In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Podsumowanie

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

Streszczenie

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

Wprowadzenie

Human amniotic epithelium cells (HAEC) originate during the early stages of embryonic development, at around eight days postfertilization. They arise from a population of squamous epithelial cells of the epiblast that derive from the innermost layer of the amniotic membrane¹. Thus, HAEC are considered remnants of pluripotent cells from the epiblast that have the potential to differentiate into the three germ layers of the embryo². In the last decade, diverse research groups have developed methods to isolate these cells from the amniotic membrane at the term of gestation to characterize their presumptive pluripotency-related properties in a culture model in vitro^3,4. 

Accordingly, it has been found that HAEC feature traits characteristic of human pluripotent stem cells (HPSC), such as the surface antigens SSEA-3, SSEA-4, TRA 1-60, TRA 1-81; the core of pluripotency transcription factors OCT4, SOX2, and NANOG; and the proliferation marker KI67, suggesting that they are self-renewing^5,6,7. Moreover, these cells have been challenged using differentiation protocols to obtain cells positive for lineage-specific markers of the three germ layers (ectoderm, mesoderm, and endoderm)^4,5,8, as well as in animal models of human diseases. Finally, HAEC express E-cadherin, which demonstrate that they retain an epithelial nature much like the HPSC^5,9.

Apart from their embryonic origin, HAEC have other intrinsic properties that make them suitable for different clinical applications, such as the secretion of anti-inflammatory and antibacterial molecules^10,11, growth factors and cytokine release¹², no formation of teratomas when they are transplanted into immunodeficient mice in contrast with HPSC², and immunological tolerance because they express HLA-G, which decreases the risk of rejection after transplantation¹³.

However, previous reports have assumed that the human amnion is a homogenous membrane, without considering that it can be anatomically and physiologically divided into three regions: placental (the amnion that covers the decidua basalis), umbilical (the part that envelops the umbilical cord), and reflected (the rest of the membrane not attached to the placenta)¹⁴. It has been shown that the placental and reflected regions of the amnion display differences in morphology, mitochondrial activity, detection of reactive oxygen species¹⁵, miRNA expression¹⁶, and activation of signaling pathways¹⁷. These results suggest that the human amnion is integrated by a heterogeneous population with different functionality that should be considered for further studies carried out in either in situ or in vitro models. While other laboratories have designed protocols for the isolation of HAEC from the whole membrane, our laboratory has established a protocol to isolate, culture, and characterize cells from different anatomical regions.

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Protokół

This protocol was approved by the ethical committee of Instituto Nacional de Perinatología in Mexico City (Registry number 212250-21041). All procedures performed in these studies were in accordance with the ethical standards of the Instituto Nacional de Perinatología, the Helsinki Declaration, and the guidelines set forth in the Ministry of Health’s Official Mexican Standard.

1. Preparation

1. Prepare a solution of 1x PBS with EDTA. To do so, add 500 μL of 0.5 M EDTA stock into 500 mL of 1x PBS for a final concentration of 0.5 mM EDTA.
2. Prepare the culture medium for HAEC. Take 450 mL of high glucose-DMEM, supplement it with 5 mL of sodium pyruvate (100 mM), 50 mL of heat-inactive fetal bovine serum qualified for stem cells, 5 mL of nonessential amino acids (100x), 5 mL of antibiotic-antimycotic (100x), 5 mL of L-glutamine (200 mM), and 500 μL of mercaptoethanol (1,000x).
3. Sterilize the containers for transporting and processing the tissue: a stainless steel container with a lid to transport the whole placenta from the operating theater to the laboratory, a tray (20 cm x 30 cm x 8 cm) to wash and remove blood from the whole placenta before the dissection of the amniotic membrane, and a plastic cutting board to separate the amniotic membrane into the three regions.
4. Sterilize the surgical instruments (scalpels, scissors, forceps, and clamps), 500 mL beakers, cotton gauzes, and saline solution.

2. Obtaining placental tissue

NOTE: The amniotic membranes were obtained from women at full-term gestation (37−40 weeks), under indication of Cesarean delivery, without any evidence of active labor, and no microbiological characteristics of infection. The complete isolation and culture procedures were carried out within a biosecurity cabinet under sterile conditions.

1. In the operating room, clamp the umbilical cord to prevent the blood flow to the rest of the tissue. Collect the entire placenta with the umbilical cord clamped in the sterile container.
2. Add 500 mL of saline solution to the container to hydrate the placenta.
3. Close the container and transport the tissue to the laboratory at room temperature.
4. Put the container with the placenta inside the biosecurity cabinet.
   NOTE: In case the dissection is not carried out within 15 min of collecting the placenta, store the container with the placenta on ice until processing. Avoid more than 1 h of elapsed time between obtaining the placenta and the start of the dissection.

3. Mechanical separation per region of the amniotic membrane

NOTE: The procedure must be carried out within a biosecurity cabinet under sterile conditions and at room temperature.

