Name: isolation, proliferation and differentiation of rhesus macaque adipose-derived stem cells
Uploaded: 2021-05-26T12:30:00
Description: Watch this Scientific Journal Video about Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells at JoVE.com

The authors would like to thank Curtis Vande Stouwe for his technical assistance. The research underlying development of the protocols was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (5P60AA009803-25, 5T32AA007577-20 and 1F31AA028459-01).

All obtained tissues and procedures were approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center and were performed in accordance with the guidelines of the National Institute of Health (NIH publication No. 85-12, revised 1996).
1. Preparation of buffers and solutions
<ol>
	<li>Prepare sterile 5-phosphate-buffered saline wash buffer (5-PBS) solution by adding 5% penicillin/streptomycin (pen/strep) and 0.25 &#956;g/mL of fungal inhi...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+regulates+insulin+sensitivity:+role+of+adipogenesis,+de+novo+lipogenesis+and+novel+lipids.">Adipose tissue regulates insulin sensitivity: role of adipogenesis, de novo lipogenesis and novel lipids.</a> Journal of Internal Medicine. 280 (5), 465-475 (2016).</li><li>Laclaustra, M., Corella, D., Ordovas, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+syndrome+pathophysiology:+the+role+of+adipose+tissue.">Metabolic syndrome pathophysiology: the role of adipose tissue.</a> Nutrition, Metabolism &amp; Cardiovascular Disease. 17 (2), 125-139 (2007).</li><li>Grundy, S. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+adipose+tissue+and+adipocyte+function+in+the+pathogenesis+of+metabolic+syndrome.">Altered adipose tissue and adipocyte function in the pathogenesis of metabolic syndrome.</a> Journal of Clinical Investigation. 129 (10), 3990-4000 (2019).</li><li>Sarjeant, K., Stephens, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipogenesis.">Adipogenesis.</a> Cold Spring Harbor Perspectives in Biology. 4 (9), 008417 (2012).</li><li>Ford, S. 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ADSC isolation, proliferation and differentiation protocols are straight-forward and reproducible, but they require careful technique to ensure adequate isolation, healthy expansion, and efficient differentiation. A sterile working environment is critical for all cell culture experiments. Bacteria or fungi may be introduced into cell cultures through contaminated tools, media or work environment. Fungal contamination is indicated by spore growth in the culture, while bacterial contamination is indicated by the presence o...

Adipose tissue is comprised of a heterogeneous mixture of cells, predominantly mature adipocytes and a stromal vascular fraction including fibroblasts, immune cells and adipose-derived stem cells (ADSCs)1,2,3. Primary ADSCs can be isolated directly from white adipose tissue and stimulated to differentiate into adipocytes, cartilage or bone cells4. ADSCs exhibit classical stem cell characteristics such as maintenance of multipotency in vitro and self-renewal; and are adherent to plastic in culture5,6. ADSCs are of important interest for the use in regenerative medicine due to their multipotency and ability to be easily harvested in large quantities using non-invasive techniques7. Adipogenic differentiation of ADSCs produces cells that functionally mimic mature adipocytes including lipid accumulation, insulin-stimulated glucose uptake, lipolysis, and adipokine secretion8. Their resemblance to mature adipocytes has led to the widespread use of ADSCs for physiological investigation of cellular characteristics and metabolic function of adipocytes. There is increasing evidence&#160;supporting the idea that the development of metabolic dysfunction and disorders originates at the cellular or tissue level9,10,11,12. Optimal ADSC differentiation is required for sufficient adipose tissue expansion, proper adipocyte function, and effective metabolic regulation13.
Protocols described in this manuscript are straightforward techniques utilizing standard laboratory equipment and basic reagents. The manuscript first describes the protocol for the isolation of primary ADSCs from fresh adipose tissue using mechanical and enzymatic digestion. Next, the protocol for proliferation and passaging of ADSCs in stromal medium is described. Lastly, the protocol for adipogenic differentiation of ADSCs is described. Following differentiation, these cells can be used for studies to better understand adipocyte metabolism and mechanisms of dysfunction. The protocols for confirmation of adipogenic differentiation and lipid droplet detection using Oil Red O and boron-dipyrromethene (BODIPY) staining are also described. The details of these protocols focused on primary ADSCs isolated from fresh omental adipose tissue of rhesus macaques. We and others have used this protocol to successfully isolate ADSCs from rhesus macaque subcutaneous and omental adipose tissues depots14,15. For the same amount of tissue used, we have observed that subcutaneous adipose tissue is more dense, tougher and yields less cells from digestion compared to omental adipose tissue. This protocol has also been used to isolate ADSCs from human adipose samples16.

