We thank the patients who volunteered to participate and the medical and nursing staff of the Hospital S&#227;o Paulo. This study was supported by the Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo&#160;(FAPESP) and Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq), Brazil.

The present study is approved by the Ethics Committee of the UNIFESP (protocol number: 0029/2015 CAAE: 40846215.0.0000.5505), performed after obtaining written informed consent from the patients according to the Declaration of Helsinki (2004). The sample of the present study is composed of nine female patients, aged 33-50 years (average age 41.5) and average initial body mass index (BMI) of 24.54 (ranging between 22.32-26.77) (Table 1) who underwent aesthetic abdominoplasty due to excess of skin after pregnancies, at the Division of Plastic Surgery of the Universidade Federal de S&#227;o Paulo (UNIFESP), Brazil. To reduce bias, the patients were selected as a homogeneous group considering sex, age, and BMI. The datasets used and/or analyzed during this study are available from the corresponding author upon reasonable request.
1. Collection of lipoaspirate
NOTE: This step needs to be performed in the surgery center.
<ol>
	<li>Use 4% chlorhexidine gluconate (see Table of Materials) for skin preparation and asepsis.
	<ol>
		<li>Perform a 2 mm subcutaneous skin incision (between the sub-dermis and aponeurosis). Insert a Klein cannula of 26 mm 3 G three-hole and 3 mm caliber and a syringe to inject a total volume of 500 mL of an adrenaline solution (1 mg/mL) (see Table of Materials) diluted in saline (1:1,000,000) in the infraumbilical area.</li>
	</ol>
	</li>
	<li>Connect a 60 mL syringe to a 26 mm 3 G three-hole and 3 mm caliber liposuction cannula and insert it through the skin incision, locking the plunger to create a vacuum.
	<ol>
		<li>Make pushing and pulling movements so that, with the vacuum created, the lipoaspirate remains in the 60 mL syringe.</li>
	</ol>
	</li>
	<li>Using a sterile connector with a valve, transfer the 100 mL of the collected lipoaspirate to a 150 mL polyvinyl chloride transfer bag (see Table of Materials).
	<ol>
		<li>Pack the transfer bag in a polystyrene box at room temperature (~25 &#176;C) and take it immediately to the laboratory. Do not take longer than 30 min to start the tissue processing.</li>
	</ol>
	</li>
</ol>
2. Processing of lipoaspirate
NOTE: This step is to be performed in the laboratory.
<ol>
	<li>First, weigh the bag, gauge the temperature with a digital non-contact infrared clinical thermometer, and leave the bag resting for 5 min inside the laminar flow chamber for precipitation of the greasier layers (bubbles) and tissue separation containing the cells of interest.
	<ol>
		<li>Perform a series of tissue washes. First wash: inject 100 mL of DPBS with calcium (1x) into the transfer bag and mix it with the hands.</li>
		<li>Let it stand for 5 min and remove most of the basal liquid that precipitates.</li>
		<li>Discard the basal liquid with a 60 mL syringe attached to the bag adapter. This process must be repeated twice.</li>
	</ol>
	</li>
	<li>Add 100 mL of digestion solution to the bag (93 mL of calcium-free DPBS + 60 &#181;L of calcium chloride (1 g/L) + 7 mL of 0.075% sterile collagenase, see Table of Materials) and leave at 37 &#176;C for 30 min under slow stirring.</li>
	<li>Transfer all the bag&#39;s content to four conical tubes of 50 mL and centrifuge them at 400 x g at 22 &#176;C for 10 min.
	<ol>
		<li>Remove and discard the supernatant and add 5 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) low glucose supplemented with 20% FBS (Fetal bovine serum) to the cell pellet (Figure 1).</li>
	</ol>
	</li>
</ol>
3. Counting of the SVF cells
<ol>
	<li>Mix a fresh solution of 10 &#181;L of trypan blue at 0.05% in distilled water with 10 &#181;L of cellular suspension for 5 min.</li>
