The authors do not have any acknowledgements.

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
<ol>
	<li>Prewash all instruments to be used in dissection with 3% hydrogen peroxide and then autoclave at 121 &#176;C for 20 min. Filter BGJb media with antibiotic and antimycotic solution using a 0.22 &#181;m pore-sized filter with vacuum suction in a hood.</li>
</ol>
2. Preparation of Culture Equipment
<ol>
	<li>Cut polycarbonate membrane filters that will support the palatal organs in small triangles, spray with 70% alcohol and allow them to dry. Prepare Center-Well Organ Culture Dish (60x15 mm) by placing a clean and sterilized equilateral triangular-shaped wire grid (20 mm) over the organ culture plate. Then fill the well with 1 ml of BGJb media culture media. &#160;NOTE: The media should be sufficient to reach the level of the grid without submerging the palates. Place the membrane filters, shiny side down, on top of the grid (3-4 membranes per grid).Depending on the experiment, treatment (proteins, antibodies or inhibitors) can be added to the media</li>
</ol>
3. Mouse Embryo Collection and Preparation for Culture
<ol>
	<li>To obtain wild type mouse embryos, use timed 13.5 pregnant CD-1 mouse genetic background strain. Clean the dissection area with 70% ethanol. &#160; 
	NOTE: Other strains of mice can be used. We prefer CD-1 mice because of the bigger litter number.</li>
	<li>Euthanize pregnant mice at E13.5 dpc (days post coitus) with 5% Isoflurane followed by cervical dislocation to insure death using approved protocols.</li>
	<li>Spray 70% ethanol on the ventral abdominal surface to avoid having the abdominal hair stick to the instruments. Then open the abdominal cavity ventrally using a sharp-blunt operating scissors and micro-dissecting forceps to locate the uterus. Using the forceps, lift the entire uterus and separate it from the body by cutting at the uterine body and at the tips of the uterine horns with a light operating scissors.</li>
	<li>Place the uterus on a clean paper towel over ice (According to the protocol guide for care and use of laboratory animals). Cover the uterus with another clean paper towel to prevent contamination. Sterilize the instruments using 70% ethanol spray before further use. The uterus and the embryos can be transported to the laboratory on ice.</li>
	<li>In an open laminar flow hood, carefully make a small opening in the yolk sac using sharp-blunt operating scissors and micro-dissecting forceps. Make the opening wider and then gently push it to exteriorize the embryo from the yolk sac.</li>
	<li>Transfer the embryos immediately to a Petri dish with cold phosphate-buffered Saline 1X (PBS) pH 7.4. To assure the proper stage, examine the embryo under a dissecting microscope for the body, head size and limb morphology. 
	NOTE: Embryos that are not at the right stage of development are not used for the experiment.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53063/53063fig2.jpg" /> 
Figure 2. Limb Bud Morphology. The fore and hind limbs are checked for the degree of webbing indentation between the digits. At E13.5, all the digit condensations are prominent, with the webbing and distal indentations between digits appearing very distinct. The hind digits are behind by a half-day. 12 Upper row: forelimb, lower row: hind limb.
4. Palate Dissection
<ol>
	<li>Place one embryo at a time in a petri dish with PBS under the dissecting microscope. Using small dissecting scissors and tweezers separate the head from the body of the embryos.</li>
	<li>Carefully holding the mouse cranium above eye level with forceps (avoiding the maxillary process) make two incisions on both sides of the lip line. Then remove the mandible and tongue. Isolate the maxillary process region by making a cut at eye level and remove the brain, eyes, nasal septum and adjoining tissue.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/53063/53063fig3.jpg" /> 
Figure 3.Palate tissue collected from 13.5 embryos, when the palate shelves had elevated but not made contact. The palatal shelves were placed in organ culture on a filter at the air media interface for 72 hr in the static model. (A) Level of dissection on E13.5 embryo. (B)&#160;Blue stars demarcate areas to hold the tissue with forceps during dissection. (C)&#160;View of dissected palatal shelves placed on a trapezoid filter membrane. (D)&#160;Side view of culture dish after placement of the dissected palates and addition of culture media.
<ol start="3">
	<li>Place the palatal oral side up and remove the nasal septum and the tissue around it, keeping the primary palate attached to help orient it from anterior to posterior. Use a spoon spatula and very carefully transfer the palatal shelves (nasal side down) to the prepared filters on the grids in the culture dishes. &#160;NOTE: Adjust volume of media to allow proper air-media interface.</li>
	<li>Under the stereo microscope, dissect the primary palate and use tweezers to place the palatal shelves close to each other. The palatal shelves will not fuse if they are not placed in contact. Cut the triangular membrane filter where the anterior part of the palate is located. The trapezoid shape will aid in localizing the anterior portion of the palate.</li>
	<li>Culture the palatal shelves individually or with two or three in the same organ culture dish using BGJb culture media.</li>
</ol>
5. Culturing Mouse Palatal Shelves
<ol>
	<li>Incubate the tissues at 37 &#176;C in a humidified gas mixture (5% CO2 and 95% air) for 72 hr, and change the medium every 24 hr.</li>
</ol>

