The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

NOTE: Placentae should be collected from singleton healthy pregnancies, with a preference for term elective caesarean sections. Written, informed consent should be given for the collection of their placenta. Your relevant human research ethics committee should approval all collection and use of human tissues.
1. Isolation of Amnion Epithelial Cells
<ol style="margin-left: 40px;">
	<li>Place the placenta onto a sterile surface within a class II biological safety cabinet.</li>
	<li>Using sterile hanks balanced salt solution (HBSS), wash as much blood as possible from the placenta surface and membranes.</li>
	<li>With the placenta placed with the umbilical cord facing up, mechanically separate the outer edge of the amnion and chorion membranes.</li>
	<li>Once separated, manually strip the amnion membrane from the chorion membrane of the placenta, working around the edge of the membrane, towards the umbilical cord. 
	NOTE: Be careful to remove any pieces of contaminating chorion membrane from the amnion.</li>
	<li>Using sterile scissors, cut the amnion membrane approximately 2 cm from the base of the umbilical cord and place in a 500 ml sealed container containing 250 ml HBSS.</li>
	<li>Wash thoroughly by shaking the sealed bottle containing the amnion membrane.</li>
	<li>Remove the amnion membrane from the collection container using sterile forceps, and place into a 15 cm Petri dish.</li>
	<li>Cut the amnion membrane into pieces approximately 5 cm long (when held vertically with forceps), discarding bloody or torn pieces.</li>
	<li>Place in a 500 ml sealed container and wash with 250 ml HBSS approximately 3-5 times or until the amnion membrane becomes translucent with no contaminating blood. 
	NOTE: Any blood present may reduce the efficiency of the enzymatic digest solution.</li>
	<li>Transfer all pieces of amnion membrane into a container containing 50 ml of the enzymatic digest solution, taking care to minimize carryover of HBSS.</li>
	<li>Incubate the amnion pieces in&#160;enzymatic digest solution in a water bath or warm room at 37 &#176;C for 15 min with gentle agitation to remove any remaining blood.</li>
	<li>Remove the amnion membrane pieces from the enzymatic digest solution and place in a new sterile container.</li>
	<li>Add 100 ml of fresh enzymatic digest solution to the amnion membrane and incubate at 37 &#176;C for 60 min&#160;in a water bath or warm room with gentle shaking (first digestion).</li>
	<li>Remove the amnion membrane pieces and place into new container taking care to allow the enzymatic digest solution to drain from each piece of membrane. 
	NOTE: This can be achieved by dragging amnion pieces along the inside rim of the container.</li>
	<li>Add 100 ml of fresh enzymatic digest solution to the amnion membrane pieces and incubate 60 min&#160;in a water bath or warm room at 37 &#176;C with gentle shaking (second digestion).</li>
	<li>Pass the cell suspension from Step 1.14 through a 70 &#181;m filter and centrifuge at 1,000 x g for 10 min.</li>
	<li>Remove and discard the supernatant, and resuspend the cell pellet in 4 ml HBSS containing 1 mg/ml soybean-based enzyme inhibitor. Gently pipette repeatedly with a 1 ml pipette to obtain a single cell suspension. 
