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

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 ...

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 experi...

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 ...

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Research

JoVE Journal

Developmental Biology

7.8K Views.  Instituto Nacional de Perinatología. Dit protocol kan helpen bij het beantwoorden van belangrijke vragen over heterogeniteit en functionele eigenschappen van menselijke vruchtwaterpitheelcellen geïsoleerd van vruchtwatermembraan. Terwijl eerdere protocollen cellen geïsoleerdn van co-vruchtwatermembraan zonder onderscheid tussen regio's, ons protocol maakt de cultuur van de celpopulatie geïsoleerd van drie verschillende anatomische regio's. Om het vruchtwatermembraan per regio te scheiden, in een bioveiligheidskast onder steri...

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 ...

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

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 ...

Biology

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 ...

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

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