1. Remove the whole placenta from the container and place it on the tray with the umbilical cord facing upward.
2. Using a sterile cotton gauze, clean the blood clots from the surface of the chorion-amnion that covers the placenta.
3. Identify the three regions of the membrane: the umbilical amnion enveloping the umbilical cord, the placental amnion covering the decidua basalis, and the reflected region, which is the rest of amnion that is not attached to the placenta (Figure 1).
4. Dissect the umbilical amnion region (Figure 2A).
   1. Using dissecting forceps, hold the portion of amnion membrane that covers the junction of the placenta and umbilical cord.
   2. With a scalpel, dissect the region that surrounds the cord while stretching to separate it from the chorion.
   3. Deposit the separated tissue in a labeled beaker with 100 mL of saline solution.
5. Dissect the placental amnion region (Figure 2B).
   1. With a sterile cotton gauze, remove the blood clots from the surface of the chorion-amnion that covers the placenta.
   2. Hold the membrane on the border between the placenta and the reflected region with dissecting forceps.
   3. Cut along the circumference of the placenta with the scalpel.
   4. Separate the placental amnion from the chorion, being careful not to cut any vessels from the placenta.
   5. Place the separated tissue in another labeled beaker with 300 mL of saline solution.
6. Separate the rest of the amniotic membrane that is not attached to the placenta (i.e., the reflected portion) from the chorion (Figure 2C).
   1. Collect the reflected region in another labeled beaker with 300 mL of saline solution.
      NOTE: Add saline solution continually during the dissection to prevent tissue from drying out.

4. Washing the membranes

NOTE: The procedure must be carried out within a biosecurity cabinet under sterile conditions at room temperature.

1. Discard the saline solution of each membrane region separately.
2. Add 100 mL of fresh saline solution to the umbilical region.
3. Add 300 mL of fresh saline solution to the placental and reflected regions respectively.
4. Stir the membranes with the help of dissecting forceps to remove blood residue.
5. Discard the saline solution.
6. Repeat the washes and agitation at least 3x until the membranes are translucent.
7. Place and extend the membranes on the board to clean with sterile gauze the blood clots that were not removed with the washes.
   NOTE: It is very important to remove as many erythrocytes as possible, as their presence affects the trypsin function and the viability of subsequent cell cultures.

5. Enzymatic digestion of the membranes from different regions

NOTE: The procedure must be carried out within a biosecurity cabinet under sterile conditions.

1. Cut the reflected and placental regions into two or three fragments.
2. Do not cut the umbilical region.
3. Place the fragments of each region in centrifuge tubes. Add 20 mL of 0.5% trypsin/EDTA to the reflected and placental regions and 5 mL of 0.5% trypsin/EDTA to the umbilical region, respectively.
   NOTE: It is important to cut the reflected and placental regions into smaller pieces because they need to be completely immersed in the trypsin solution.
4. Shake the centrifuge tubes lightly for 30 s. Discard the trypsin.
5. Add 30 mL of new 0.5% trypsin/EDTA to the reflected and placental regions and 15 mL of new 0.5% trypsin/EDTA to the umbilical region, respectively.
6. Place the tubes in a rotator inside the incubator.
7. Incubate with rotation (20 rpm) for 40 min at 37 °C.
   NOTE: If a tube rotator is not available, shake the tubes lightly manually every 10 min.
8. Transfer the trypsin/cell solutions from each region into new centrifuge tubes.
9. Add 2x the volume of HAEC media prewarmed at 37 °C per tube to inactivate the enzyme.
10. Store the first digestion on ice.
11. Repeat steps 5.6−5.8 for a second digestion period.
12. For each region, hold one end of the amnion portion using dissecting forceps, and with another pair squeeze along the tissue to remove rows of epithelial cells that did not completely peel off during previous incubation periods.
13. Collect the second digestion into another set of centrifuge tubes and inactivate with 2x the volume of HAEC media.
14. Discard the digested membranes into a biohazard container.

6. Isolation of the HAEC

NOTE: The procedure must be carried out within a biosecurity cabinet under sterile conditions.

1. Centrifuge all tubes at 200 x g for 10 min at 4 °C.
2. Discard the supernatant and add 10 mL of prewarmed HAEC media (to 37 °C) per tube and pipet gently to disaggregate each pellet.
3. Combine the cellular suspensions of the two digestions in an individual tube for each membrane region.
4. Filter the cellular suspensions using 100 μm cell strainers to remove the extracellular matrix debris and obtain single cells.
5. Prepare three aliquots with 90 µL of trypan blue in a microcentrifuge tube.
6. Add 10 μL of each cell suspension per membrane region into the microcentrifuge tubes and mix.
7. Count the cells with a hemocytometer under a light field microscope.