6. Palate Processing for Histological Analysis
<ol>
	<li>Fix the palates after 72 hr in culture with 4% formaldehyde/phosphate-buffered saline O/N at 4&#176;&#160;C. Then, gently with a transfer pipette wash the tissue in the culture dish with PBS. Wrap the palates in a piece of laboratory wipes and place in embedding cassettes. &#160; 
	NOTE: Wrapping the palates in laboratory wipes prevent their loss during the dehydration process.</li>
	<li>Dehydrate the tissue following 70%, 80% and up to 100% ethanol washes for 1 hr, followed by the regular paraffin embedding procedure. For embedding, place the anterior part of the palate facing the tray.</li>
	<li>Cut Serial 6 &#181;m sections in the coronal orientation from anterior to posterior. A complete section palate will yield 350-450 sections. Stain the sections with hematoxylin and eosin (H&#38;E).10 Score every 20th section using the previously described Mean Fusion Score (MFS) scale (Table 1). 8</li>
</ol>

<ol><li>Nawshad, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Palatal+seam+disintegration+to+die+or+not+to+die+that+is+no+longer+the+question%5BTitle%5D)+AND+%22Developmental+dynamics+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">Palatal seam disintegration to die or not to die that is no longer the question.</a> Developmental dynamics an official publication of the American Association of Anatomists. 237, 2643-2656 (2008).</li>
<li>Strong, E. B., Buckmiller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+of+the+cleft+palate%5BTitle%5D)+AND+%22Facial+plastic+surgery+clinics+of+North+America%22%5BJournal%5D)">Management of the cleft palate.</a> Facial plastic surgery clinics of North America. 9, 15-25 (2001).</li>
<li>Witt, P. D., Marsh, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+assessing+outcome+of+surgical+repair+of+cleft+lip+and+cleft+palate%5BTitle%5D)+AND+%22Plastic+and+reconstructive+surgery%22%5BJournal%5D)">Advances in assessing outcome of surgical repair of cleft lip and cleft palate.</a> Plastic and reconstructive surgery. 100, 1907-1917 (1997).</li>
<li>4miloro, principles of oral and maxillofafcial surgery. 209, 231-249 (1984).</li>
<li>Mima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+the+epithelial+adhesion+molecule+CEACAM1+is+important+for+palate+formation%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Regulation of the epithelial adhesion molecule CEACAM1 is important for palate formation.</a> PloS one. 8, e61653(2013).</li>
<li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro+an+analysis+by+DiI+labelling+and+confocal+microscopy%5BTitle%5D)+AND+%22Development+%28Cambridge%2C+England%29%22%5BJournal%5D)">The fate of medial edge epithelial cells during palatal fusion in vitro an analysis by DiI labelling and confocal microscopy.</a> Development (Cambridge, England). 114, 379-388 (1992).</li>
<li>Ferguson, M. W., Honig, L. S., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+cultured+palatal+shelves+from+alligator+chick+and+mouse+embryos%5BTitle%5D)+AND+%22The+Anatomical+record%22%5BJournal%5D)">Differentiation of cultured palatal shelves from alligator chick and mouse embryos.</a> The Anatomical record. 209, 231-249 (1984).</li>
<li>San Miguel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ephrin+reverse+signaling+controls+palate+fusion+via+a+PI3+kinase+dependent+mechanism%5BTitle%5D)+AND+%22Developmental+dynamics+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">Ephrin reverse signaling controls palate fusion via a PI3 kinase dependent mechanism.</a> Developmental dynamics an official publication of the American Association of Anatomists. 240, 357-364 (2011).</li>
<li>Kang, P., Svoboda, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PI+3+kinase+activity+is+required+for+epithelial+mesenchymal+transformation+during+palate+fusion%5BTitle%5D)+AND+%22Developmental+dynamics+an+official+publication+of+the+American+Association+of+Anatomists%22%5BJournal%5D)">PI 3 kinase activity is required for epithelial mesenchymal transformation during palate fusion.</a> Developmental dynamics an official publication of the American Association of Anatomists. 225, 316-322 (2002).</li>
<li>RD, L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRD%2C+L+%22Histopathologic+Technic+and+Practical+Histochemistry%22">Histopathologic Technic and Practical Histochemistry</a>. , 3rd edn, McGraw Hill Book Co. New York. (1965).</li>
<li>Kaufman, M. H. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKaufman%2C+MH+%22The+Atlas+of+Mouse+Development%22">The Atlas of Mouse Development</a>. , Elsevier. (1992).</li>
<li>Taher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+gene+expression+analysis+of+murine+limb+development%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Global gene expression analysis of murine limb development.</a> PloS one. 6, e28358(2011).</li>
</ol>

