Name: Video: In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane
Uploaded: 2019-11-28T14:00:00.000+00:00
Duration: 10 min
Description: 7.7K Views. Instituto Nacional de Perinatología. 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.

Our research was supported by grants from Instituto Nacional de Perinatolog&#237;a de M&#233;xico (21041 and 21081) and CONACYT (A1-S-8450 and&#160;252756). We thank Jessica Gonz&#225;lez Norris and Lidia Yuriria Paredes Vivas for the technical support.

This protocol was approved by the ethical committee of Instituto Nacional de Perinatolog&#237;a 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 Perinatolog&#237;a, the Helsinki Declaration, and the guidelines set forth in the Ministry of Health&#8217;s Official Mexican Standard.
1. Preparation
<ol>
	<li>Prepare a solution of 1x PBS with EDTA. To do so, add 500 &#956;L of 0.5 M EDTA stock into 500 mL of 1x PBS for a final concentration of 0.5 mM EDTA.</li>
	<li>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 &#956;L of mercaptoethanol (1,000x).</li>
	<li>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.</li>
	<li>Sterilize the surgical instruments (scalpels, scissors, forceps, and clamps), 500 mL beakers, cotton gauzes, and saline solution.</li>
</ol>
2. Obtaining placental tissue
NOTE: The amniotic membranes were obtained from women at full-term gestation (37&#8722;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.
<ol>
	<li>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.</li>
	<li>Add 500 mL of saline solution to the container to hydrate the placenta.</li>
	<li>Close the container and transport the tissue to the laboratory at room temperature.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Remove the whole placenta from the container and place it on the tray with the umbilical cord facing upward.</li>
	<li>Using a sterile cotton gauze, clean the blood clots from the surface of the chorion-amnion that covers the placenta.</li>
	<li>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).</li>
	<li>Dissect the umbilical amnion region (Figure 2A).
	<ol>
		<li>Using dissecting forceps, hold the portion of amnion membrane that covers the junction of the placenta and umbilical cord.</li>
		<li>With a scalpel, dissect the region that surrounds the cord while stretching to separate it from the chorion.</li>
		<li>Deposit the separated tissue in a labeled beaker with 100 mL of saline solution.</li>
	</ol>
	</li>
	<li>Dissect the placental amnion region (Figure 2B).
	<ol>
		<li>With a sterile cotton gauze, remove the blood clots from the surface of the chorion-amnion that covers the placenta.</li>
		<li>Hold the membrane on the border between the placenta and the reflected region with dissecting forceps.</li>
		<li>Cut along the circumference of the placenta with the scalpel.</li>
		<li>Separate the placental amnion from the chorion, being careful not to cut any vessels from the placenta.</li>
		<li>Place the separated tissue in another labeled beaker with 300 mL of saline solution.</li>
	</ol>
	</li>
	<li>Separate the rest of the amniotic membrane that is not attached to the placenta (i.e., the reflected portion) from the chorion (Figure 2C).
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Washing the membranes
NOTE: The procedure must be carried out within a biosecurity cabinet under sterile conditions at room temperature.
<ol>
	<li>Discard the saline solution of each membrane region separately.</li>
	<li>Add 100 mL of fresh saline solution to the umbilical region.</li>
	<li>Add 300 mL of fresh saline solution to the placental and reflected regions respectively.</li>
