The authors thank Katharina Schallmoser for providing pHPL, Eva Rohde and Nicole Hofmann for useful comments to the manuscript, Daniela Thaler and Margaretha Fr hwirth for technical assistance, Monica Farrell for linguistic editing, Uwe Lang, Margit Holzapfel and Sandra Eppich at Dept. of Obstetrics for providing the cords.
This work has been supported by the Austrian Research Foundation (FWF, grant N211-NAN to DS), the Austrian Research Promotion Agency (FFG, grant N200 to DS) and by The Adult Stem Cell Research Foundation (A.R.). A.R is a fellow of the PhD program Molecular Medicine of the Medical University of Graz.

Part 1: Setting up
1. Preparation of cell culture medium
Before starting the medium preparation collect all the materials and tools you will need. 
 Thaw 2 x 56 ml aliquots of pooled human platelet lysate (pHPL, self-made: reference 1 &amp; JoVE #1523), 10 ml of a 100x Penicillin/Streptomycin solution, 2 x 5 mL of L-Glutamine (both Sigma).
Thaw the cytokine and growth factor aliquots (VEGF, bFGF, EGF, IGF, hydrocortisone, ascorbic acid, Heparin, Amphotericin provided as 'single quots') adjusted for supplementing 1 bottle (500 mL) of EGM-2 medium (Lonza).
From the fridge take one 500 ml bottle each of (i) alpha-modified minimal essential medium (a-MEM) and (ii) endothelial basal medium (EBM).
All of the following steps are performed in a laminar flow tissue culture hood under sterile conditions.
Prepare preservative-free heparin (e.g., Biochrom) by dissolving the powder to a final concentration of 1000 IU/mL with sterile water.
MSC-Medium:
Use 500 mL of a-MEM, add 56 mL of thawed pHPL (see also reference 1 for further details) and 2 IU/mL (=224 &mu;l of stock solution) of preservative-free Heparin (avoids coagulation of the medium through clumping of the fibrinogen in the plasma) to reach a final concentration of 10% pHPL. Additionally add Penicillin (100U/mL) /Streptomycin (100&mu;g/mL) solution and 2mM of L-Glutamin (both Sigma).
Filter the medium through a 20 &mu;m-pore size vacuum filter (Millipore). Label the bottle appropriately (content, date).
ECFC-Medium:
Use one bottle (500 mL) of EBM, add the cytokine-aliquots, 56 mL of pHPL, 10 IU/ml (=1120&mu;l of stock solution) of preservative-free Heparin, Penicillin (100U/mL) /Streptomycin (100 g/mL) solution and 2mM of L-Glutamin to the basal medium and filter with a 20 &mu;l-pore size vacuum filter (Millipore). Label the bottle appropriately (content, date).
2. Sterilization of surgical instruments
Forceps, sharp pair of surgical scissors and scalpel holder must be heat sterilized ore sterile disposables.
3. Preparation of tubes for cord collection
Add 500 IU of preservative - free heparin to 50 ml polypropylene tubes (Falcon) and adjust to a final volume of 20 mL with PBS. Transfer the tubes into the delivery room.
4. Informed consent (confirmed by your local ethical committee or IRB).
Ask the parents prior to delivery if they would like to donate a piece of the cord and adequately inform them of the purposes it will serve.
5. Cord donation
After delivery and inspection of placenta and cord cut off a 10 cm piece and immediately transfer it to your tube prefilled with heparin to avoid coagulation of the remaining blood inside the cord vessels. Continue on with cell isolation as soon as possible. 
Part 2: Cell isolation
<ol>
<li>Prepare laminar flow tissue culture hood by cleaning surfaces with Incidin or 70% EtOH. Place sterile 150 cm2 cell culture plates, 75 cm cell culture flasks (both Corning), PBS, sterilized surgical instruments, cell scrapers, tube holder, 25 mL stripettes and 5 ml syringes equipped with blunt end needles appropriately in your cleaned hood.</li>
<li>Pre-warm your prepared &alpha;-MEM and EGM-2 cell culture medium to 37&deg;C in a water bath.</li>
<li>Bring the cord from the delivery room to your lab equipped with the cell culture hood. After transferring the cord into the laminar flow wash it twice in PBS (by transferring to a new plate) to get rid of contaminating blood cells. Rinse the umbilical vein after canulating it on one side with a sterile 5mL syringe equipped with a blunt end needle with PBS until the fluid coming out the other end of the cord becomes completely transparent (reddish indicates blood contamination).</li>
</ol>
ECFCs:
<ol start="4">
<li>Prepare a 75 cm2 flask (Corning), label it and fill in 15-20 mL of pre-warmed EGM-2 medium.</li>
<li>Place the cord into a new plate filled with sterile PBS. 
Take the surgical scissors and cut the umbilical cord into two pieces of about 5 cm in length (short pieces are easier to process, because the umbilical vessels are twisted along their axis).</li>
<li>Try to find the umbilical vein (large vessel), insert one blade of your surgical scissors and cut the vein longitudinally. At this point it could be helpful if an assistant can hold the already cut vein with two forceps while you cut further.</li>
<li>Place the cord piece with the cut vein in a new plate without PBS, take the cell scraper and scrape of the inner (endothelial) surface of the umbilical vein by rubbings from one end to the other. Transfer the cells to your prepared flask by cleaning the tip of the scraper in the medium. Repeat this procedure at least 10 times.</li>
<li>Close your flask and put it into an incubator (37&deg;C, 5% CO2, 95% Humidity).</li>
</ol>
MSCs:
<ol start="4">
<li>Prepare a 150 cm2 culture plate (Corning) and label it.</li>
