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 cells, maintaining high viability, and extracting the maximum number of cells from the initial material while accounting for the long-term culture and manipulation of the cells. Therefore, straight culture maintenance is required to keep the cells away from apoptosis, senescence, or genetic instability, since cell therapy is likely to be effective and safe for patients.
To the best of our knowledge, there is no previous article using this set of steps for the isolation of mesenchymal stem cells derived from lipoaspirate, which results in a time-saving and cost-benefit technique. In this study, each methodological step was reasoned according to the literature that showed the highest final cell yield in cellular isolation. The novelty of the technique performed in this study was the use of manual aspiration associated with a series of lipoaspirate washes, with the subsequent bag resting. The collection bag used to transport and process lipoaspirate allowed undigested tissue fragments to not participate in collagenase digestion. The most critical step is adding calcium chloride to the fresh digestion solution as it potentiates the action of collagenase. The time gain is not yet reported in the literature with a cell thawing method that allows viable cells even after long cryopreservation time. The SVF cellular yield found in this study varied broadly from 6.15 to 26.2 x 105 cells/mL with an average of 15.7 x 105 cells/mL. This may be due to the presence of a more significant amount of adrenaline solution suctioned, which may have been higher or lower according to the stage of the surgical procedure and to the number of other known cell types generally found in the SVF. Although some studies have found negative correlations between BMI and ADSC yield36,37, this study found no significant correlation, like the other two studies38,39, decreasing the possibility of that being the cause of the incredible variety of SVF cellular yield found in this study. These data show that the lowest SVF cellular yield obtained was 6.15 x 105 cells/mL. Some studies had already measured the efficiency of ADSC isolation according to the surgical technique for obtaining lipoaspirate. One study obtained 0.087 x 105 cells/mL in freshly isolated SVF for the liposuction technique using adrenaline solution (as used in this study) and 0.143 x 105 cells/mL without it26. This work highlighted the significance of the adrenaline solution injection due to the vasoconstrictive effect that decreases intraoperative bleeding and bruising, as the majority of surgeons choose to perform in clinical practice. Another study demonstrated that live ADSCs isolated ranged from 0 to 0.59 x 105 cells/g lipoaspirate harvested, with an average of 0.295 (&#177;0.25) x 105 cells/g tissue31. Some studies have tested different ways to achieve higher ADSC yield. One of these studies achieved about 350 x 105 for the method that presented various constituents in collagenase digestion buffer and the use of an orbital shaker40. Another study showed 29.7 (&#177;0.2) x 105 cells/mL as the total number of SVF cells from the abdominal area41. The abdominal area chosen in this study is still the reference area for the best availability and accessibility of lipoaspirate42. The body region for lipoaspirate collection, among other factors as donor age and method of the collection chosen, is a strong determinant of the quality of the ADSC yields.
The possibility of microorganisms&#39; contamination causing the unavailability of cells to continue the experiments was the only execution problem that limited the completion of this study. Even using antibiotics and Good Manufacturing Practice requirements, contamination can occur due to the lack of a total aseptic environment to perform the aspiration of fat.
SVF immunophenotyping 
According to The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy34, one of the three minimal criteria to define human mesenchymal stem cells is that the cells must express CD10534, CD7334, and CD9034 and should not express CD4534, CD3434, CD1434, or CD11b34, CD79a34 or CD1934, and HLA-DR34 surface membrane molecules. Mitchell43 tested fresh SVF cells by immunophenotyping and found a maximum of 54% of cells with ADSC surface markers. In this study, the immunophenotyping revealed a higher percentage of confirmed ADSC (non-hematopoietic cells) of up to 78.91% in the SVF after long-term cryopreservation (ranging from 37.95%-78.91% with a mean of 53.68%). Evidence shows that the progenitors of a stem cell population not yet committed do not express the CD34 marker34,44,45. Depending on the stage of differentiation, the CD3434 negative stem cells can generate not only hematopoietic progenitors but also more specific mesenchymal precursors, such as osteoclasts, chondrocytes, myocytes, adipocytes, and others. Some studies demonstrated the striking plasticity of the primitive stem cell population, composed of cells with stromal cell function and hematopoietic and mesenchymal progenitors45. According to the literature, the complete CD3434 functional role in the tissue formation in SVF cells is still unknown46. Mitchell43 showed a mean of 60% cells of SVF expressing CD3434 marker, whereas, in this study, the mean was 78.81%. It is known that the expression of CD3434 surface marker decreases along passages.