	<li>Stir&#160;the membranes with the help of dissecting forceps to remove blood residue.</li>
	<li>Discard the saline solution.</li>
	<li>Repeat the washes and agitation at least 3x until the membranes are translucent.</li>
	<li>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.</li>
</ol>
5. Enzymatic digestion of the membranes from different regions
NOTE: The procedure must be carried out within a biosecurity cabinet under sterile conditions.
<ol>
	<li>Cut the reflected and placental regions into two or three fragments.</li>
	<li>Do not cut the umbilical region.</li>
	<li>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.</li>
	<li>Shake the centrifuge tubes lightly for 30 s. Discard the trypsin.</li>
	<li>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.</li>
	<li>Place the tubes in a rotator inside the incubator.</li>
	<li>Incubate with rotation (20 rpm) for 40 min at 37 &#176;C. 
	NOTE: If a tube rotator is not available, shake the tubes lightly manually every 10 min.</li>
	<li>Transfer the trypsin/cell solutions from each region into new centrifuge tubes.</li>
	<li>Add 2x the volume of HAEC media prewarmed at 37 &#176;C per tube to inactivate the enzyme.</li>
	<li>Store the first digestion on ice.</li>
	<li>Repeat steps 5.6&#8722;5.8 for a second digestion period.</li>
	<li>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.</li>
	<li>Collect the second digestion into another set of centrifuge tubes and inactivate with 2x the volume of HAEC media.</li>
	<li>Discard the digested membranes into a biohazard container.</li>
</ol>
6. Isolation of the HAEC
NOTE: The procedure must be carried out within a biosecurity cabinet under sterile conditions.
<ol>
	<li>Centrifuge all tubes at 200 x g for 10 min at 4 &#176;C.</li>
	<li>Discard the supernatant and add 10 mL of prewarmed HAEC media (to 37 &#176;C) per tube and pipet gently to disaggregate each pellet.</li>
	<li>Combine the cellular suspensions of the two digestions in an individual tube for each membrane region.</li>
	<li>Filter the cellular suspensions using 100 &#956;m cell strainers to remove the extracellular matrix debris and obtain single cells.</li>
	<li>Prepare three aliquots with 90 &#181;L of trypan blue in a microcentrifuge tube.</li>
	<li>Add 10 &#956;L of each cell suspension per membrane region into the microcentrifuge tubes and mix.</li>
	<li>Count the cells with a hemocytometer under a light field microscope.</li>
</ol>
7. Culture of HAEC
<ol>
	<li>Seed the HAEC from the three regions separately at a density of 3 &#215; 104 cells/cm2 with prewarmed HAEC media, supplemented with 10 ng/mL of human epidermal growth factor (EGF).
	<ol>
		<li>Seed the cells in 100 mm plates to maintain them in vitro or in 24 well plates for immunochemical analysis.</li>
	</ol>
	</li>
	<li>Incubate the dishes at 37 &#176;C under normoxic conditions (5% CO2) in a humidified incubator.</li>
	<li>Add EGF daily and change the medium every third day. 
	NOTE: The cells will become confluent after 4&#8722;6 days.</li>
	<li>Use the cells for conventional immunohistochemistry, cell sorting analysis, cryopreserving, RNA and protein extraction, or to continue the passage.</li>
</ol>
8. Passage of HAEC
<ol>
	<li>Remove the HAEC medium and wash 2x with PBS/EDTA 0.01M solution.</li>
	<li>Incubate with a PBS/EDTA 0.01M solution for 15 min at 37 &#176;C.</li>
	<li>Remove the PBS/EDTA and add 1.5 mL of 0.5% trypsin/EDTA.</li>
	<li>Incubate for 5&#8722;8 min at 37 &#176;C.</li>
	<li>Inactivate the enzyme with two volumes of HAEC media.</li>
	<li>Collect the mixed solution and centrifuge for 5 min at 200 x g.</li>
	<li>Count the cells and continue with following passages or use the cells for further analysis.</li>
</ol>

