The authors have no acknowledgments.

hASCs were isolated from the abdominal adipose tissue of a healthy woman who underwent breast reconstruction using abdominal autologous flaps (deep inferior epigastric perforator flaps, [DIEP]) at the University Hospital of Lausanne, CHUV, Lausanne, Switzerland. The discarded part of the flap and the adipose tissue was obtained after the patient signed informed consent. All protocols were reviewed and approved by the hospital&#39;s Biobank Department DAL (number 314 GGC) and ethics committees in accordance with the Declaration of Helsinki.
NOTE: All steps must be performed under a laminar flow hood and with aseptic conditions. Gloves and lab coats for personal protection should always be worn, and all surfaces should be washed with appropriate biocides. Excess tissue should be disposed of properly as biomedical waste.
1. Materials needed and preparation of the culture solutions
<ol>
	<li>For cell isolation, expansion, and cryopreservation, obtain the following instruments: sterile scissors; sterile scalpels; sterile tweezers; a 10 cm Petri dish; 15 mL tubes; T25 flasks; a Burker chamber; cryovials; a cell freezing container; an optical microscope with 4x and 10x magnification objectives; and a swinging bucket centrifuge.</li>
	<li>For immunophenotyping, obtain the following instruments and reagents: FACS tubes, sterile phosphate-buffered saline (PBS); Dulbecco&#39;s minimal essential medium (DMEM); hPL; dimethyl sulfide; and animal-free trypsin.</li>
	<li>Prepare the complete culture medium by supplementing DMEM with 5% heparin-free hPL. Store the medium at 4 &#176;C for up to 3 weeks, and always use warm (between room temperature and 37 &#176;C). Following GMP guidelines for the production of cell medicines intended for human therapy, do not add any antibiotics to the culture medium.</li>
	<li>Prepare freezing medium containing 20% hPL and 10% dimethyl sulfide in DMEM.</li>
</ol>
2. Isolation of hASCs from the adipose tissue sample
<ol>
	<li>Process the adipose tissue sample within 6 h of obtaining it. Before proceeding to the established mechanical xenogeneic-free isolation method, wash the tissue 2x with PBS for 10 min until connective tissue and blood are released.</li>
	<li>Store the obtained adipose tissue sample from abdominoplastic surgery in PBS at 4&#176; C until processing and, in any case, no longer than 24 h, in order to not damage the hASC viability.</li>
	<li>Cut the adipose tissue into smaller pieces of approximately 1 cm2 with sterile scissors.</li>
	<li>With a scalpel, micro-dissect each piece into little fragments with diameters &#60;5 mm, and disperse five of them in a 10 cm Petri dish (Figure 2A).</li>
	<li>In order to attach each fragment, use the scalpel to create incisions on the plastic surface, dragging each fragment until it is firmly stuck to the Petri dish. Use tweezers to help handle the fragment (Figure 2B).</li>
	<li>Gently add 10 mL of complete culture medium in order to cover all the fragments without detaching them.</li>
	<li>Incubate the Petri dishes at 37 &#176;C and 5% CO2. The hASCs will migrate out of the tissue and be selected due to their plastic adherence without enzymatic digestion.</li>
	<li>Until cell confluence, monitor the hASC expansion under an optical microscope at 4x magnification once per day. As the hASCs begin to exit from the tissue, they appear as small clusters around the tissue pieces with a typical fibroblast-like morphology. The hASCs are selected due to their ability to adhere to the plastic surface. Every 72 h, remove as much medium as possible, and replace it with fresh complete medium in order to remove cell debris.</li>
	<li>Leave the fragments of tissue in place until the first passage of cells, which is usually after 7 days.</li>
</ol>
3. hASC culture after the isolation phase
<ol>
	<li>When the hASC culture reaches 70%-80% confluency, the hASCs are ready to be detached and further expanded to be either used for other experiments or long-term storage.</li>