	NOTE: At this stage cells can be left on ice until cells from the second digest are at the same stage of processing.</li>
	<li>Remove the amnion membrane pieces and place into new container taking care to allow the enzymatic digest solution to drain from each piece of membrane. 
	NOTE: This can be achieved by dragging amnion pieces along the inside rim of the container.</li>
	<li>Pass the cell suspension from Step 1.18 through a 70 &#181;m filter and centrifuge at 1,000 x g for 10 min.</li>
	<li>Remove and discard the supernatant, and resuspend the cell pellet in 4 ml&#160;HBSS containing 1 mg/ml&#160;soybean-based enzyme inhibitor. Gently pipette repeatedly with a 1 ml pipette to obtain a single cell suspension.</li>
	<li>Pool cells from the two digests or alternatively keep cell digests separated until after cell counts. 
	NOTE: This prevents mixing of potentially low viability cells with high viability cells.</li>
	<li>Add HBSS to a final volume of 10-20 ml and perform cell counts using trypan blue to measure viability.</li>
	<li>Confirm the epithelial cell phenotype by flow cytometry. Make up appropriate antibody cocktails using anti-EpCAM-PE (1:2 dilution) anti-CD90-PeCy5 (1:250) and anti-CD105-APC (1:100) in HBSS.</li>
	<li>Resuspend a subset of cells from one or both digests in the antibody cocktail at a concentration of 25 x 106 cells/ml.</li>
	<li>For flow cytometry, stain 1 x 106 cells with antibody cocktails containing isotype controls (eg. IgG1 isotype control-PE + anti-CD90-PeCy5 + anti-CD105-APC). Keep ~1 x 106 cells unstained for flow cytometer set-up.</li>
	<li>Incubate cells in antibody cocktails at 4 &#176;C for 60 min.</li>
	<li>Wash cells 3x in HBSS and resuspend at 5 to 10 million cells per ml for flow cytometry.</li>
	<li>Perform flow cytometry and note results. The purity of the cell isolate (the percentage of EpCAM positive and CD90/CD105 negative cells) is expected to be 90-95% and &#60;1% respectively.</li>
	<li>Set aside a suitable number of cells for testing for appropriate quality control or release criteria as determined by final application. 
	NOTE: This can include disease/viral screening, or other safety or biological criteria.</li>
	<li>For the remaining cells, follow the cryopreservation protocol, or alternatively place directly into culture (direct to culture step 3.6).</li>
</ol>
2. Cryopreservation of hAECs
<ol style="margin-left: 40px;">
	<li>Determine cell number and viability using flow cytometry data or manual counting with trypan blue exclusion.</li>
	<li>Centrifuge at 700 x g for 5 min.</li>
	<li>Resuspend cells between 1-10 million cells per ml in animal product-free cryopreservation media.</li>
	<li>Pipette a suitable volume of cells into O-ringed cryopreservation vials and place into a controlled rate freezing apparatus where cooling occurs at approximately 1&#176;C/min&#160;until -80&#176;C is achieved.</li>
	<li>Transfer vials into liquid nitrogen for long-term storage.</li>
</ol>
3. Thawing and Culture of Cyropreserved hAECs
<ol style="margin-left: 40px;">