Adherent cells and differentiation assay 
Depending on the number of cells obtained after isolation, the number of cells seeded in the first culture varied. The first culture time for reaching 80%-90% confluence in 75 cm2 flasks took an average of 8.4 days and a standard deviation of 7.5 (&#177;4.5) days ranging from 6.6 to 16.1 x 105 cells/mL. It is to be noted that even for the cases with lower cellular yield, the first culture time led to high cellular yield indexes compared to the literature, probably due to the best availability of viable cells maintained during the entire collection and isolation process. One study obtained a cellular yield of 3.75 (&#177;1.42) x 105 ADSC per mL of lipoaspirate within a 4.1 (&#177;0.7) day culture period47. Another study demonstrated a yield of nucleated SVF cells of 3.08 (&#177;1.40) x 105 per millimeter with a mean of 6.0 (&#177;2.4) days in the first culture period43. In this study, by estimating the number of ADSC seeded in culture, which varied as a function of the number of cells observed in the SVF from the collection of the same volume of 100 mL of lipoaspirate, it was verified that the number of days to reach P1 had no relation to cell volume. For example, for Case 9, in which 12.2 x 105 cells were incubated, 6 days were required to reach 80%-90% confluence. For Case 6, in which 14.6 x 105 cells were seeded in culture, a more extended period was necessary (10 days) to reach up to the same level of confluence. Perhaps, a minimum ADSC number is enough for the first adhesion period. There may be significant interindividual variation, such as comorbidities, age, and general health status. Some studies in the literature questioned the importance of considering the patients&#39; inter-individual variability for SVF cell yield48,49.
According to The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy34, mesenchymal cell must have the ability to differentiate into three different cell types as osteogenic, adipogenic and chondrogenic lineages as it was demonstrated in Figure 4.
Cell viability, cryopreservation, and genetic instability 
Different methods have been used to determine viability loss, defined as plasma membrane integrity damage50. However, the cultures can also present early apoptotic cells that these approaches can ignore because they maintain an intact plasma membrane, but they are nonviable. The results reported here show a great range in viability marker,&#160;from 23.06%-72.34%, with a mean of 47.6% after long-term cryopreservation. Considering that cryopreservation is a significant step concerning cell therapy, recovery of the maximum number of viable and functional stem cells after thawing is one of the priority issues for the success of cell therapy. The literature has shown that at least 50% of cell viability is lost from 1-4 months after cryopreservation43. Notably, the lowest indexes presented in this study are from Case 5, Case 4, and Case 2, which are the oldest cryopreserved samples (about 2 years). Although they presented the lowest viability indexes, they demonstrated high cellular yield in trypan blue dye exclusion assay in fresh SVF. Although the literature supports causes of viability loss, these rates are lower than expected. Temperature fluctuations in cell storage due to technical reasons can cause the increase and accumulation of stress and favor the accumulation of aqueous portions, generating crystals that damage the plasmatic membrane during long-term cryopreservation51. The literature shows more than 70% viability for samples with the same or longer freezing time. However, the viability check was performed with a different technique than that carried out in this study52.&#160;Another study showed that longer cryopreservation negatively affects cellular viability31, which can be explained by temperature variations in the -80 &#176;C freezer. Hence, cells often need to stay too long in culture, which increases cell cycle stress, bringing risks for genetic instability and consequently compromising cellular therapy. There is also a consensus in the literature indicating some stress factors and how they affect cell cytogenetic stability, which is essential to maintain the prolonged stem cell cultivation required for cell therapy35,53.
Methodology benefit 
To date, literature shows no standardized protocol to isolate ADSC aiming for clinical applications. Most of the studies demonstrate complex, time-consuming protocols24. In this study, the efficiency of the method versus the time demanded to complete the initial cellular yield must be emphasized: about 1.5 h. According to literature, isolation of adipose-derived stem cells can take about 3 h to 8 h54,55. Thus, the gain of time allied to the high cellular income is critical for regenerative medicine therapeutics advancement. More cell viability assessments should be carried out parallel to those performed in this work to improve this method. Further randomized controlled trials incorporating a more extensive sample using this methodology are required to countersign these results.