The authors do not have any competing financial interests.

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 &#176;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 taken to start the organ culture; embryos need to be at embryonic day 13.5 as the secondary palate shelves are horizontal, but have not started to fuse (Figure&#160;1).
Another critical point during the procedure is the exteriorization of embryos from the yolk sac. This step requires great care to prevent damage to the facial structures. Once the embryo is removed from the sac, the head is removed from the body and placed in a petri dish to be viewed with a stereomicroscope with fiber optic illumination from both sides. The mandible and the tongue are removed, exposing the palate. The brain and residual tissue are removed at eye level. At this point it is important to consider the epithelial layer around the shelves. It is necessary to manipulate the tissue very carefully to keep the integrity of these cells. Epithelial cells are responsible for adherence and formation of the MES.
Each dissected palate is turned oral side down to clean any cartilage from the nasal septum. This tissue is on the anterior side of the palate and appears whiter and more condensed. Palatal shelves free of unwanted tissue are moved to a culture dish and placed on top of the filter membrane. The position of the shelves is checked again under the stereo dissecting microscope. They must be pushed together to allow adherence and further on palatal fusion.

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 implications for patients who have this anomaly. The estimated cost for each patient with oral cleft is approximately $100,0004. The treatment of a patient with cleft lip and palate requires a team of doctors including craniofacial surgeons, otolaryngologists, geneticists, anesthesiologists, speech-language pathologists, nutritionists, orthodontists, prosthodontists, psychologists, neurosurgeons, and ophthalmologists.
In palatogenesis, the secondary palate arises as paired outgrowths, which initially grow vertically and undergo palatal shelf elevation above the dorsum of the tongue. Following elevation, the paired palatal shelves grow towards the midline (at E14.5 &#8211;E15 in mice and week 9 in humans). The medial edge epithelium (MEE) that covers the shelf tip adheres forming the midline epithelial seam.
This is followed by epithelial to mesenchymal transition and/or apoptosis to allow mesenchymal confluence. Adhesion of opposing MEE is an essential event whose alteration causes cleft palate. However, only few studies investigated the initial adhesion of palatal shelves5. The hard palate forms by differentiation of mesenchymal cells into osteoblast. The abnormal development of the palate can produce cleft palates with or without involvement of the lip.
Palate organ culture techniques had been used widely for many labs over the past 30 years6,7.
In this protocol we describe in details a method of palatal dissection and static organ culture. The advantage of a motionless organ culture is that it allows palatal shelves to fuse. This technique had been used successfully in our laboratory for many fusion and signaling experiments8,9. However the scope of the technique is vast and can be used whenever static organ culture system is required, including the evaluation of responses to exogenous chemical agents, the effects of regulatory growth factors in different pathways and specific proteins.
<img alt="Figure 1" src="/files/ftp_upload/53063/53063fig1.jpg" /> 
Figure 1. Mouse Palatogenesis. Developmental stages of the mice palate. (B-F) Scanning electron micrographs (SEM) of the secondary palate at representative developmental times. Red arrows: show the initial part of palatal adhesion and fusion. Yellow arrows: point to the space between the primary and secondary palates that will disappear after fusion (reprinted from Kaufman11 with permission from PLOS one).