<table>
	<tbody>
		<tr>
			<td>0.4 % trypan blue</td>
			<td>Thermo-Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5-ml microcentrifuge tube</td>
			<td>Dot Scientific</td>
			<td>707-FTG</td>
			<td></td>
		</tr>
		<tr>
			<td>100 % isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>PX1838-P</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm cell culture dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL plastic conical tube</td>
			<td>Fisher Scientific</td>
			<td>50-465-232</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#181;m cell strainer</td>
			<td>Corning</td>
			<td>CLS431751</td>
			<td></td>
		</tr>
		<tr>
			<td>a-MEM</td>
			<td>Thermo-Fisher</td>
			<td>12561056</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds Wrap</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY</td>
			<td>Thermo-Fisher</td>
			<td>D3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>05470</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type I</td>
			<td>Thermo-Fisher</td>
			<td>17100017</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone-Water Soluble</td>
			<td>Sigma-Aldrich</td>
			<td>D2915</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide, DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Thermo-Fisher</td>
			<td>15230162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, USDA-approved</td>
			<td>Sigma-Aldrich</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone/Amphotericin B (250 ug/mL)</td>
			<td>Thermo-Fisher</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Thermo-Fisher</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer with cover slip</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Nikon</td>
			<td>DIAPHOT-TMD</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Thermo-Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory rocker, 0.5 to 1.0 Hz</td>
			<td>Reliable Scientific</td>
			<td>Model 55 Rocking</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral buffered formalin (10 %)</td>
			<td>Pharmco</td>
			<td>8BUFF-FORM</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>O0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformeldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo-Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Thermo-Fisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell (RBC) lysis buffer</td>
			<td>Qiagen</td>
			<td>158904</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes, 2 to 25 mL</td>
			<td>Costar Stripettes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard humidified cell culture incubator, 37 &#176;C, 5 % CO2</td>
			<td>Sanyo</td>
			<td>MCO-17AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo-Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, proliferation and differentiation of rhesus macaque adipose-derived stem cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

The ADSCs isolated from rhesus macaque adipose tissue samples were seeded on culture plates and is shown in Figure 1. On the day of plating, cells are non-adherent and float in the culture dish as shown in Figure 1A. Within 72 h, ADSCs will become 80% confluent and are ready for adipocyte differentiation (Figure 1B). ADSCs exhibit strong adipogenic characteristics after chemical induction. After 14 days of differentiation, mature ad...

Watch this Scientific Journal Video about Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells at JoVE.com

