Name: primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos
Uploaded: 2016-03-10T13:00:00
Description: Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Funding for the development of current protocol is particularly supported by &#8220;Aim for the Top University Plan&#8221; of the National Taiwan University, Taiwan (grant ID-104R350144), as well as the Ministry of Science and Technology of Taiwan (grant ID: MOST 104-2313-B-002-039-MY3). We especially thank the Center for Biotechnology of National Taiwan University for providing an animal room and laboratory space for the current study.

NOTE: This procedure is a modification of a chicken model culture protocol developed by Bauer et al. 2013 and Nakazawa et al., 2011. 2,9
1. Prepare Healthy Embryonic Day 5 Embryos from Japanese Quail
<ol>
	<li>Place 1 male and 3 female sexually mature Japanese quail together in the same cage. Supply feed and water ad libitum. Adjust the light in the animal room to 14 hr of light and 10 hr of darkness.</li>
	<li>Collect fertilized eggs in plastic bags eve...

<ol>
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Because the previous culture system has only limited success, a better culture system is needed. The Japanese quail YSM endoderm requires treatment with proteolytic enzymes such as collagenase to loosen the cell-cell junctions to achieve better growth performance in the ex-vivo explants. Our data show that cell numbers from partial digestion treatment were greater than from the undigested tissue culture after seeding for 2 days (Figure 1). Therefore, the partial proteolytic digestion is a critical step t...

The major nutritional resource of the avian embryo is yolk, composed of 33% lipids, 17% protein, and 1% ash.1 During embryonic development, the yolk sac membrane (YSM) grows from within the embryonic abdominal cavity and gradually covers the yolk surface. Beginning at embryonic day 2, gene expression associated with lipid metabolism and angiogenesis is gradually increased in YSM, and the YSM slowly develops villus-like projections.8,9 These projections increase absorption of yolk nutrients to support embryonic development. The YSM is an extraembryonic tissue that contains three germ layers, endoderm, mesoderm and ectoderm.14 The yolk sac ectoderm faces the albumen and links with the vitelline membrane to gently cover the yolk sac. The endodermal epithelial cells are faced directly toward the egg yolk and serve as nutrient utilization portals.6 As the YSM expands, endodermal epithelial cell (EECs) can be divided by shape and functionality into two groups, area vitelline and area vasculosa.7
Area vitelline is composed of endodermal cells and is distant from the embryo; area vasculosa is composed of mesodermal cells and covers differentiated EECs with blood vessels and connective tissues. By embryonic day 5, the yolk is totally covered by ectoderm and endoderm of YSM and the vascular area has grown rapidly. The YSM absorbs, recomposes and releases lipids (as yolk-derived very low density lipoprotein) and proteins into the embryonic circulatory system.9, 2 Therefore, we established a primary Japanese quail embryonic endodermal epithelial cell culture system, to study the mechanisms of lipid utilization in YSM during avian embryonic development.
Lipids such as triacylglycerol, lecithin, phospholipid and cholesterol ester (CE) are the primary energy sources for avian embryos. At the early stages of development, yolk lipids are composed of only 1.3% CE and it rises to 10-15% at mid-term of avian embryonic development 3, 11. Cholesterol ester is synthesized from cholesterol by sterol O-acyltransferase 1 (SOAT1) in avian embryo YSM.4
The storage form of cholesterol is CE, CE is carried in lipoproteins, and lipoproteins are transported by circulation to tissues.13 A week before hatch there is rapid growth of avian embryos. Approximately 68% of the remaining lipid contents in yolk are absorbed during this stage.10 The mechanism by which yolk lipids are utilized can be clarified by an EEC research model. A chicken EEC culture protocol was established to achieve this research goal.2, 9 However, due to the low success rate of tissue explants, an improved EEC cell culture procedure is needed to study the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development.