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><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&#039;s modified Eagle&#039;s medium)</td><td>Gibco</td><td>11966025</td><td></td></tr><tr><td>DPBS no calcium, no magnesium (Dulbecco&#039;s Phosphate Buffered Saline Gibco Cell Therapy Systems)</td><td>Gibco</td><td>A1285801</td><td></td></tr><tr><td>DPBS with calcium (Dulbecco&#039;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>&amp;nbsp;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 was determined: 1 mL for samples with higher cellular yield, 1.1 mL for samples with intermediate cellular yield, and 2 mL for samples with lower cellular yield so as to perform more similar cell seeding between cases. When the culture reached about 80%-90% confluence (Figure 2A) (about 7.5 &#177; 4.5 days), trypsinization of adherent cells was carried out (Table 2 and Figure 2B).
The cellular yield before passage 1 broadly varied even when the same confluence before trypsinization was observed (Table 2). This can be explained by the fact that cells may have grown in layers. Different parameters from the patients&#39; ADSC were also assessed at different periods, as demonstrated in Table 2.
Some samples (Case 1, Case 2, Case 7) could not be evaluated regarding the percentage of confirmed ADSC and the estimated number of ADSC in culture due to bacteria contamination and lack of available cells to perform cryopreserved SVF immunophenotyping. According to the Spearman&#39;s Rho Calculator33, no statistical differences were found between SVF cellular yield and days to P1 (r = 0.37816, p = 0.31561), between SVF cellular yield before and after going to P1 (r = -0.33333, p = 0.38071), and between days to P1 and cellular yield before going to P1 (r = -0.53783, p = 0.13529). Furthermore, no significant differences were observed when correlating the SVF cellular yield with the average percentage of confirmed ADSC (r = -0.02857, p = 0.95716) and between the average percentage of confirmed ADSC and the cellular yield before going to P1 (r = 0.42857, p = 0.3965). Also, the correlation between BMI and SVF cellular yield could not be considered statistically significant (r = -0.46667, p = 0.20539). Table 3 shows flow cytometric data performed on SVF cells cryopreserved. The initial SVF cells contained a subset of positive cells for hematopoietic markers (CD45, CD11b, CD19, HLA-DR)34. From the initial SVF cell population, a particular subgroup expressed CD11b34 and CD1934 stromal cell-associated markers. The levels of CD7334, CD9034, and CD10534 were intermediate between these values. The initial SVF contained a subpopulation of cells positive for stem cell-associated markers (Figure 3). A mean of 79% of SVFs expressed the HSC-associated marker CD3434.
In total, 21 min were necessary for the three washes, 30 min for collagenase digestion, 10 min for centrifugation, and 5 min for cell counting and plating.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63036/63036fig01.jpg" /> 
Figure 1: Steps from the protocol adipose-derived stem cells isolation. (A) Bag for lipoaspirate transport in a closed system. (B) The step of resting bag repeated three times, after washing. (C) Lipoaspirate after three washes with DPBS. (D) Lipoaspirate after collagenase digestion. (E) Lipoaspirate after digestion is distributed in a 50 mL tube. (F) Digested lipoaspirate after centrifugation. (G) Final process isolation with the pellet with the stromal vascular fraction (SVF). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63036/63036fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63036/63036fig02.jpg" /> 
Figure 2: Morphology and viability of ADSCs. (A) Plastic adherent mesenchymal adipose-derived stem cells at the first passage after isolation at light microscopy. The cells show adhesion to the plastic and fibroblast-like morphology. (B) Trypan blue assay showing viable cells counted in Neubauer chamber using a light microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63036/63036fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63036/63036fig03.jpg" /> 