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The authors have nothing to disclose.

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 with excessive erythrocytes and few adherent epithelial cells. If no attachment is observed in the first 24&#8722;48 h after seeding the cells, we recommend discarding the culture. Another critical point is the incubation time for the enzymatic digestion. If the incubation period is too long, cell viability may decrease and/or cell cultures with mesenchymal instead of epithelial morphology may be obtained (Figure 4). Also, if membrane digestion is not done with the proper agitation, the probability of obtaining a low number of cells per membrane increases.
In this protocol, we can maintain populations of HAEC in vitro without losing their epithelial phenotype, as demonstrated by the presence of specific epithelial cell marker E-cadherin for most cells along the passages (Figure 6). However, in our experience the HAEC can be only be expanded for three passages (P1&#8722;P3). The limited number of passages should be taken into consideration for experiments that require long periods of culture or multiple passages. In addition, it has been reported that after P3 the cells begin to change their morphology and reduce the expression of E-cadherin, so the prolonged culture could induce epithelial-mesenchymal transition (EMT)18. Recently, it was demonstrated that progesterone prevents EMT in ovine amniotic epithelial cells19,20, so it would be interesting to analyze the effect of different concentrations of progesterone in the HAEC culture medium to avoid the mesenchymal phenotype after the third passage.
In all previous reports where HAEC were isolated, the amnion has been assumed to be a uniform tissue despite the possible heterogeneity of epithelial populations that comprise the whole membrane. In contrast, this protocol divides the membrane into its three anatomical areas to separately isolate and culture HAEC considering the previous morphological and gene expression reports that suggest functional differences. It is also unnecessary to purify through cell sorting to obtain only epithelial populations, as demonstrated by detection of the E-cadherin marker during passages until P2.
It has been shown that isolated HAEC from each of the membrane regions have a similar expression of pluripotency core, being a latent reservoir of cells with stemness. In this context, these cells can be used in vitro to study molecular mechanisms, such as the dynamic localization of pluripotency-related transcription factors or their ability to remain quiescent despite expressing cancer-associated genes, regardless of their anatomical region of origin. However, future research must consider molecular differences reported (global expression genes, metabolic activity, signaling pathways) between each region, which would have repercussions for their application in regenerative medicine. We previously reported a different expression of genes involved in PI3K/AKT and focal adhesion signaling pathways between the different amniotic regions by RNA-seq6. PI3K/AKT regulates self-renewal and maintains pluripotency in HPSC, while activation of focal adhesion kinase promotes their differentiation21,22. Thus, we propose to characterize the role of both pathways in HAEC isolated from different anatomical regions during lineage-specific differentiation protocols. In addition, several studies demonstrate the differences between both regions regarding cellular components, molecular function, and biological processes. For example, the region-specific gene expression and protein distribution of aquaporins, which are involved in the transport process of intramembranous absorption, have been reported23. Another study compared the miRNome by microarrays, showing region-specific expression of miR-145 and miR-143, the latter being able to posttranscriptionally regulate the prostaglandin-endoperoxidase synthase 2 under specific conditions such as labor at term16. Moreover, the placental region presents higher mitochondrial respiration but lower reactive oxygen species detection as compared with reflected tissue15. Dissecting the amnion in reflected and placental regions is encouraged to isolate HAEC and analyze their peculiar properties separately, such as the release of growth factors and cytokines, their low immunogenicity, production of extracellular matrix, the effect of their conditioned medium in coculture with other cell types, and novel functions such as their capacity to maintain HPSC lines as a feeder layer24.