<li>Take the piece of cord (after scraping off the endothelial layer) and cut the whole cord into very small pieces by using sterile scissors and sterile scalpel. Maximum size of the cord pieces should be 1-2 mm.</li>
<li>Transfer your cord pieces with the sterile forceps into your pre-labeled culture plate. By leaving the plate open for 5 minutes before filling up with medium, the attachment of the pieces to the plastic surface is strengthened.</li>
<li>Once the cord pieces are connected appropriately to the plastic, gently add in 30-35 mL of pre-warmed alpha modified MEM. Try to use the lowest speed to be sure that all pieces remain attached.</li>
<li>Close the plate and put it into an incubator (37&deg;C, 5% CO2, 95% Humidity).</li>
</ol>
Part 3: Cell expansion and confirmation of immune-phenotype by flow cytometry
Cell Expansion
EFCFs:
<ol><li>Check the attachment of endothelial cells the next day on an inverted microscope and exchange whole EGM-2 medium to get rid of all non-attached cells and particles.</li>
<li>Expand the cells until confluence and exchange one third of the medium twice a week. When confluence is reached, detach the cells with 0.05% Trypsin/0.7 mM EDTA and transfer them to new culture vessels. Depending on the number of cells within your T75 flask, you should choose the size and number of your new flasks to guarantee low seeding density for further expansion.</li>
<li>Exchange one third of the medium twice a week during the expansion of the cells.</li>
</ol>
MSCs:
<ol>
<li>The outgrowth of MSCs from the cord will take more time, so you can wait at least 3-4 days till you check on an inverted microscope for the appearance of spindle-shaped cells in the surroundings of the attached tissue pieces. When there is sufficient cell out-growth, the pieces tend to detach because they lose contact with the plastic.</li>
<li>After 10-12 days remove the tissue pieces, detach the MSCs from the plastic with 0.05% Trypsin/0.7 mM EDTA and transfer them to new culture vessels. Depending on the amount of cells within your plate you should decide the size and number of your new flasks to guarantee low seeding density for further expansion.</li>
<li>Exchange one third of the medium twice a week during the expansion of the cells.</li>
</ol>
Flow cytometry
Equipment needed:
Flow cytometer (BD FACSCalibur), micronic tubes (Corning), tube holder, sheep serum (blocking reagent), Eppifuge (Eppendorf), monoclonal antibodies 
 MSCs: CD45, CD14, CD19, HLA-DR, CD31, CD73, CD90, CD105, isotype controls 
 ECFCs: CD45, CD14, CD19, HLA-DR, CD31, CD34, CD90, CD105, CD144, CD146, isotype controls
<ol>
<li>After reaching confluence, detach the cell with 0.05% Trypsin/0.7 mM EDTA and centrifuge at 300g for 7 min at 4&deg;C.</li>
<li>Resuspend the cells to a concentration of 1x106 cells/mL in modified PBS and block unspecific antibody binding with 10% v/v sheep serum for 20 min at 4&deg;C.</li>
<li>Label the micronic tubes and add the appropriate concentration of fluorescent labeled antibodies.</li>
<li>Add 50 &mu;l of cell suspension (blocked) to each tube and incubate for 30 min at 4&deg;C.</li>
<li>Wash the cells by adding modified PBS to remove non-binding antibodies.</li>
<li>Go with your labeled cell to the flow cytometer and perform the analysis.</li>
</ol>
Part 4: ECFC colony growth, fixation, staining and hierarchy analysis
<ol>
 <li>Seed the virtually pure harvested ECFCs at very low plating density of 3 or 10 cells per square centimeter in cell culture plates to allow for single colony growth.</li>
<li>Exchange one third of the medium twice a week.</li>
 <li>After 14 days (end of culture) remove medium, wash twice with PBS and fix the colonies with ice cold fixation solution (Acetone : Methanol = 3 : 2) for 15 minutes in the refrigerator. </li>
<li>Discard the fixation solution and leave the plates open to dry for 10 minutes. </li>
 <li>Rehydrate the cells with distilled water for 10 minutes. </li>
<li>Add Harris Hematoxylin solution and stain the fixed colonies for 12 minutes. </li>
 <li>Remove H matoxylin solution and rinse the plates with tap water to get rid of the remaining staining solution. </li>
<li>Take photos of each colony by using a stereomicroscope and import *.jpg or *.tif format files onto your computer. </li>
 <li>Open the photos with ImageJ software and analyze the accurate cell number of each colony as described in the animation. </li>
 <li>Open the ImageJ software and import a colony image using the &#x2018;File\Open&#x2019; function in the pull down menu. </li>
 <li>Proceed by using the &#x2018;Process\Subtract Background&#x2019; function</li>
 <li>Use the &#x2018;*elliptical* or brush selection&#x2019; function in the ImageJ menu to mark the colony margin.</li>
 <li>Clear the surrounding image using the &#x2018;Edit\Clear Outside&#x2019; function and crop the picture by selecting &#x2018;Image\Crop&#x2019;. </li>
 <li>Adjust the threshold manually using the &#x2018;Image\Adjust\Threshold&#x2019; function, so that all cells are completely colored in red. </li>
<li>Count the cell number of the colony by selecting &#x2018;Analyze\Analyze Particles&#x2019;. Choose appropriate settings (optimized for each cell type) and select &#x2018;OK&#x2019;. The results box, including cell count and a new image containing the corresponding outlines of the counted cells will appear. 
<img src="/files/ftp_upload/1525/1525op2.jpg" alt="Figure 1" /></li>
</ol>