	<li>Count viable cells in a Neubauer cell counting chamber32 using an inverted light microscope (see Table of Materials) at 20x magnification.</li>
	<li>Resuspend the cell pellet in a cryoprotective medium (5 mL of FBS + 10% of Dimethyl Sulfoxide - DMSO) at a concentration of 1 x 106 cells/mL.</li>
	<li>Place 1 mL of this mix in cryovials. Use a freezing container (see Table of Materials) with a cooling rate of (1 &#176;C/min to -80 &#176;C).
	<ol>
		<li>Store at -80 &#176;C for 1 year.</li>
		<li>After this time, store in standard cassette boxes immersed in the liquid nitrogen vapor phase (-165 &#176;C).</li>
	</ol>
	</li>
</ol>
4. Thawing process of the cells
<ol>
	<li>Remove the vials from liquid nitrogen and place them immediately in the 37 &#176;C water bath for 1 min.</li>
	<li>Place the SVF cells in a conical tube with 4 mL of DMEM (low glucose supplemented with 20% FBS) preheated at 37 &#176;C.</li>
	<li>Centrifuge at 400 x g at 22 &#176;C for 5 min.</li>
	<li>Remove the supernatant and add 1 mL of DMEM (low glucose) + 10% FBS. Perform immunophenotyping following the steps below.</li>
</ol>
5. Flow cytometry technique (immunophenotype multiple labeling)
<ol>
	<li>Place 1 mL of cell pellet (concentration of 1,000 cells/&#181;L) in five cytometry tubes (200 &#181;L each).</li>
	<li>Centrifuge at 400 x g at 22 &#176;C for 5 min and discard the supernatant with a pipette.</li>
	<li>Add 300 &#181;L of Phosphate-Buffered Saline (PBS) (10x), centrifuge at 400 x g at 22 &#176;C and discard the supernatant with a pipette.</li>
	<li>Prepare five tubes for different marker combinations as follows: 5 &#181;L of CD11B/5 &#181;L of CD19/20 &#181;L of CD45; 5 &#181;L of CD73/20 &#181;L of CD90/5 &#181;L of CD105/20 &#181;L of CD45; 20 &#181;L of CD34/5 &#181;L of HLA-DR/20 &#181;L of CD45; Cell viability assay-5 &#181;L of fluorescent reactive dye. (see Table of Materials) and a tube with unstained cells and PBS as the negative control. Homogenize in a vortex and incubate at 4 &#176;C for 30 min.
	<ol>
		<li>Centrifuge at 400 x g at 22 &#176;C for 5 min, discard the supernatant with a pipette, add 500 &#181;L of PBS (10x), and proceed with cell sorting. 
		&#8203;NOTE: Five thousand events are acquired per antibody set in the Flow Cytometer of four colors and five parameters and analyzed with CellQuest software.</li>
	</ol>
	</li>
</ol>
6. Seeding of passage 1 (P1)
<ol>
	<li>Seed 2 x 105 cells in a 75 cm2 culture flask.</li>
	<li>Add 12 mL of DMEM low glucose + 20% of FBS + 10% antibiotic/antimycotic (with 10,000 units penicillin, 10 mg streptomycin, and 25 &#181;g amphotericin B per mL, 0.1 &#181;m).</li>
	<li>When the cells reach between 80%-90% confluence, perform trypsinization of adherent cells with 2 mL of 0.25% EDTA-trypsin for 3 min.</li>
	<li>Count cells again (as mentioned in step 3).</li>
	<li>Perform immunophenotyping again (as mentioned in step 5).</li>
</ol>
7. Statistical analysis
<ol>
	<li>Use Spearman&#39;s Rho Calculator33 to measure the strength of association between the following variables with P &#60; 0.05, as mentioned below.
	<ol>
		<li>Select SVF cellular yield and the number of days SVF stays in culture in the first passage (P1) until 80%-90% confluence (days to P1).</li>
		<li>Select SVF cellular yield before and after going to P1.</li>
		<li>Consider days to P1 and cellular yield before going to P1.</li>
		<li>Select SVF cellular yield with the average percentage of confirmed ADSC.</li>
		<li>Calculate the percentage of confirmed ADSC and the cellular yield before going to P1.</li>
		<li>Determine the BMI and SVF cellular yield.</li>
	</ol>
	</li>
</ol>
8. Differentiation assay
<ol>
	<li>Perform the differentiation assay following a differentiation kit protocol (see Table of materials). Figure 4 demonstrates the results for Case 1.</li>
</ol>