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Adipose-derived stem cell isolation and proliferation produces a large number of stem cells that can be used for several downstream applications and experimental techniques. A major intended use for differentiated adipocytes in this protocol is metabolic assays such as insulin stimulated glucose uptake, lipogenesis, and stimulated lipolysis. To begin, create a sterile working environment by disinfecting the biosafety hood and all the tools.Pipette approximately 10 milliliters of five PBS buffer into four 100 millimeter cell culture dishes. Transfer a 50 gram adipose sample into one of the dishes and wash it four times by sequentially transferring it across the other dishes containing five PBS. Then transfer the washed adipose tissue into a clean 100 millimeter culture dish and thoroughly mince it using scissors or sterile forceps.Transfer a one to three centimeter section of the minced tissue to a 50 milliliter conical tube containing 13 milliliters of collagenase buffer. Rinse the remaining tissue from the culture dish with two milliliters of collagenase buffer to ensure a total volume of 15 milliliters. Thoroughly mix the sample using a 25 milliliter serological pipette and incubate the tube at 37 degrees Celsius on a rocker for 30 to 60 minutes.In order to neutralize the enzyme activity, add 10 milliliters of ADSC growth medium into the tube after incubation and mix it well with a pipette to separate any tissue aggregates. Transfer the liquid portion to a new sterile 50 milliliter conical tube, leaving the solid tissue. Wash the tissue three times using seven milliliters of two PBS and transfer the liquid to the new tube.Next, centrifuge the tube at 500 times G for five minutes and carefully remove as much supernatant as possible without disturbing the pellet. Resuspend the pellet in one milliliter of 1X red blood cell or RBC lysis buffer and incubate it at room temperature for 10 minutes. Then wash the cells twice by adding five milliliters of ADSC growth medium to the tube and centrifuging to remove the supernatant.After the last wash, resuspend the pellet in two milliliters of ADSC growth medium and filter it through a 70 micron cell strainer into a sterile 50 milliliter conical tube. Rinse the strainer with an additional two milliliters of the medium and transfer four milliliters of the suspension into a sterile 100 milliliter culture dish. Rinse the conical tube twice with three milliliters of medium to collect a total volume of 10 milliliters in the culture dish.Observe the collected cells under an inverted microscope at 10X magnification to check for floating cells. Incubate the cells for 24 hours in a carbon dioxide incubator at 37 degrees Celsius, 5%carbon dioxide and 100%relative humidity. To ensure sterility of the culture media, include a plate with media alone.After 24 hours, view the cells under the inverted microscope to check for cell adherence. Remove and replace the medium with warm medium every 48 hours until cells are 80 to 90%confluent. For proliferation, remove the growth medium from the confluent ADSC cells and wash them twice with two milliliters of sterile room temperature PBS.Aspirate the PBS and add two milliliters of 0.25%trypsin EDTA into each culture plate, covering the entire surface area of the plate. Incubate the plate for seven minutes at 37 degrees Celsius and 5%carbon dioxide, then mechanically dislodge the trypsinized cells by forceful pipetting. Add two milliliters of ADSC growth medium and gently mix the cells.Transfer the cells into a 50 milliliter conical tube, rinsing the dish with an additional two milliliters of medium to ensure maximal cell recovery from the plate. Next, centrifuge the tube at 500 times G for five minutes at room temperature and decant the supernatant to obtain the cell pellet. Resuspend the cell pellet in five to six milliliters of ADSC growth medium per million cells.Count the cells using a hemocytometer and plate them as described in the text manuscript. Once the cells reach 80 to 90%confluency, aspirate the growth medium and rinse the cells with sterile room temperature PBS, then add 10 milliliters of ADSC differentiation medium. Replace the medium every three days for approximately 14 to 21 days.Observe the cells for the presence of lipid droplets under the inverted microscope at 40X magnification. Mature adipocytes are generally observed after 14 days. On the day of plating, the ADSCs were non-adherent and floated in culture.The cells became 80%confluent after 72 hours and were ready for adipocyte differentiation. After 14 days of ADSC differentiation, mature adipocytes exhibited strong adipogenic characteristics which were observed under 40X magnification. On day 14, cells were stained and fixed with Oil Red O and BODIPY for lipid droplet visualization.Differentiated adipocytes could be observed under 20X magnification. When attempting this protocol, it is important to remember that a sterile working environment is critical for healthy isolation, adequate expansion, and efficient differentiation of adipose-derived stem cells. Following this procedure, these cells can be used for several metabolic assays such as glucose uptake, lipogenesis, and lipolysis which is a major focus of our research.This technique allows for the investigation of how chronic alcohol and SIV impact adipocyte metabolic capacity.

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