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.