The authors declare no competing financial interests.

Isolation yield 
It is well established that the cryopreservation process, frequently required in cellular therapy, results in significant cell loss, sometimes greater than 50%29,30,35. Thus, a technical improvement for obtaining high initial cellular yield in isolation is fundamental. The collecting method of lipoaspirate and the isolation method of the cells must focus on preserving a greater number of ce...

Human mesenchymal stem cells are advantageous in both basic and applied research. The use of this adult cell type overpasses ethical issues-compared to the use of embryonic or other cells-being one of the most promising areas of study in autologous tissue regeneration engineering and cell therapy1, such as the neoplastic area, the treatment of degenerative diseases, and therapeutic applications in the reconstructive surgery area2,3,4,5. It has been previously reported that there is an abundant source of mesenchymal multipotent and pluripotent stem cells in the stromal vascular cell fraction of adipose tissue6,7. These ADSC are considered great candidates for use in cell therapy and transplantation/infusion since a considerable number of cells with a strong capacity for expansion ex vivo can be easily obtained with a high yield from a minimal invasive procedure5,8.
It was also demonstrated that adipose tissue presents a greater capacity to provide mesenchymal stem cells than two other sources (bone marrow and umbilical cord tissue)9. Besides being poorly immunogenic and having a high ability to integrate into the host tissue and to interact with the surrounding tissues4,10, ADSC has a multipotent capacity of differentiation into cell lines, with reports of chondrogenic, osteogenic, and myogenic differentiation under appropriate culture conditions11,12,13, and into cells, such as pancreatic, hepatocytes, and neurogenic cells14,15,16.
The scientific community agrees that the mesenchymal stem cells&#39; immunomodulatory effect is a more relevant mechanism of action for cell therapy17,18,19 than their differentiation property. One of the most significant merits of the ADSC use is the possibility of autologous infusion or grafting, becoming an alternative treatment for several diseases. For regenerative medicine, ADSC have already been used in cases of liver damage, reconstruction of cardiac muscle, regeneration of nervous tissue, improvement of skeletal muscle function, bone regeneration, cancer therapy, and diabetes treatment20,21.
To this date, there are 263 registered clinical trials for the evaluation of ADSC&#39;s potential, listed on the website of the United States National Institutes of Health22. Different protocols to harvest adipose tissue have been established, but there is no consensus in the literature about a standardized method to isolate ADSC for clinical use23,24. Lipoaspirate processing methods during and after surgery can directly affect cell viability, the final cellular yield25, and the quality of the ADSC population20. Regarding the surgical pre-treatment, it is not well established which surgical pre-treatment technique yields a more significant number of viable cells after isolation or whether the anesthetic solution injected into adipose tissue affects cell yield and its functions26. Similarly, the difference between techniques for obtaining adipose cells can lead to as much as a 70% decrease in the number of viable ADSC20. According to the literature, mechanical treatments to obtain cell populations with high viability-including ultrasound-should be avoided, for they can break down the adipose tissue20. However, the manual fat aspiration method with syringes is less harmful, causing less cell destruction, with tumescent liposuction yielding a significant number of cells with the best quality26.
This technique uses a saline solution with lidocaine and epinephrine that is injected into the liposuction area. For each 3 mL volume of solution injected, 1 mL is aspirated. In this study, the wet liposuction technique was performed, in which for each 1 mL of adrenaline and saline solution injected, 0.2 mL of adipose tissue is aspirated. The use of digestive enzymes, especially collagenase, is common for the process of isolating ADSC.
After the first isolation step in the laboratory, the final pellet is called stromal vascular fraction (SVF). It contains different cell types27, including endothelial precursor cells, endothelial cells, macrophages, smooth muscle cells, lymphocytes, pericytes, pre-adipocytes, and ADSCs, which are capable of adhesion. Once the final isolation is concluded from in vitro cultures, cells that did not adhere to the plastic are eliminated in medium exchanges. After eight weeks of expansion, medium changes, and passages, ADSCs represent most of the cell population in the flasks20. One of the most significant advantages of using isolated adipose-derived stem cells for a possible future therapy is the possibility of cryopreservation. It was demonstrated that cryopreserved lipoaspirate is a potential source of SVF cells even after 6 weeks of freezing28, with biological activity even after 2 years of cryopreservation29, and full capability to grow and differentiate in culture30. However, during the thawing process, a considerable percentage of cells is usually lost31. Therefore, the lipoaspirate removal process and the following methods of cell isolation must ensure the highest cell yield.
This study describes a faster methodology for collecting and isolating ADSC, demonstrating high cellular yield and viability for better efficiency of cellular therapeutics. Furthermore, the effect of this improved technique after long-term SVF cryopreservation was evaluated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.8 mL cryovials</td>
			<td>Nunc Thermo Fisher Scientific</td>
			<td>340711</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mL polyvinyl chloride transfer bag</td>
			<td>JP FARMA</td>
			<td>80146150059</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Alizarin Red S Solution, pH 4.2</td>
			<td>Sigma Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Adrenaline (1 mg/mL)</td>
			<td>Hipolabor</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue solution</td>
			<td>Sigma Aldrich</td>
			<td>1,01,647</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic 100x</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCalibur Flow Cytometer using BD CellQues Pro Analysis</td>
			<td>BD BioSciences</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride 10%</td>
			<td>Merck</td>
			<td>102379</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhexidine gluconate 4%</td>
			<td>VIC PHARMA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type I, powder</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s modified Eagle&#39;s medium)</td>
			<td>Gibco</td>
			<td>11966025</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS no calcium, no magnesium (Dulbecco&#39;s Phosphate Buffered Saline Gibco Cell Therapy Systems)</td>
			<td>Gibco</td>
			<td>A1285801</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS with calcium (Dulbecco&#39;s Phosphate Buffered Saline Gibco Cell Therapy Systems)</td>
			<td>Gibco</td>
			<td>A1285601</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10500056</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 4%</td>
			<td>Sigma Aldrich</td>
			<td>1,00,496</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Nikon Eclipse TS100</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Live and Dead Cell Assay</td>
			<td>Thermofisher</td>
			<td>01-3333-41 | 01-3333-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: CD105</td>
			<td>BD BioSciences</td>
			<td>745927</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: CD11B</td>
			<td>BD BioSciences</td>
			<td>746004</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: CD19</td>
			<td>BD BioSciences</td>
			<td>745907</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: CD34</td>
			<td>BD BioSciences</td>
			<td>747822</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: CD45</td>
			<td>DAKO</td>
			<td>M0701</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: CD73</td>
			<td>BD BioSciences</td>
			<td>746000</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: CD90</td>
			<td>BD BioSciences</td>
			<td>553011</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal antibody: HLA-DR</td>
			<td>BD BioSciences</td>
			<td>340827</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline) 1x pH 7.4</td>
			<td>Gibco</td>
			<td>&#160;10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Adipogenesis Differentiation Kit</td>
			<td>Gibco</td>
			<td>A1007001</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Chondrogenesis Differentiation Kit</td>
			<td>Gibco</td>
			<td>A1007101</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Osteogenesis Differentiation Kit</td>
			<td>Gibco</td>
			<td>A1007201</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile connector with one spike with needle injection site</td>
			<td>Origen Biomedical Connector, USA</td>
			<td>NA</td>
			<td>Code mark: IBS</td>
		</tr>
		<tr>
			<td>Trypan blue solution 0.4%</td>
			<td>Sigma Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25% 1x, phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