	<li>With sterile tweezers, remove and discard all the pieces of tissue from the Petri dish. Remove and discard the exhausted medium, being careful not to touch the hASCs in order not to detach them.</li>
	<li>Carefully wash the cells once with 10 mL of PBS. Add 1 mL of 1x animal-free trypsin, and leave the Petri dish in the incubator at 37 &#176;C for 5 min.</li>
	<li>Check under an optical microscope at 4x magnification if all the cells have detached; otherwise, leave the Petri dish for an additional 2 min in the incubator, and eventually gently tap the plastic surface to support cell detachment.</li>
	<li>Stop the trypsin action by adding 2 mL of complete medium, and collect all the cells in a 15 mL tube.</li>
	<li>In order to remove any remaining trypsin, centrifuge the tube at 300 x g for 5 min. Discard the supernatant, suspend the cells with 5 mL of complete medium, and transfer the cell suspension into a new T25 flask. Keep the hASCs in culture, and expand them until needed.</li>
</ol>
4. Immunophenotype investigation by flow cytometry
<ol>
	<li>When the hASC culture reaches 70%-80% confluency, detach the hASCs as previously described in section 3.</li>
	<li>After collecting the cells, suspend them in 1 mL of complete medium, and count an aliquot with the cell counter under an optical microscope at 10x magnification.</li>
	<li>Centrifuge the hASCs at 300 x g for 5 min, and suspend them at 1 x 106 cells/mL of FACS buffer. Transfer 100 &#181;L of the suspension containing 1 x 105 cells into one FACS tube. Use one FACS tube for each investigated antigen, plus one more for each isotypic control.</li>
	<li>Stain the cells with 100 &#181;L of the following antibodies diluted in FACS buffer: anti-CD73 (1:1,000, IgG1, FITC-labeled), anti-CD105 (1:200, IgG1, PE-labeled); anti-CD34 (1:66, IgG1, FITC-labeled); anti-CD45 (1:66, IgG1, FITC-labeled); and isotypic controls for each fluorophore, IgG1 FITC-labeled (1:1,000) and IgG1 PE-labeled (1:50).</li>
	<li>Incubate the samples for 1 h in the dark with agitation at 4 &#176;C. Centrifuge the tubes at 300 x g for 5 min, discard the supernatant, and suspend the cells in 500 &#181;L of FACS buffer.</li>
	<li>Assess the cell immunophenotype with a flow cytometry instrument, recording 50,000 events for each tube inside the gate selected to exclude cell debris. Calculate the percentage of expressing cells as the difference from the specific isotypic control.</li>
</ol>
5. Proliferation assay of hASC
<ol>
	<li>When the hASC culture reaches 70%-80% confluency, detach the hASCs as described in section 3.</li>
	<li>After collecting the cells, suspend them in 1 mL of complete medium, and count an aliquot with the cell counter under an optical microscope at 10x magnification.</li>
	<li>Seed 5 x 102 hASCs in 200 &#181;L of complete medium in 96-well transparent plates. Seed the cells in technical replicates, with one well for each time point.</li>
	<li>At 3 days after seeding, start detecting the cell proliferation using a proliferation assay kit following the manufacturer&#39;s instructions. In brief, replace the culture medium with 100 &#181;L of fresh medium added to 20 &#181;L of assay reagent per well. Incubate the hASCs for 2.5 h at 37 &#176;C, 5% CO2 for reagent development, and then detect absorbance at 490 nm with a spectrophotometer.</li>
	<li>Express the hASC proliferation as the percentage absorbance after removing the blank absorbance.</li>
</ol>
6. Cryopreservation and storage of the hASCs
<ol>
	<li>Detach the cells as described in section 3, and count an aliquot with the cell counter under an optical microscope at 10x magnification.</li>
	<li>Centrifuge the cells at 300 x g for 5 min, and discard the supernatant. Suspend the cells with freezing mix at a density of 1 x 106 hASCs/mL, and transfer 1 mL of the cell solution into one cryovial.</li>
	<li>Put the cryovials at &#8722;80 &#176;C in a freezing container that decreases the temperature by 1 &#176;C/min, and then move the cryovials to liquid nitrogen for long-term storage.</li>
</ol>