	<li>Remove cryopreservation vials from liquid nitrogen and place on ice. 
	NOTE: For transfer only - proceed rapidly to next step.</li>
	<li>Thaw the cryopreservation vials rapidly at 37 &#176;C until only a small piece (~5 mm x 5 mm) of ice remains. 
	NOTE: It is important that the cell solution does not warm to reduce the toxic effects of DMSO in the cryopreservation media.</li>
	<li>Dilute cryopreservation media 10-fold with cold (~4 &#176;C) serum-free media, added drop-wise in a suitable sized tube or container.</li>
	<li>Centrifuge at 700 x g for 5 min&#160;at RT.</li>
	<li>Resuspend in serum-free medium, to an approximate concentration of 1 million cells per ml. Determine cell number and viability using automated or manual counting with trypan blue exclusion. Expected viability post-thaw will be 5-10% less than pre-cryopreservation viability.</li>
	<li>Plate cells on standard cell culture-treated plastic ware, or alternatively use Type I collagen to coat culture surfaces.</li>
	<li>For Type I Collagen coating follow the protocol below
	<ol>
		<li>Prepare acid soluble Type I Collagen from human placenta (15 mg collagen with 25 ml deionized water and 50 &#181;l glacial acetic acid).</li>
		<li>Cover beaker with Parafilm and stir moderately at 37 &#176;C until collagen strands are dissolved.</li>
		<li>Sterile filter collagen solution using a 0.22 &#181;m filter.</li>
		<li>Dilute the filtered collagen stock 1:10 with deionized water. This diluted stock is the working concentration solution for coating plastic and membrane surfaces.</li>
		<li>Collagen coat plastic, glass and membrane surfaces for 2 hr at RT at 37&#176;C, or O/N at 2-8 &#176;C.</li>
		<li>Remove excess fluid from the coated surface, and allow it to dry O/N.</li>
		<li>Rinse with sterile tissue culture grade water or HBSS before introducing cells and medium.</li>
	</ol>
	</li>
	<li>Plate hAECs at a density of 25,000 cells/cm2 in serum-free medium, and allow attachment to occur for 72-96 hr.</li>
	<li>Following attachment, change media by carefully aspirating and replacing with a suitable volume of serum-free medium every 2-3 days.</li>
	<li>Passage cells using the protocol below
	<ol>
		<li>Aspirate cell culture media and wash with 1x volume of sterile HBSS</li>
		<li>Add 1x volume of enzymatic digest solution and incubate for 5-15 min, or until cells are observed detaching from the cell culture surface.</li>
		<li>Collect enzymatic digest solution containing cells, ensuring cells are detached from the cell culture surface by repeatedly pipetting the enzymatic digest solution against the cell culture surface</li>
		<li>Centrifuge at 700 x g for 5 min.</li>
		<li>Resuspend the cell pellet in a suitable volume of HBSS containing 1 mg/ml soybean-based enzyme inhibitor. Gently pipette repeatedly with a 1ml pipette to obtain a single cell suspension.</li>
		<li>Add serum-free medium to an approximate concentration of 1 million cells/ml. Determine cell number and viability using automated or manual counting with trypan blue exclusion.</li>
		<li>Plate hAECs at a density of 25,000 cells/cm2 in serum-free medium, and continue with standard culturing methods.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