Figure 3: Subpopulation of cells positive for stem cell-associated markers in SVF of Case 9 after 8 months of cryopreservation. R1: Total cellular region analyzed in FSC (Forward Scatter) x SSC (Side Scatter) (Size x Complexity); R2: CD45 negative region, whose populations CD73, CD90, and CD105 are positive in this region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63036/63036fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63036/63036fig04.jpg" /> 
Figure 4: Differentiation assay. (A) ADSC differentiation in chondrocytes. (B) ADSC differentiation in osteocytes. (C) ADSC differentiation in adipocytes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63036/63036fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Patient</td>
			<td>Age at collection (years)</td>
			<td>Weight (kg)</td>
			<td>Height (meter)</td>
			<td>BMI*</td>
		</tr>
		<tr>
			<td>Case 1</td>
			<td>35</td>
			<td>68</td>
			<td>1.64</td>
			<td>25.28</td>
		</tr>
		<tr>
			<td>Case 2</td>
			<td>33</td>
			<td>65</td>
			<td>1.65</td>
			<td>23.88</td>
		</tr>
		<tr>
			<td>Case 3</td>
			<td>35</td>
			<td>70</td>
			<td>1.68</td>
			<td>24.8</td>
		</tr>
		<tr>
			<td>Case 4</td>
			<td>34</td>
			<td>72</td>
			<td>1.64</td>
			<td>26.77</td>
		</tr>
		<tr>
			<td>Case 5</td>
			<td>36</td>
			<td>72</td>
			<td>1.69</td>
			<td>25.21</td>
		</tr>
		<tr>
			<td>Case 6</td>
			<td>36</td>
			<td>67</td>
			<td>1.64</td>
			<td>24.91</td>
		</tr>
		<tr>
			<td>Case 7</td>
			<td>38</td>
			<td>62</td>
			<td>1.53</td>
			<td>26.49</td>
		</tr>
		<tr>
			<td>Case 8</td>
			<td>50</td>
			<td>63</td>
			<td>1.68</td>
			<td>22.32</td>
		</tr>
		<tr>
			<td>Case 9</td>
			<td>37</td>
			<td>65</td>
			<td>1.58</td>
			<td>26.04</td>
		</tr>
	</tbody>
</table>
Table 1: Data from the samples of the individuals studied. *BMI: body mass index.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Patient</td>
			<td>Volume collected (mL)</td>
			<td>SVF Cellular yield (cell/mL) (x 105)</td>
			<td>Volume in culture (mL)</td>
			<td>Average percentage of confirmed ADSC (%)</td>
			<td>Number of cells in initial culture (x 105)</td>
			<td>Estimated number of ADSC in culture (x 105)</td>
			<td>Days to P1</td>
			<td>Cellular yield before going to P1 (x 105)</td>
		</tr>
		<tr>
			<td>Case 1</td>
			<td>96</td>
			<td>9.2</td>
			<td>2</td>
			<td>na</td>
			<td>18.4</td>
			<td>na</td>
			<td>10</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Case 2</td>
			<td>100</td>
			<td>25.2</td>
			<td>1</td>
			<td>38</td>
			<td>25.2</td>
			<td>9.6</td>
			<td>12</td>
			<td>10.8</td>
		</tr>
		<tr>
			<td>Case 3</td>
			<td>100</td>
			<td>26.2</td>
			<td>1</td>
			<td>na</td>
			<td>26.2</td>
			<td>na</td>
			<td>12</td>
			<td>6.6</td>
		</tr>
		<tr>
			<td>Case 4</td>
			<td>105</td>
			<td>21.1</td>
			<td>1</td>
			<td>55.9</td>
			<td>21.1</td>
			<td>11.8</td>
			<td>3</td>
			<td>13.1</td>
		</tr>
		<tr>
			<td>Case 5</td>
			<td>110</td>
			<td>23.7</td>
			<td>1</td>
			<td>61.4</td>
			<td>23.7</td>
			<td>14.5</td>
			<td>4</td>
			<td>16.1</td>
		</tr>
		<tr>
			<td>Case 6</td>
			<td>100</td>
			<td>13.3</td>
			<td>1.1</td>
			<td>78.9</td>
			<td>14.6</td>
			<td>11.5</td>
			<td>10</td>
			<td>13.5</td>
		</tr>
		<tr>
			<td>Case 7</td>
			<td>98</td>
			<td>6.8</td>
			<td>2</td>
			<td>na</td>
			<td>13.6</td>
			<td>na</td>
			<td>8</td>
			<td>10.5</td>
		</tr>
		<tr>
			<td>Case 8</td>
			<td>100</td>
			<td>9.7</td>
			<td>1.1</td>
			<td>44.2</td>
			<td>10.7</td>
			<td>4.7</td>
			<td>11</td>
			<td>6.9</td>
		</tr>
		<tr>
			<td>Case 9</td>
			<td>100</td>
			<td>6.1</td>
			<td>2</td>
			<td>43.8</td>
			<td>12.2</td>
			<td>5.3</td>
			<td>6</td>
			<td>15.9</td>
		</tr>
		<tr>
			<td>SD</td>
			<td>3.89</td>
			<td>7.81</td>
			<td>0.46</td>
			<td>13.75</td>
			<td>5.55</td>
			<td>3.53</td>
			<td>3.2</td>
			<td>3.79</td>
		</tr>
	</tbody>
</table>
