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

Summary

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.

Abstract

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 °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.

Introduction

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.

Protocol

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

1. 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.
2. Collect fertilized eggs in plastic bags every day in the afternoon and keep them in a 16 °C refrigerator to retard the growth rate of embryos.
3. After collecting a sufficient number of eggs within a two-week period, incubate the fertilized eggs in a 37 °C incubator with good ventilation for five days.

2. Prepare Basal Medium and Wash Solutions

1. Use DMEM/ F-12 medium (adjusted to pH 7.2) as the growth medium, and supplement it with 10% new born calf serum (NBCS) and 1% Pen-Strep Ampho. Solution (PSA; Penicillin, 10⁵ units/L; Streptomycin, 100 mg/L; Amphotericin B, 0.25 mg/L).
2. Prepare phosphate buffered saline (PBS) solution at a 10x concentration for storage; use 1x PBS solution (containing NaCl, 137.93 mM; KCl, 2.667 mM; KH₂PO_4, 1.471 mM; Na₂HPO₄-7H₂O, 8.06 mM; adjust to pH 7.2) to wash and moisten the yolk sac membrane while separating the endoderm from egg yolk and albumin.
3. Use DMEM medium (adjusted to pH 7.2) to re-suspend endoderm pellets.

3. Collection of Primary EECs from Japanese Quail YSMs

1. Incubate the collected eggs for 5 days (step 1.3). Examine the eggs by an egg candler to confirm normal embryonic development.
   NOTE: On embryonic day 5, the boundary of YSM mesoderm is very clear and the 3 embryonic layers are less tightly-linked together, so it is easy to obtain endoderm. Only use the eggs showing normal capillary circulation development for YSM collections.
2. Clean the eggshell carefully using fresh water and 75% ethanol. Open the eggshell from the air cell position by a pair of scissors, gently peel out and collect the whole YSM by forceps and place in a 10 cm culture dish filled with 37°C 1x PBS.
3. Using a pair of scissors remove the embryo, yolk and egg-white, and gently wash the YSM with 1x PBS three times and place YSM in PBS solution in a 10 cm culture dish.
4. Use forceps and a pair of scissors to remove the ectoderm from YSM. Use 2 forceps, one to firmly hold the endodermal cell layer, and the other to hold the capillary mesoderm.
5. Under a dissecting microscope, pull apart and separate the endoderm from the edge of the mesoderm in the direction towards the embryo. Collect the light-yellow endoderm in 50 ml centrifuge tubes using a dropper and place in 15 ml of growth medium in a 37 °C water bath until all tissue collections are completed.

4. Digestion of the Endoderm Slices by Collagenase Digestion

1. Freshly dissolve 6.5 units of type IV collagenase in 10 ml of DMEM.
2. Centrifuge the 50 ml tubes (3.4) at 130 x g for 3 min at RT. Aspirate the medium and place all the endoderm in a 6 cm culture dish using a pipette. Add 1 ml collagenase solution to this culture dish.
3. Slice the endoderm collected from 2 quail embryos with a pair of curved scissors until the size of each slice is approximately 2 to 3 mm. Use a dropper, collect all the slices into a 50 ml centrifuge tube along with 9 ml of fresh collagenase solution.
4. Digest the tissue with collagenase for 30 min in a 37 °C shaking water bath at 175 rpm. This partial digestion with collagenase is used to improve the explant cell proliferation.
5. Centrifuge at 130 x g for 3 min at RT and gently remove the supernatant fraction using a pipette. Wash the pellet by suspension in 20 ml DMEM and remove the supernatant fraction after 130 x g centrifugation for 3 min.
6. Re-suspend the pellet with 12 ml of growth medium (DMEM/F12 with 10% NBCS and 1% PSA) and seed gently and equally into a 24-well plate (each well with 500 µl cell solution).
7. Incubate tissue explants at 37 °C in 5% CO₂ in air for 2 days to allow cells to proliferate out of the tissues.
8. Incubate cells for another 2 days and characterize for cell viability by a cell viability assay protocol.¹⁵
   NOTE: Living cells maintain a reducing environment within the cytosol. Resazurin, the active ingredient, is a non-toxic, cell permeable compound that is blue in color and virtually non-fluorescent. When added to cell cultures, resazurin is reduced to resorufin in the cytosol by mitochondrial enzymes. Resorufin is red in color and highly fluorescent. Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence and color of the culture medium surrounding cells.
9. Perform endoderm epithelial cell total RNA isolation, reverse transcription and quantitative real-time PCR of marker gene mRNA expression as described. ^16,17
   1. Use reverse transcriptase PCR to detect SOAT1 mRNA (sense: 5'-GAAGGGGCCTATCTGGAACG-3'; antisense: 5'-ATCTGCACGTGACATGACCA-3'; PCR product = 168 bp) as a specific marker for the EEC cells. Use β-Actin mRNA (sense: 5'- TGGTGAAGCTGTAGCCTCTC-3'; antisense: 5'- GTGATGGACTCTGGTGATGG-3'; PCR product = 151 bp) as a housekeeping gene and internal control.
   2. Use gel electrophoresis to detect the product size of RT-qPCR. Prepare 1% agarose gel, and mix 10 μl qPCR product with 2 μl loading dye. Load 10 μl mixture into each well in the 1% agarose gel, and run the gel by 100V for 30 min.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium	Gibco by Life technologies	12800-017 10 X 1 L	For wash the EECs pellets
D-MEM/F-12	Gibco by Life technologies	12400-024 10 X 1 L	As the basal medium in culturing EECs
NBCS	Gibco by Life technologies	16010-159	As the supplyment serum in culturing EECs
Pen-Strep Ampho. Solution	BI (Biological Industries)	03-033-1B 100 ml	For attenuating the possible infection
Collagenase Type IV	Gibco by Life technologies	17104-019 1 g	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.
24-well plate	FALCON®	REF-353047	For EECs to attach and extension
50 ml PP centrifuge tubes	Corning® CentriStarTM	430829	For transportion of membranes and enzyme digestion
50 ml Conical bottomed Tube with Cap	PRO TECH	CT-50-PL-TW	For transportion of membranes and enzyme digestion
Reciprocal shaking bath	DEAGLE	SB302	For better enzymatic digestion on membranes