Name: technique for obtaining mesenchymal stem cell from adipose tissue and stromal vascular fraction characterization in long-term cryopreservation
Uploaded: 2021-12-30T16:00:00
Description: 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

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 pr...

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 ...

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