<ol>
	<li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+lysate+can+replace+fetal+bovine+serum+for+clinical-scale+expansion+of+functional+mesenchymal+stromal+cells.">Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.</a> Transfusion. 47, 1436-1446 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+system+to+propagate+cord+blood-derived+multipotent+mesenchymal+stromal+cells+for+clinical+application.">Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.</a> Regen Med. 2, 371-382 (2007).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. 113, 6716-6725 (2009).</li><li>Strunk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+preclinical+production+of+human+lineage-negative+cells+for+regenerative+stem+cell+therapy.">Phenotypic characterization and preclinical production of human lineage-negative cells for regenerative stem cell therapy.</a> Transfusion. 45, 315-326 (2005).</li></ol>

The authors don't have any financial conflicts of interest to declare.

There is a variety of sources from which to get MSCs or ECFCs including bone marrow, adipose tissue, cord blood, etc.
It is necessary to get both types of progenitors from the same source to directly compare the involvement of the different cell types in processes like tissue and vessel regeneration or contribution to tumor progression.
The ease with which to isolate and subsequently study autologous pairs of ECFCs and MSCs from the same donor makes this method ad...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>alpha modified MEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-GLutamin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EBM</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Single cytokine quots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>75 cm2 flasks</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>150 cm2 plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell scraper</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin/EDTA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Monoclonal antibodies</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>500 mL filter 20 &mu;m pore size </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Preservative free heparin</td>
<td>&nbsp;</td>
<td>Biochrom</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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isolation and animal serum free expansion of human umbilical cord derived mesenchymal stromal cells (mscs) and endothelial colony forming progenitor cells (ecfcs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Watch this Scientific Journal Video about Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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15.4K Views.  Medical University of Graz, Austria. This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Video: Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

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

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord Wharton's jelly, as well as from the umbilical arteries and umbilical vein. They are known as perivascular stem cells obtained from umbilical arteries (UCA-PSCs), perivascular stem cells obtained from the umbilical vein (UCV-PSCs), and mesenchymal stem cells obtained from Wharton's jelly (WJ-MSCs). UCA-PSCs and UCV-PSCs are pericytes derived from perivascular regions that are progenitors of MSCs. Isolation and culture of the three kinds of cells is an important source for studying stem cell transplantation and repair. The present protocol focuses on the isolation and culture of cells through mechanical separation, adherent culture, and cell crawling out. Through this technique, the three different types of stem cells can be derived. Cell surface markers were detected by flow cytometry. The stem cells were detected for multilineage differentiation potential by adipogenic, osteogenic, and neural-like differentiation, which is consistent with the phenotype of MSCs. This experimental protocol expands the source of UC-MSCs. In addition, the cell isolation method provides a basis for further study of regenerative medicine and other applications.

The present protocol describes the isolation and culture of mesenchymal stem cells from the umbilical cord arteries, vein, and Wharton's jelly.

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Closed System Production of Adherent Cells Based on Dissolvable Microcarriers

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

Author Spotlight: Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers 

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...

Bioengineering

Functional Analysis of BAMBIhighMFGE8high UC-MSC Subpopulations

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...