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12800-017 10 X 1 L</td>
			<td style="width:167px;">For wash the EECs pellets</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">D-MEM/F-12</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12400-024 10 X 1 L</td>
			<td>As the basal medium in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">NBCS</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">16010-159</td>
			<td>As the supplyment serum in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Pen-Strep Ampho. Solution</td>
			<td style="width:199px;">BI (Biological Industries)</td>
			<td style="width:148px;">03-033-1B 100ml</td>
			<td>For attenuating the possible infection&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Collagenase Type IV</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">17104-019 1g</td>
			<td>&#160;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">24 well plate</td>
			<td style="width:199px;">FALCON&#174;</td>
			<td style="width:148px;">REF-353047</td>
			<td>For EECs to attach and extension</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">50 ML PP centrifuge tubes</td>
			<td style="width:199px;">Corning&#174; CentriStarTM</td>
			<td style="width:148px;">430829</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50ML Conical bottomed Tube with Cap</td>
			<td style="width:199px;">PRO TECH</td>
			<td>CT-50-PL-TW</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Reciprocal shaking bath</td>
			<td>DEAGLE</td>
			<td style="width:148px;">SB302</td>
			<td>For better enzymatic digestion on membranes</td>
		</tr>
	</tbody>
</table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

In order to achieve the goal of establishing a consistent and useful cell model, we need to extend and stabilize the proliferation rate and performance of avian EECs. We compared direct incubation of the endoderm with no enzyme digestion with endoderm partially digested with proteolytic enzymes, such as collagenase or collagenase plus 0.6 U of dispase. Dispase is an amino-endopeptidase that hydrolyzes the N-terminal peptide bonds of non-polar amino acid residues. Whereas protease digestio...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

