Method of Studying Palatal Fusion using Static Organ Culture

Summary

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Abstract

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate ¹. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Introduction

Orofacial clefts are the most predominant craniofacial birth defects. Also, taking in consideration all possible craniofacial defects, these are the second most common birth anomaly in newborns ². Cleft palates occur at approximately 1 in every 700 births in the United States (US) every year, the incidence of cleft palate is equal to 475 children born with cleft palates per month or 15 children with clefts per day ³. Approximately 1% of kids born around the world each year exhibit some form of craniofacial dysmorphology.

Clefts of the palate and the lip need a very expensive and complicated procedure with lifelong impl....

Protocol

All procedures described must be performed in accordance with the guidelines and regulations for the use of vertebrate animals, including prior approval by the local Institutional Animal Care and Use Committee.

1. Preparation of Dissecting Instruments and Culture Media

1. Prewash all instruments to be used in dissection with 3% hydrogen peroxide and then autoclave at 121 °C for 20 min. Filter BGJb media with antibiotic and antimycotic solution using a 0.22 µm pore-sized filter with vacuum suction in a hood.

2. Preparation of Culture Equipment

1. Cut polycarbonate membrane filters that wil....

Results

The technique can be applied to different kinds of experiments.The following study was designed to test the teratogenic effect of nicotine in vitro on palatal fusion. Two strains of mice were utilized for this experiment, CD1 and C57.Palatal tissue was treated with 0.06 and 6 mM concentrations of nicotine hemisulfate. It was observed that palatal fusion patterns and timing were different in the two different strains. In CD1 mice, palatal shelves from the control group continued fusion in time dependent manner. H.......

Discussion

The protocol in this article provides a method of dissecting palatal shelves from embryos at embryonic day 13.5. Palatal shelves from mouse embryos were cultured in a serum-free culture media in an atmosphere of 95% O₂ 5% CO₂ at 37 °C. Successful palatal dissection is critically dependent on multiple factors at each step during the procedure from the embryonic time of the euthanized mice to the completion of the culture. One of the most important factors influencing palatal fusion is the time t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BGJb	Invitrogen
Penicillin/Streptomycin	Lonza Inc.	17-602E	100X
Petri dishes	VWR	25384-088	100 x15 mm
Center-well organ culture dish	Falcon	#353037	60 x15 mm
Wire triangular grid	Custom made with stainless steel wire mesh to fit the organ culture dish well.
Polycarbonate filter	GE& Water and Process Technologies	K04BP04700	Black 0.4 micron, 47 mm
Stereo microscope	ZEISS  Stemi SR
Culture hood	NUAIRE Laminar Flow
Microdissecting forceps	Dumont Medical		#5
Microdissecting scissors	Kent Scientific Corporation	INS14003-G	Vannas Scissors, straight, 8 cm long, 0.1mm tips, 5 mm blades, German made
Microdissecting scissors	Kent Scientific Corporation	INS500086	Vannas Scissors, straight, 8.5 cm long, 0.025 mm x 0.015 mm tips, 7 mm blades
Microdissecting scissors	Kent Scientific Corporation	INS14127-G	Spring scissors curved curved, 10.5 cm long, 8 mm blades, German made
Surgical currete (spoon spatula)	Hu-friedy		CM 2/4
Protector laboratory hood	Labconco
Incubator	Thermo Scientific		Farma
Series11 water Jacket
CO2 incubator
PBS	GIBCO	10010-023	1X
Fiber optic Light source	Fiber-light  Dolan-Jenner	PL-750
Embedding cassette	Statlab	EC301
Kim Wipes	VWR	470173-504