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The authors have nothing to disclose.

Adipose-derived stem cells have attracted the interest of translational research in the last decade due to their abundance, quick and affordable isolation methods, high in vitro/in vivo proliferation rate, and stemness/differentiation properties18,19,20. As a result, hASCs are considered an excellent candidate for cell-based strategies in regenerative medicine21. After isolation from adi...

In the last decades, the increasing demand for innovative therapeutic treatments has given rise to significant efforts and resource investment in the translational medicine field1. Cell-based products are associated with risks determined by the cell source, the manufacturing process (isolation, expansion, or genetic modification), and the non-cellular supplements (enzymes, growth factors, culture supplements, and antibiotics), and these risk factors depend on the specific therapeutic indication. The quality, safety, and efficacy of the final product could be deeply influenced by the above-indicated elements2. Stem cell therapy requires adherence to biosafety principles; the potential risks of pathogenic transmission with animal-derived products in cell culture could be problematic, and the thorough testing of any product introduced in the manufacturing is essential3.
The traditional method to isolate human adipose-derived stem cells (hASCs) involves an enzymatic digestion performed with collagenase followed by washing steps through centrifugation4. While enzymatic isolation is generally considered more efficient than other mechanical techniques in terms of cell yields and viability, the animal-derived components used, such as collagenase, are considered more than minimally manipulated by the U.S. Food and Drug Administration. This means there is a significantly increased risk of immune reactions or disease transmission, thus limiting the translation of hASC therapy to clinical settings5,6.
Trypsin-based digestion is another enzymatic protocol to isolate ASCs. Different techniques have been described with slight modifications in terms of the trypsin concentration, centrifugation speed, and incubation time. Unfortunately, this method is not well described, and a lack of comparison exists in the literature, particularly with the mechanical isolation protocols7. However, in terms of the translatability of the approach, trypsin has the same drawbacks of collagenase.
Alternative isolation methods to obtain ASCs, based on mechanical forces and without enzyme addition, involve high-speed centrifugation (800 x g or 1,280 x g, 15 min) of the adipose tissue fragments. Then, the pellet is incubated with a red blood cell lysis buffer (5 min), followed by another centrifugation step at 600 x g before resuspension in culture medium. Despite a greater number of cells being isolated in the first days compared to the explant methods, a previous study showed lower or absent proliferation beyond the second week of culture8.
Besides that, further manipulation with xenogeneic added medium, such as fetal bovine serum (FBS), which is used as a growth factor supplement for cell culture, is associated with an increased risk of immune reactivity and exposure to viral, bacterial, or prion infections of the host patient9,10. Immune reactions and urticariform rash development have been already described in individuals receiving several doses of mesenchymal stem cells produced with FBS11. Furthermore, FBS is subjected to batch-to-batch variability, which can have an impact on the final product quality12.
In accordance with good manufacturing practice (GMP) guidelines, enzymatic tissue dissociation should be avoided, and FBS should be substituted with xenogeneic-free supplements. These steps, together with more reliable and reproducible protocols, are essential to support the application of cell therapy3,13.
In this context, human platelet lysate (hPL) has been suggested as a substitute for FBS since it is a cell-free, protein-containing, growth factor-enriched supplement, and it was earlier introduced among clinical-grade cell-based products as an additive of growth medium for in vitro cell culture and expansion14,15. As hPL is a human-derived product, it is frequently used as a substitute for FBS during the in vitro culture of hASCs intended for clinical applications, thus reducing issues regarding immunological reactions and infections related to FBS translatability15,16.
Despite the higher production costs, it has already been demonstrated that compared to FBS, hPL supports cell viability for many cell types, increases proliferation, delays senescence, assures genomic stability, and conserves the cellular immunophenotype even in late cell passages; all these elements support the switching toward this culture supplement11.
The aim of this work was to develop a standardized protocol to isolate and culture hASCs with a complete animal-free method, without modifying the cell physiology and stemness properties in comparison to classical FBS-cultured hASCs (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL tubes</td>
			<td>euroclone</td>
			<td>et5015b</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD105</td>
			<td>BD Biosciences</td>
			<td>BD560839</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD34</td>
			<td>BD Biosciences</td>
			<td>BD555821</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45</td>
			<td>BD Biosciences</td>
			<td>BD555482</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD73</td>
			<td>BD Biosciences</td>
			<td>BD561254</td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution (FACS buffer)</td>
			<td>Miltenyi</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Accuri C6 apparatus (flow cytometry instrument)</td>
			<td>BD accuri</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Burker chamber</td>
			<td>Blaubrand</td>
			<td>717810</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell freezing container</td>
			<td>corning</td>
			<td>CLS432002</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter 96 AQueous One Solution Cell Proliferation Assay</td>
			<td>Promega</td>
			<td>G3582</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolCell Freezing container</td>
			<td>Corning</td>
			<td>CLS432002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>clearline</td>
			<td>390701</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfide</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>disposabile blade scalpel</td>
			<td>paragon</td>
			<td>bs 2982</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>GIBCO</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Platelet Lysate FD (GMP grade)</td>
			<td>Stemulate</td>
			<td>PL-NH-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite F50 spectrophotometer</td>
			<td>Tecan</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope with 4x and 10x magnification objectives</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 10 cm</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpels</td>
			<td>Reda</td>
			<td>07104-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scissors</td>
			<td>Bochem</td>
			<td>4071</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tweezers</td>
			<td>Bochem</td>
			<td>1152</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging bucket centrifuge</td>
			<td>Sigma</td>
			<td>3-16K</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks</td>
			<td>Greiner bio-one</td>
			<td>6910170</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLe (animal free trypsin substitute)</td>
			<td>GIBCO</td>
			<td>12604-013</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Applying the isolation method detailed above, hASCs were successfully obtained from abdominal adipose tissue samples without the use of collagenase. Moreover, the hASCs were expanded in complete xenogeneic-free conditions in the presence of hPL and without any other components of animal origin. The following results support the protocol and are obtained from hASCs cultured in parallel with hPL and with FBS as the control condition.
After the initial cluster appearance, the hASCs showed the cla...

Watch this Scientific Journal Video about Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy at JoVE.com

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

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 ...

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JoVE Journal

Medicine

2.9K Views.  Centre Hospitalier Universitaire Vaudois (CHUV. 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.

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

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

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 ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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

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

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

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

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 ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...