technique for obtaining mesenchymal stem cell from adipose tissue and stromal vascular fraction characterization in long-term cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The characterization of the nine individuals studied, including their age, weight, height, and BMI, are shown in Table 1.
According to the cellular yield initially presented, the cell volume inoculated in culture was calculated to be as close as possible to the capacity of the 75 cm2 culture flask. The sample volume seeded in each case is described in Table 2. Then, according to the initial cellular yield, a variable volume of cells for each sample ...

Watch this Scientific Journal Video about Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation at JoVE.com

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

technique-for-obtaining-mesenchymal-stem-cell-from-adipose-tissue

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2.8K Views.  Universidade Federal de São Paulo (UNIFESP). The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Video: Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

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 ...

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 ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

Hickman Catheter for Long-Term Vascular Access in Swine

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood monitoring and reliable intravenous fluid and drug administration. Specifically, the tunneled multi-lumen Hickman catheter (HC) is commonly used in swine models due to its lower extrication and complication rates. Despite fewer complications relative to other CVCs, HC-related morbidity presents a significant challenge, as it can significantly delay or otherwise negatively impact ongoing studies. The proper insertion and maintenance of HCs is paramount in preventing these complications, but there is no consensus on best practices. The purpose of this protocol is to comprehensively describe an approach for the insertion and maintenance of a tunneled HC in swine that mitigates HC-related complications and morbidity. The use of these techniques in &gt;100 swine has resulted in complication-free patent lines up to 8 months and no catheter-related mortality or infection of the ventral surgical site. This protocol offers a method to optimize the lifespan of the HC and guidance for approaching issues during use.

Author Spotlight: Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model  

A reliable and reproducible approach for the insertion and maintenance of a tunneled Hickman catheter for long-term vascular access in swine is described. Placement of a central venous catheter allows for convenient daily sampling of whole blood from awake animals and intravenous administration of medication and fluids.

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood ...

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 ...

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...