There are several critical parameters that can have a significant impact in the success of this methodology. Storage of the placenta or amnion for up to 3 hr before isolation of hAECs may be desirable for logistic or scheduling purposes, however it is recommended that the tissue is processed as soon as possible. If tissue is to be stored, it is recommended that storage be performed following dissection and washing of the amnion membrane. Amnion can be stored in sterile HBSS containing antibiotics at 4 &#176;C, however ce...

Cells derived from perinatal sources, such as the placenta, placental membranes, umbilical cord and amniotic fluid have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine1,2. The reason for this interest is that these cell types all possess some degree of plasticity and immunomodulatory capability3, properties that are fundamental to their potential therapeutic applications.
hAECs are a heterogeneous epithelial population that can be derived from term or pre-term amnion membrane4, providing an abundant potential source of regenerative cellular material. The properties that make hAECs appealing as a cellular therapy include their multipotency, low immunogenicity, and anti-inflammatory properties. hAECs have been found to be highly multipotent both in vitro and in vivo, capable of differentiating into mesodermal lineages (cardiomyocytes, myocytes, osteocytes, adipocytes), endodermal lineages (pancreatic cells, hepatic cells, lung cells) and ectodermal lineages (hair, skin, neural cells and astrocytes)5-10.
Reassuringly, despite their multipotency hAECs do not appear to either form tumors or promote tumour development in vivo. Furthermore, hAECs are also immune privileged, expressing low levels of class II human leukocyte antigens (HLAs)8. This property likely underlies their ability to evade immune rejection after allogeneic and xenogenic transplantation, as demonstrated in studies using immune competent monkeys, rabbits, guinea pigs, rats, and pigs11-13. hAECs display potent immunomodulatory and immunosuppressive properties and thus offer significant practical advantages for potential clinical applications in autoimmune disease therapy. hAECs are believed to exert immunomodulatory functions on both the innate and adaptive immune systems. One of the mechanisms suggested, is through the secretion of immunomodulatory factors14.
Current applications of hAECs in pre-clinical animal disease models include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Researchers have shown interest in using hAECs to treat post-stroke brain inflammation due to their unique properties. There is evidence that hAECs can cross the blood brain barrier where they can engraft, survive for up to 60 days, differentiate into neurons, decrease inflammation and promote regeneration of damaged central nervous system tissue in animal models of neurological diseases15.
hAECs offer the ability to target and reverse multiple pathological pathways that contribute to the development and progression of multiple sclerosis. For example, results from pre-clinical animal studies suggest that hAECs are strongly immunosuppressive and can potentially induce peripheral immune tolerance and reverse ongoing inflammatory responses. hAECs have also been shown to have the capacity to differentiate into neural cells in vivo and enhance endogenous neuroregeneration through the secretion of a vast array of neurotrophic factors16.
Human and rodent amnion epithelial cells have already demonstrated their therapeutic efficacy for the treatment of liver disease in animal models. In a carbon tetrachloride damage induction model of liver disease, hAEC transplantation lead to engraftment of viable hAECs in the liver, accompanied with reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis17.
hAECs can be stimulated to expressed pancreatic factors including insulin and glucose transporters. Several studies have investigated the potential for hAECs to restore blood glucose levels in diabetic mice18. In mice receiving hAECs, both animal body weight and blood glucose levels decreased to normal levels following injection of cells. These studies present a strong case for the use of hAECs for the treatment of diabetes mellitus.
hAECs have a proven role in the prevention and repair of experimental acute and chronic lung injury in both adult and neonatal models19. These studies found that hAECs differentiate in vitro into functional lung epithelial cells expressing multiple lung-associated proteins, including Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the ion channel that is mutated in patients with cystic fibrosis20. Additionally, when hAECs are delivered to the injured adult and neonatal lung, they exert their reparative effects via the modulation of host immune cells, reducing pulmonary leucocyte recruitment, including neutrophils, macrophages and lymphocytes21-23.
Given their abundance, safety record, and proven clinical applications for multiple diseases, clinical trials using hAECs is inevitable. With the goal of accelerating the translation of hAEC therapies into clinical-trials, we developed methods to isolate, cryopreserve and culture hAECs in a manner suitable for clinical trials, using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.
We based this protocol a previously published protocol that we were using successfully to isolate hAECs using animal-derived reagents6. We altered the original protocol to replace animal-derived products with animal product-free reagents, and subsequent optimization was performed to optimize cell yield, viability and purity. Our goal was to develop a protocol that would comply with regulatory standards for cell manufacturing for human clinical trials.