Human amniotic epithelium&#160;cells&#160;(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&#160;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&#160;research groups have developed methods to isolate&#160;these cells from the amniotic membrane at the term of gestation to characterize their presumptive pluripotency-related properties in a culture model&#160;in vitro3,4.&#160;
Accordingly, it&#160;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-renewing5,6,7. 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 HPSC5,9.
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 molecules10,11, 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.

<table><tbody><tr><td>&lt;strong&gt;Culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Thermo Fisher Scientific/Gibco</td><td>21985023</td><td>55 mM</td></tr><tr><td>Animal-Free Recombinant Human EGF</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Thermo Fisher Scientific/Gibco</td><td>15240062</td><td>100X</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Thermo Fisher Scientific/Gibco</td><td>12430054</td><td>Supplemented with high glucose and HEPES</td></tr><tr><td>EDTA</td><td>Thermo Fisher Scientific/Ambion</td><td>AM9260G</td><td>0.5 M</td></tr><tr><td>Embryonic stem-cell FBS, qualified</td><td>Thermo Fisher Scientific/Gibco</td><td>10439024</td><td></td></tr><tr><td>Non-Essential Amino Acids</td><td>Thermo Fisher Scientific/Gibco</td><td>11140050</td><td>100X</td></tr><tr><td>Paraformaldehyde</td><td>any brand</td><td></td><td></td></tr><tr><td>Phosphate-Buffered Saline</td><td>Thermo Fisher Scientific/Gibco</td><td>10010023</td><td>1X</td></tr><tr><td>Saline solution (sodium chloride 0.9%)</td><td>any brand</td><td></td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Thermo Fisher Scientific/Gibco</td><td>11360070</td><td>100 mM</td></tr><tr><td>Trypsin/EDTA 0.05%</td><td>Thermo Fisher Scientific/Gibco</td><td>25300054</td><td></td></tr><tr><td>&lt;strong&gt;Disposable material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>100 &amp;micro;m Cell Strainer</td><td>Corning/Falcon</td><td>352360</td><td></td></tr><tr><td>100 mm TC-Treated Culture Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>24-well Clear TC-treated Multiple Well Plates</td><td>Corning/Costar</td><td>3526</td><td></td></tr><tr><td>6-well Clear TC-treated Multiple Well Plates</td><td>Corning/Costar</td><td>3516</td><td></td></tr><tr><td>Non-Pyrogenic Sterile Centrifuge Tube</td><td>any brand</td><td></td><td>with conical bottom</td></tr><tr><td>Non-Pyrogenic sterile tips of 1,000 &amp;micro;l, 200 &amp;micro;l and 10 &amp;micro;l.</td><td></td><td></td><td></td></tr><tr><td>Sterile cotton gauzes</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes of 5, 10 and 25 mL</td><td>any brand</td><td></td><td></td></tr><tr><td>Sterile surgical gloves</td><td>any brand</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biological safety cabinet</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Micropipettes</td><td></td><td></td><td></td></tr><tr><td>Motorized Pipet Filler/Dispenser</td><td></td><td></td><td></td></tr><tr><td>Sterile beakers of 500 mL</td><td></td><td></td><td></td></tr><tr><td>Sterile plastic cutting board</td><td></td><td></td><td></td></tr><tr><td>Sterile scalpels, scissors, forceps, clamps</td><td></td><td></td><td></td></tr><tr><td>Sterile stainless steel container</td><td></td><td></td><td></td></tr><tr><td>Sterile tray</td><td></td><td></td><td></td></tr><tr><td>Tube Rotator</td><td>MaCSmix</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibodies and Kits&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Antibody ID&lt;/strong&gt;</td></tr><tr><td>Anti-E-cadherin</td><td>BD Biosciences</td><td>610181</td><td>RRID:AB_3975</td></tr><tr><td>Anti-KI67</td><td>Santa Cruz</td><td>23900</td><td>RRID:AB_627859)</td></tr><tr><td>Anti-NANOG</td><td>Peprotech</td><td>500-P236</td><td>RRID:AB_1268274</td></tr><tr><td>Anti-OCT4</td><td>Abcam</td><td>ab19857</td><td>RRID:AB_44517</td></tr><tr><td>Anti-SOX2</td><td>Millipore</td><td>AB5603</td><td>RRID:AB_2286686</td></tr><tr><td>Anti-SSEA-4</td><td>Cell Signaling</td><td>4755</td><td>RRID:AB_1264259</td></tr><tr><td>Anti-TRA-1-60</td><td>Cell Signaling</td><td>4746</td><td>RRID:AB_2119059</td></tr><tr><td>Goat Anti-Mouse Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-11029</td><td>RRID:AB_2534088</td></tr><tr><td>Goat Anti-Rabbit Alexa Fluor 568</td><td>Thermo Fisher Scientific</td><td>A-11036</td><td>RRID:AB_10563566</td></tr><tr><td>Tunel Assay Kit</td><td>Abcam</td><td>66110</td><td></td></tr></tbody></table>

