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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.
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.
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 pres....
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
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.......
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 .......
Our research was supported by grants from Instituto Nacional de Perinatología de México (21041 and 21081) and CONACYT (A1-S-8450 and 252756). We thank Jessica González Norris and Lidia Yuriria Paredes Vivas for the technical support.
....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 |
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