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

method of studying palatal fusion using static organ culture

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.

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. However, in the treated group nicotine delayed fusion especially at 6mM nicotine concentration (Figure 4A). Histology sections of CD1 mouse palates showed total fusion after 72 hr in the control specimens. At lower doses of nicotine the palatal shelves adhere but the epithelial cells remain after 3 days in culture. Palatal shelves incubated at high doses of nicotine did not make contact after 72 hr&#160;of incubation.
In C57 mice, palatal shelves from the control group continued fusion in time dependent manner. However, palatal shelves of C57 mice showed higher score at 6 mM concentration when the specimens were incubated for 48 hr in comparison with the specimens incubated at 0.6 mM concentration of nicotine for the same period of time (Figure 4B). Histology sections of C57 mice palates showed that in treatment groups a continuous epithelial seam persisted in the midline. Fusion was observed just in the control group at 72 hr.
We observed that palatal fusion in both kinds of mice was affected by high doses of nicotine. However, palates from C57 were more sensible to nicotine. In conclusion, our results demonstrate that genetic background of mice influences nicotine responses in palatal tissues.
<img alt="Figure 4" src="/files/ftp_upload/53063/53063fig4.jpg" /> 
Figure 4.&#160;Two strains of mouse palates cultured in the presence of nicotine for 24, 48, or 72 hr. The mouse strains responded to the nicotine in different degrees. All palates exposed to the high dose of nicotine (6 mM) failed to fuse and had low mean fusion score (MFS) .Note that at 24 hr, none of the palates had closed and the MFS ranged from 1 (palatal shelves not touching) to 3 (partial fusion) in all groups. By 48 hr, many of the control palates had partially fused (scores over 3) and by 72 hr, the CD1 palates were completely fused (score of 5), but the C57 controls had not completely fused (3.67). (Unpublished data from Maria Serrano and Dr Kathy Svoboda).
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Score</td>
			<td>Criteria</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Non-fusion with no adhesion.</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Non-fusion with some apparent adhesion.</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Adhesion with some disintegration of MEE layers and clear partial mesenchymal confluence.</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Complete fusion with some traces of MEE cells or seam remaining.</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Complete fusion with no evidence of MEE cells or seam visible.</td>
		</tr>
	</tbody>
</table>
Table 1:Mean Fusion Score (MFS) scale (Kang and Svoboda, 2002). Calculate the scores for each palate for the anterior (sections 1-150), middle (sections 150-300) and posterior (sections 300-450). 8

Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

Method of Studying Palatal Fusion using Static Organ Culture

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.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.7K Views.  Texas A&M University Baylor College of Dentistry.  Studies van gehemelte ontwikkeling worden gemotiveerd door de incidentie van gespleten gehemelte, een aangeboren afwijking die een enorme gezondheid last oplegt en kunnen verlaten blijvende verminking. We tonen hier een techniek om cultuur palatini die kunnen worden gebruikt om verschillende signaalwegen betrokken bij palatinale ontwikkeling en fusie bestuderen. 

Video: Method of Studying Palatal Fusion using Static Organ Culture

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Medicine

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Neuroscience

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biology

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Method of Studying Palatal Fusion using Static Organ Culture

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