<table><tbody><tr><td>Collection Kit</td><td></td><td></td><td></td></tr><tr><td>Stripping tray</td><td>Fisher Scientific</td><td>13-361B</td><td></td></tr><tr><td>Liberty Dressing Forcep, pointed 13 cm</td><td>Fisher Scientific</td><td>S17329&amp;nbsp;</td><td></td></tr><tr><td>Scissors - Sharp/Blunt Straight</td><td>Fisher Scientific</td><td>NC0562592&amp;nbsp;</td><td></td></tr><tr><td>Sterile latex gloves&amp;nbsp;</td><td>Fisher Scientific</td><td>19-014-643&amp;nbsp;</td><td></td></tr><tr><td>Protective Apparel</td><td></td><td></td><td></td></tr><tr><td>Protective Apparel (Gown)</td><td>U-line</td><td>S-15374-M</td><td></td></tr><tr><td>Isolation gowns</td><td>U-line</td><td>S-15374-M</td><td></td></tr><tr><td>Sterile latex gloves&amp;nbsp;</td><td>Fisher Scientific</td><td>19-014-643&amp;nbsp;</td><td></td></tr><tr><td>General purpose face mask</td><td>Cardinal Health</td><td>AT7511</td><td></td></tr><tr><td>Bonnets</td><td>Medline</td><td>CRI1001</td><td></td></tr><tr><td>Shoe covers</td><td>U-line</td><td>S-7873W</td><td></td></tr><tr><td>Media and Reagents</td><td></td><td></td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14175095</td><td>without calcium or magnesium</td></tr><tr><td>TrypZean (animal product&amp;ndash;free recombinant trypsin)</td><td>Sigma Aldrich</td><td>T3449</td><td></td></tr><tr><td>Soybean Trypsin Inhibitor&amp;nbsp; 1g/50mL</td><td>Sigma Aldrich</td><td>T6522</td><td></td></tr><tr><td>Cryostor CS5</td><td>BioLife Solutions</td><td>205102</td><td></td></tr><tr><td>Trypan blue reagent</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Anti-EpCam-PE</td><td>Miltenyi Biotec</td><td>130 - 091-253</td><td></td></tr><tr><td>PE-isotype control</td><td>Miltenyi Biotec</td><td>130-098-845</td><td></td></tr><tr><td>Anti-CD90-PeCy5</td><td>BD Pharmingen</td><td>555597</td><td></td></tr><tr><td>PeCy5-isotype control</td><td>BD Pharmingen</td><td>557224</td><td></td></tr><tr><td>Anti-CD105-APC</td><td>BD Pharmingen</td><td>562408</td><td></td></tr><tr><td>APC-isotype control</td><td>BD Pharmingen</td><td>340754</td><td></td></tr><tr><td>Collagen Type VI</td><td>Sigma Aldrich</td><td>C7521</td><td></td></tr><tr><td>Consumables</td><td></td><td></td><td></td></tr><tr><td>50 ml graduated pipette</td><td>BD/Falcon</td><td>356550</td><td></td></tr><tr><td>10 ml graduated pipette</td><td>BD/Falcon</td><td>356551</td><td></td></tr><tr><td>5 ml graduated pipette</td><td>BD/Falcon</td><td>356543</td><td></td></tr><tr><td>50 ml falcon tubes</td><td>BD/Falcon</td><td>352070</td><td></td></tr><tr><td>15 ml falcon tubes</td><td>BD/Falcon</td><td>352096</td><td></td></tr><tr><td>15 cm Petri dishes</td><td>Corning</td><td>351058</td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>BD/Falcon</td><td>352350</td><td></td></tr><tr><td>0.22 &amp;mu;m filters</td><td>Millipore</td><td>SLGV033RS</td><td></td></tr><tr><td>1 ml Pipette tips</td><td>Fisherbrand</td><td>02-707-401</td><td></td></tr><tr><td>200 &amp;mu;l Pipette tips</td><td>Fisherbrand</td><td>02-707-409</td><td></td></tr><tr><td>20 ml Syringe</td><td>BD/Medical</td><td>309661</td><td></td></tr><tr><td>Plastic spatula</td><td>Fisher Scientific</td><td>14-245-97&amp;nbsp;</td><td></td></tr><tr><td>Plastic weighing boat</td><td>Fisher Scientific</td><td>02-202-102&amp;nbsp;</td><td></td></tr><tr><td>Cryo vials</td><td>Nunc</td><td>377267</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Mr Frosty</td><td>Fisher Scientific</td><td>A451-4&amp;nbsp;</td><td></td></tr><tr><td>Biohazard Cabinet</td><td></td><td></td><td></td></tr></tbody></table>

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.

When this procedure is followed correctly, an average yield of 120 million hAECs should be expected, with a typical range of 80-160 million cells. From these yields, an average viability of 83 &#177; 4% can be expected. The increased average yield and slightly lower viability in the clinical method may be due to higher trypsin activity than the animal-derived product, and perhaps also due to the lack of serum proteins. Isolated hAECs have an average cell surface profile of 92% EpCAM positive cells with &#60;1% CD90, CD10...

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

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-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K Views.  Wake Forest University Health Sciences. 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. 

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

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 ...

Developmental Biology

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

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 ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Biology

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.

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 ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Bioengineering

Advancing Cryostorage of Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Protocol for Isolating Mammary Epithelial Cells from Human Tissue

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Author Spotlight: Integrating Eastern and Western Medicine for Treatment of Granulomatous Mastitis

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...

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

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