The overall goal of this ex vivo explant procedure is to improve the collection and extend the application of avian endodermal epithelial cells in order to study the function of enzymes and proteins in mediating nutrient utilization by avian embryos. This method can help answer key questions regarding lipid utilization during avian embryogenesis, such as how the yolk lipids are packaged and transported from the yolk to the embryo during the later stages of development. The main advantage of this technique is that we can successfully explain functional endodermal epithelial cells from Japanese quail yolk sac membrane, which can be utilized as a model to study avian embryonic development.Begin by making a 10x PBS solution as described in the text protocol. Dilute this stock solution one to ten to prepare 1x PBS, which will be used to wash the yolk sac membrane during endoderm dissection. To prepare growth medium for endodermal epithelial cells, or EECs, first supplement DMEM/F-12 with 10%newborn calf serum and 1%penicillin streptomycin amphotericin solution.Afterwards, insert the probe of a pH meter into previously prepared DMEM. Add three normal HCl dropwise to the medium until the pH has been adjusted to 7.2. After collecting approximately two dozen fertilized quail eggs, place them vertically in egg containers so that their wide, untapered ends, where the air cells are located, are pointing upward.Then, transfer the eggs to a well-ventilated incubator set at 37 degrees Celsius. After five days of incubation, examine the eggs with an egg candler. If embryos have developed normally, expect to see a circular structure of capillary vessels beneath the air cell.Select an egg demonstrating normal capillary development, and clean the shell with fresh water and 75%ethanol. Next, open the egg shell from the air cell position using scissors. Use forceps to gently help pour out the contents of the egg into an empty 10 cm culture dish.Then, use scissors to detach the embryo, yolk, and any albumin from the yolk sac membrane, which is abbreviated as YSM. Once isolated, wash the YSM three times in 1x PBS, and then, transfer it to a new 10 cm dish containing PBS. Afterwards, place the dish under a dissecting scope.Then, proceed to remove any residual egg white, which abuts the outer ectoderm layer of the YSM, with scissors. Continue by using forceps to lift the translucent ectoderm and red capillary structural mesoderm from the edge of the mesoderm on the yolk sac membrane. Then, gently peel the ectoderm and mesoderm together away to isolate the underlying endoderm cell layer.Then, with one pair of forceps, firmly hold the yellow endodermal cell layer of the membrane. With another pair, grip the capillary mesoderm, and proceed to pull the mesoderm away from the junction edge of the mesoderm and endoderm. Using a pair of scissors, cut off the remaining yellow endoderm layer, and transfer it to the six centimeter culture dish filled with growth medium.Using a dropper, transfer the light yellow endoderm from the six centimeter culture dish to a 15 mL centrifuge tube after YSM collection, and to it, add 15 mL of previously prepared growth medium. Place the conical in a 37 degrees Celsius water bath until all tissue collections are completed. Begin by dissolving 6.5 units of Type IV collagenase in 10 mL of DMEM.Next, centrifuge the endoderm-containing tubes at 130 time g for three minutes at room temperature. Following centrifugation, aspirate the medium, and use a dropper to transfer all the endoderm samples to a six centimeter culture dish. Then, add one milliliter of collagen solution to the dish.With curved scissors, proceed to cut the endoderm collected from four quail embryos into slices approximately two to three millimeters in size. Afterwards, collect the slices into a 50 mL centrifuge tube, and with nine milliliters of fresh collagenase solution, which is already inside of the 50 mL centrifuge tube. To improve cell proliferation during the explant stage, place the tube in a shaking water bath set to 37 degrees Celsius.Then, incubate the conical for 30 minutes to partially digest the tissue with collagenase. The partial proteolytic digestion is the most important step to improve the cell yolk from yolk sac membrane endoderm collection. Our data shows a significant improvement of the successful rate of cell culture and cell proliferation by the collagenase digestion procedure.Following collagenase digestion, centrifuge the tissue at 130 times g for three minutes at room temperature, and then remove the supernatant fraction with a pipette. Proceed to wash the pellet by resuspending it in 20 mL of DMEM. Centrifuge the tube as previously described, then afterwards, remove the supernatant, and resuspend the pellet into 12 mL of growth medium.Then, gently add 500 microliters of the suspension to each well of a 24-well plate. Proceed to incubate the endoderm explants for two days at 37 degrees Celsius in 5%CO2. During this time, expect cells to proliferate out of the tissues.Next, incubate the cells for another two days. Then, subject them to a cell viability assay, and visualize the results using a plate reader. Afterwards, perform RT-qPCR to evaluate expression levels of markers of interest in EECs and check the product size from RT-qPCR by gel electrophoresis as described in the text protocol.Compared to untreated explants, those partially digested with collagenase exhibited significantly increased cell proliferation, as shown in these graphs and determined by a paired t-test, demonstrating the success of the culture system. To evaluate the effects of other enzymes, cell viability assays were performed for tissues treated with collagenase and dispase or with collagenase alone. Over the first five days of incubation, cell growth was similar in both conditions.However, from days six to nine, only collagenase-treated EECs proliferated steadily, suggesting that partial digestion with this enzyme improves growth and should be preferentially used. When cultured EECs were visualized, they demonstrated the adipocyte-like features of large circular lipid droplets as shown here. Such cells were also positive for Oil Red O staining, which is typically used to identify adipocytes.Supporting these morphological data, real-time quantitative PCR revealed that cultured EECs demonstrate high levels of SOAT1, a protein that synthesizes cholesterol ester from cholesterol. This further suggests that cultured EECs demonstrate the correct physiological characteristics of adipocytes. Importantly, when EECs were exposed to IBMX and Forskolin, factors that increase cyclic adenosine monophosphate, or cAMP levels, and cultured for 24 hours, both treatments stimulated SOAT1 expression, suggesting possible mechanisms of SOAT1 regulation and lipid utilization in the YSM.Once mastered, this technique can be done in about four hours, during which time, at least three samples can be collected. Following this procedure, methods like SOAT1 or other enzymatic activity assays as well as similar visual confirmation can be performed. Such assay may help answer additional questions, like how certain protein and macronutrients are involved in the later stages of embryonic development.After its development, this technique paved the way for researchers in the field of embryonic nutrient utilization to explore the possible mechanism by which nutrients are utilized and transported in an avian embryonic model. After watching this video, you should be able to learn how to collect endoderm tissues from avian embryos, and also know how to culture EECs for studying embryonic nutrient transportation and utilization using the avian model.

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