Table 2: Data from different steps of the procedure from the nine patients analyzed. SVF: stromal vascular fraction; ADSC: adipose-derived stem cell; P1: passage 1; na: data not available.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">SAMPLE</td>
			<td colspan="5">% Of ADSC DETERMINED BY MONOCLONAL ANTIBODIES</td>
			<td colspan="4">% OF HEMATOPOIETIC CELLS DETERMINED BY MONOCLONAL ANTIBODIES</td>
			<td colspan="3">CELL VIABILITY ASSAY AND TIME OF SVF CRYOPRESERVATION</td>
		</tr>
		<tr>
			<td>CD45-(*)</td>
			<td>CD73+/CD90+</td>
			<td>CD73+/CD105+</td>
			<td>CD105+/CD90+</td>
			<td>Mean</td>
			<td>CD34+</td>
			<td>HLA-DR+</td>
			<td>CD11b+</td>
			<td>CD19+</td>
			<td colspan="3">LIVE/DEAD +</td>
		</tr>
		<tr>
			<td>Case 2</td>
			<td>52.34%</td>
			<td>31.97%</td>
			<td>25.36%</td>
			<td>56.52%</td>
			<td>37.95%</td>
			<td>63.16%</td>
			<td>12.87%</td>
			<td>2.41%</td>
			<td>0.21%</td>
			<td colspan="3">39.54% (2 years)</td>
		</tr>
		<tr>
			<td>Case 4</td>
			<td>48.02%</td>
			<td>61.62%</td>
			<td>40.93%</td>
			<td>65.25%</td>
			<td>55.93%</td>
			<td>82.94%</td>
			<td>26.62%</td>
			<td>0.00%</td>
			<td>0.16%</td>
			<td colspan="3">38.30% (2 years)</td>
		</tr>
		<tr>
			<td>Case 5</td>
			<td>27.74%</td>
			<td>54.02%</td>
			<td>49.72%</td>
			<td>80.42%</td>
			<td>61.38%</td>
			<td>73.33%</td>
			<td>51.31%</td>
			<td>0.05%</td>
			<td>0.00%</td>
			<td colspan="3">23.06% (2 years)</td>
		</tr>
		<tr>
			<td>Case 6</td>
			<td>55.52%</td>
			<td>79.52%</td>
			<td>67.70%</td>
			<td>89.52%</td>
			<td>78.91%</td>
			<td>86.86%</td>
			<td>8.83%</td>
			<td>0.18%</td>
			<td>1.06%</td>
			<td colspan="3">56.76% (2 years)</td>
		</tr>
		<tr>
			<td>Case 8</td>
			<td>57.28%</td>
			<td>46.84%</td>
			<td>30.88%</td>
			<td>57.65%</td>
			<td>45.12%</td>
			<td>78.47%</td>
			<td>26.97%</td>
			<td>0.03%</td>
			<td>0.00%</td>
			<td colspan="3">55.56% (1 year)</td>
		</tr>
		<tr>
			<td>Case 9</td>
			<td>56.14%</td>
			<td>47.52%</td>
			<td>36.30%</td>
			<td>47.69%</td>
			<td>43.83%</td>
			<td>88.10%</td>
			<td>26.94%</td>
			<td>0.05%</td>
			<td>0.24%</td>
			<td colspan="3">72.34% (8 months)</td>
		</tr>
	</tbody>
</table>
Table 3: Flow cytometry data from six of the patients. (*) From these CD45- cells, % of ADSC with different combinations of stem cell markers was determined. ADSC: adipose-derived stem cell; SVF: stromal vascular fraction; (*) From these CD45- cells, % of ADSC with different combinations of stem cell markers was determined.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

3.0K Views.  Universidade Federal de São Paulo (UNIFESP). Hier hebben we het protocol gedemonstreerd voor het verkrijgen en behouden van de goede en levensvatbare cellen voor gebruik van therapeutische stappen, zoals de infusie van pluripotente mesenchymale cellen. Het belangrijkste voordeel hiervan is dat we met behulp van deze techniek het levensvatbare celvolume kunnen verkrijgen versus de tijd die is gewonnen in de isolatiestap van de cellen uit het verzamelde weefsel. Deze techniek kan worden uitgebreid naar alle ziekten of aandoeningen waarbij...

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

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Developmental Biology

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Biology

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. ...

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 ...

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

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

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

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

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

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 ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Primary Culture of Adipose Tissue Derived Stem Cells from Mouse Epididymis

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...

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

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