceps, remove the brain. Leave the beak intact.

4. Culture Setup

1. Place 2 of the 35 mm Petri dishes inside one 100 mm Petri dish.
2. Place a steel wire mesh inside each 35 mm Petri dish.
3. Using forceps, transfer 1 of the bisected heads to a piece of 2.5 cm x 2.5 cm semi-porous filter paper with the eye facing up. Ensure the tissue is secure and does not slip off by tilting it slightly.
4. Using forceps, carefully place the eye tissue and the filter paper on top of the steel wire mesh in 1 of the 35 mm Petri dishes, creating a raised stage with the eye tissue on top.
5. Repeat steps 4.3 and 4.4 with the other half head.
6. Using a sterile glass pipette, carefully add nutrient medium directly into each 35 mm Petri dish until it reaches the level of the filter paper. Do not submerge the half head in the nutrient medium.
7. Add 50 µL of 10,000 U/mL penicillin-streptomycin to the nutrient medium in each Petri dish.
8. Fold a piece of tissue paper into a small square and place it inside the 100 mm Petri dish. Moisten it with autoclaved distilled water.
9. Place the culture dish into the sterile, dark incubator at 37 °C ± 1 °C and ~40% humidity for up to 4 days.
10. Repeat steps 3 and 4 for each embryo.
    NOTE: The number of embryos needed will depend on the specific experiment; here, we describe the protocol for 12 embryos.

5. Culture Maintenance

1. Once per day, top up the plastic containers in the incubator with fresh, autoclaved distilled water.
2. Daily check all cultures for bacterial or fungal infections. Cultures that have disintegrated or in which the medium has changed color are infected. Discard all cultures that are infected.

6. Fixation

1. Following culturing, remove all dishes from the incubator.
2. Using a pair of forceps, gently remove the eye from the filter paper, taking care not to tear the tissue.
3. Fix the eye tissue in 4% paraformaldehyde in 1x phosphate-buffered saline overnight at 4 °C or in 10% neutral-buffered formalin overnight at room temperature.
4. Store the tissue at 4 °C in 1x phosphate-buffered saline.

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