7. Culture of HAEC

1. Seed the HAEC from the three regions separately at a density of 3 × 10⁴ cells/cm² with prewarmed HAEC media, supplemented with 10 ng/mL of human epidermal growth factor (EGF).
   1. Seed the cells in 100 mm plates to maintain them in vitro or in 24 well plates for immunochemical analysis.
2. Incubate the dishes at 37 °C under normoxic conditions (5% CO₂) in a humidified incubator.
3. Add EGF daily and change the medium every third day.
   NOTE: The cells will become confluent after 4−6 days.
4. Use the cells for conventional immunohistochemistry, cell sorting analysis, cryopreserving, RNA and protein extraction, or to continue the passage.

8. Passage of HAEC

1. Remove the HAEC medium and wash 2x with PBS/EDTA 0.01M solution.
2. Incubate with a PBS/EDTA 0.01M solution for 15 min at 37 °C.
3. Remove the PBS/EDTA and add 1.5 mL of 0.5% trypsin/EDTA.
4. Incubate for 5−8 min at 37 °C.
5. Inactivate the enzyme with two volumes of HAEC media.
6. Collect the mixed solution and centrifuge for 5 min at 200 x g.
7. Count the cells and continue with following passages or use the cells for further analysis.

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Wyniki

HAEC were isolated from each of the three anatomical regions of the amniotic membrane and individually cultured in vitro. After 48 h of culture, cells with an epithelial phenotype adhered to the surface of the plate, although the media also contained cell debris and floating cells, which were removed once the medium was changed (Figure 3).

During the processing of primary culture (passage zero, P0), some complications could arise that can interfere with the experi...

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Dyskusje

We implemented a new protocol to isolate HAEC from term membranes. It differs from previous reports in that each membrane was divided into its three anatomical regions prior to isolation to analyze cells from each one.

One of the most critical steps in the protocol is the washing of the membrane to remove all blood clots, because they can interfere with the activity of trypsin when separating the epithelial cells. Failure to carry out this step properly can lead to obtaining a primary culture ...

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Materiały

Name	Company	Catalog Number	Comments
Culture reagents
2-Mercaptoethanol	Thermo Fisher Scientific/Gibco	21985023	55 mM
Animal-Free Recombinant Human EGF	Peprotech	AF-100-15
Antibiotic-Antimycotic	Thermo Fisher Scientific/Gibco	15240062	100X
Dulbecco's Modified Eagle Medium	Thermo Fisher Scientific/Gibco	12430054	Supplemented with high glucose and HEPES
EDTA	Thermo Fisher Scientific/Ambion	AM9260G	0.5 M
Embryonic stem-cell FBS, qualified	Thermo Fisher Scientific/Gibco	10439024
Non-Essential Amino Acids	Thermo Fisher Scientific/Gibco	11140050	100X
Paraformaldehyde	any brand
Phosphate-Buffered Saline	Thermo Fisher Scientific/Gibco	10010023	1X
Saline solution (sodium chloride 0.9%)	any brand
Sodium Pyruvate	Thermo Fisher Scientific/Gibco	11360070	100 mM
Trypsin/EDTA 0.05%	Thermo Fisher Scientific/Gibco	25300054
Disposable material
100 µm Cell Strainer	Corning/Falcon	352360
100 mm TC-Treated Culture Dish	Corning	430167
24-well Clear TC-treated Multiple Well Plates	Corning/Costar	3526
6-well Clear TC-treated Multiple Well Plates	Corning/Costar	3516
Non-Pyrogenic Sterile Centrifuge Tube	any brand		with conical bottom
Non-Pyrogenic sterile tips of 1,000 µl, 200 µl and 10 µl.
Sterile cotton gauzes
Sterile serological pipettes of 5, 10 and 25 mL	any brand
Sterile surgical gloves	any brand
Equipment
Biological safety cabinet
Centrifuge
Micropipettes
Motorized Pipet Filler/Dispenser
Sterile beakers of 500 mL
Sterile plastic cutting board
Sterile scalpels, scissors, forceps, clamps
Sterile stainless steel container
Sterile tray
Tube Rotator	MaCSmix
Antibodies and Kits			Antibody ID
Anti-E-cadherin	BD Biosciences	610181	RRID:AB_3975
Anti-KI67	Santa Cruz	23900	RRID:AB_627859)
Anti-NANOG	Peprotech	500-P236	RRID:AB_1268274
Anti-OCT4	Abcam	ab19857	RRID:AB_44517
Anti-SOX2	Millipore	AB5603	RRID:AB_2286686
Anti-SSEA-4	Cell Signaling	4755	RRID:AB_1264259
Anti-TRA-1-60	Cell Signaling	4746	RRID:AB_2119059
Goat Anti-Mouse Alexa Fluor 488	Thermo Fisher Scientific	A-11029	RRID:AB_2534088
Goat Anti-Rabbit Alexa Fluor 568	Thermo Fisher Scientific	A-11036	RRID:AB_10563566
Tunel Assay Kit	Abcam	66110