in vitro culture of epithelial cells from different anatomical regions of the human amniotic membrane

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.

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 experimental data analysis (Figure 4): it is advisable to discard the cultures and process another membrane upon identifying the presence of bacteria due to contamination of the reagents or during the isolation process (Figure 4A); excessive erythrocytes due to insufficient washing of the membranes (Figure 4B); deficient or no adhesion of the cells to the plates (Figure 4C); or cells with fibroblast morphology (Figure 4D), suggesting that human amniotic mesenchymal cells (HAMC) were isolated instead of HAEC.
HAEC morphology depends on the origin of these cells: cells from the reflected zone have a cuboidal morphology and grow in a cobbled monolayer, unlike cells from the placental and umbilical regions, which are flatter and squamous (Figure 5). These data support that the epithelial layer from the amnion is not uniform throughout the membrane. During the passages, the size of the cells from all regions increases, but they maintain their epithelial nature and do not acquire a fibroblast morphology. Indeed, immunofluorescence against E-cadherin showed that the primary cultures (P0) and subcultures (P1-P2) maintained their epithelial phenotype (Figure 6), suggesting that there is no contamination of another cell type, such as mesenchymal stromal cells, or epithelial-mesenchymal transition. In addition, these cells show no evidence of cell death according to the TUNEL assay (Supplementary Figure 1) but are positive for the KI-67 proliferation marker (Figure 6), although our previous results found no significant differences for this marker between each passage5.
The number of cells obtained varies according to the region: 61.6 x 106 and 71.8 x 106 cells from the reflected and placental regions, respectively, and less than 1 x 106 per sample from the umbilical region (Table 1). It has been reported with this protocol that placental and umbilical region are very similar in their expression profiles, as opposed to the reflected and placental regions, especially in genes that participate in ECM receptor interaction, focal adhesion, and the PI3K-Akt signaling pathway through RNA-seq6. In agreement, a previous study reported the differential expression of mitogen-activated protein kinase and transforming growth factor beta pathways, as well as proinflammatory cytokines between both regions17.
Although the evidence showed that the subpopulations from the amnion differ in their morphology and physiological properties, we previously demonstrated that the expression and presence of the core of the pluripotency factors does not change in HAEC derived from placental and reflected regions6. In this context, in addition to the relatively limited number of cells from the umbilical region, subsequent studies should focus on isolated HAEC from the placental and reflected amnion independently, considering the physiological implications, because the different subpopulations would not respond equally to specific events, such as prolonged pregnancy or inflammatory processes during labor, although there are no specific positive cells to the main pluripotency markers (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60551/60551fig01.jpg" /> 
Figure 1: Anatomical regions from the amniotic membrane at term.&#160;Umbilical amniotic membrane (yellow), placental amniotic membrane (white), and reflected amniotic membrane (black). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60551/60551fig02.jpg" /> 
Figure 2: Mechanical dissection of the amniotic membrane.&#160;(A) Umbilical region, (B) placental region, and (C) reflected region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60551/60551fig03.jpg" /> 
Figure 3: Representative primary culture of HAEC derived from the amniotic membrane.&#160;Culture 48 h after isolation without (A) and with (B) a change of media. Scale bars = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60551/60551fig04.jpg" /> 
Figure 4: Representative micrographs of negative results of primary culture of HAEC.&#160;(A) Excess of erythrocytes. (B) Bacterial contamination. (C) HAEC not adhered after 48 h of isolation. (D) Primary culture composed of cells with fibroblast morphology. Scale bars = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60551/60551fig05.jpg" /> 
Figure 5: Morphology of HAEC from different anatomical regions in vitro.&#160;Representative micrographs of confluent HAEC from reflected (upper panel), placental (middle panel), and umbilical (lower panel) regions cultured though P0&#8722;P2. Scale bars 200 = &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60551/60551fig06.jpg" /> 
Figure 6: Expression of E-cadherin and KI-67 in HAEC.&#160;Representative epifluorescence microscopy images of E-cadherin+ and KI-67+ HAEC from reflected (left panel) and placental (right panel) amnion cultured through P0&#8722;P2. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60551/60551fig07.jpg" /> 
Figure 7: HAEC from different anatomical regions display a pluripotency marker panel.&#160;Representative confocal microscopy images of double immunofluorescence amnion for NANOG with TRA-1-60, OCT4 with E-cadherin, and SOX2 with SSEA-4 in HAEC (P1) from reflected (upper panel) and placental (lower panel) regions. Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td># MEMBRANE</td>
			<td>REFLECTED</td>
			<td>PLACENTAL</td>
			<td>UMBILICAL</td>
			<td>COMPLETE MEMBRANE</td>
		</tr>
		<tr>
			<td>1</td>
			<td>75 X 106</td>
			<td>130 X 106</td>
			<td>0.2 X 106</td>
			<td>205.2 X 106</td>
		</tr>
		<tr>
			<td>2</td>
			<td>38.5 X 106</td>
			<td>52 X 106</td>
			<td>0.53 X 106</td>
			<td>91.03 X 106</td>
		</tr>
		<tr>
			<td>3</td>
			<td>59 X 106</td>
			<td>53 X 106</td>
			<td>0.2 X 106</td>
			<td>112.2 X 106</td>
		</tr>
		<tr>
			<td>4</td>
			<td>42 X 106</td>
			<td>27 X 106</td>
			<td>0.36 X 106</td>
			<td>69.36 X 106</td>
		</tr>
		<tr>
			<td>5</td>
			<td>44.8 X 106</td>
			<td>22.3 X 106</td>
			<td>NP</td>
			<td>76.1 X 106</td>
		</tr>
		<tr>
			<td>6</td>
			<td>100 X 106</td>
			<td>140 X 106</td>
			<td>1 X 106</td>
			<td>241 X 106</td>
		</tr>
		<tr>
			<td>7</td>
			<td>72 X 106</td>
			<td>78 X 106</td>
			<td>NP</td>
			<td>150 X 106</td>
		</tr>
		<tr>
			<td>AVERAGE</td>
			<td>61.6 X 106</td>
			<td>71.8 X 106</td>
			<td>0.45 X 106</td>
			<td>133.8 X 106</td>
		</tr>
	</tbody>
</table>
Table 1: Number of HAEC isolated per region from amniotic membranes. (NP = not processed).
Supplementary Figure 1: TUNEL staining in HAEC (passage 2) from reflected region and in positive control cells (HL-60 cells treated with camptothecin). Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60551/60551supfig01.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane at JoVE.com

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

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 ...

in-vitro-culture-epithelial-cells-from-different-anatomical-regions

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.7K Views.  Instituto Nacional de Perinatología. 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.

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

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Medicine

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioengineering

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Biology

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Advanced Two-Layered 3D Co-Culture System for Epithelial and Fibroblast Cells

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

Author Spotlight: Advancing 3D Coculture Systems with PVA-PCL Nanofibrous Membranes 

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...