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>

<ol>
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Med. 31 (1), 62-68 (2013).</li><li>Parolini, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+isolation+and+characterization+of+cells+from+human+term+placenta:+outcome+of+the+first+international+Workshop+on+Placenta+Derived+Stem+Cells.">Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.</a> Stem Cells. 26 (2), 300-311 (2008).</li><li>Lim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preterm+human+amnion+epithelial+cells+have+limited+reparative+potential.">Preterm human amnion epithelial cells have limited reparative potential.</a> Placenta. 34 (6), 486-492 (2013).</li><li>Tamagawa, T., Ishiwata, I., Saito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+pluripotent+stem+cell+line+derived+from+human+amniotic+membranes+and+initiation+of+germ+layers+in+vitro.">Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro.</a> Hum Cell. 17 (3), 125-130 (2004).</li><li>Miki, T., Marongiu, F., Ellis, E., Strom, C. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion-isolated+cells+normalize+blood+glucose+in+streptozotocin-induced+diabetic+mice.">Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice.</a> Cell Transplant. 12 (5), 545-552 (2003).</li><li>Murphy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion+epithelial+cells+prevent+bleomycin-induced+lung+injury+and+preserve+lung+function.">Human amnion epithelial cells prevent bleomycin-induced lung injury and preserve lung function.</a> Cell Transplant. 20 (6), 909-923 (2011).</li><li>Murphy, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion+epithelial+cells+induced+to+express+functional+cystic+fibrosis+transmembrane+conductance+regulator.">Human amnion epithelial cells induced to express functional cystic fibrosis transmembrane conductance regulator.</a> PLoS One. 7 (9), e46533 (2012).</li><li>Murphy, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amnion+epithelial+cells+do+not+abrogate+pulmonary+fibrosis+in+mice+with+impaired+macrophage+function.">Human amnion epithelial cells do not abrogate pulmonary fibrosis in mice with impaired macrophage function.</a> Cell Transplant. 21 (7), 1477-1492 (2012).</li><li>Hodges, R. J., Lim, R., Jenkin, G., Wallace, E. 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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 border="0" cellpadding="0" cellspacing="0" width="641">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Collection Kit</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Stripping tray</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:119px;">13-361B</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:220px;">Liberty Dressing Forcep, pointed 13cm</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>S17329&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Scissors - Sharp/Blunt Straight</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>NC0562592&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Protective Apparel (Gown)</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protective Apparel</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolation gowns</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">General purpose face mask</td>
			<td style="width:119px;">Cardinal Health</td>
			<td style="width:119px;">AT7511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bonnets</td>
			<td style="width:119px;">Medline</td>
			<td style="width:119px;">CRI1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shoe covers</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-7873W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Media and Reagents</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td style="width:119px;">Life Technologies</td>
			<td align="right" style="width:119px;">14175095</td>
			<td>without calcium or magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T3449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostor CS5</td>
			<td style="width:119px;">BioLife Solutions</td>
			<td align="right" style="width:119px;">205102</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-EpCam-PE</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130 - 091-253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-isotype control</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD90-PeCy5</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">555597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PeCy5-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">557224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD105-APC</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">340754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm petri dishes</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#956;m filters</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
			<td style="width:119px;">Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">20ml Syringe</td>
			<td style="width:119px;">BD/Medical</td>
			<td align="right" style="width:119px;">309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic spatula</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>14-245-97&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>02-202-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cryo vials</td>
			<td>Nunc</td>
			<td>377267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:119px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></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...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

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, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

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 ...