isolating adipose-derived stem cells from murine periaortic adipose tissue

Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue

Transfer the harvested adipose tissue into a new 2-milliliter microcentrifuge tube, containing 1 milliliter of freshly prepared digestion medium, and use surgical scissors to mince the tissues. Transfer the tissue slurry into a 50-milliliter tube containing 9 milliliters of fresh digestion medium, and use a 1-milliliter pipette to triturate the tissues 10 times.
When a homogeneous solution has been obtained, incubate the tube for 30 to 45 minutes at 37 degrees Celsius and 100 revolutions per minute, checking every 5 to 10 minutes to prevent over-digestion. When a light yellow homogeneous adipose tissue solution can be observed upon gentle swirling of the tube, stop the digestion with 5 milliliters of HDMEM supplemented with 10% fetal bovine serum. After a thorough mixing, centrifuge the adipose tissue sample.
The stromal vascular cell fraction will be visible as a brownish pellet. Resuspend the pellet in 10 milliliters of culture medium, and filter the cell suspension through a 70-micrometer cell strainer.
Collect the cells by another centrifugation, and gently resuspend the pellet in 5 milliliters of erythrocyte lysis buffer. Transfer the suspension to a 15-millimeter tube. After 10 minutes, stop the reaction with two centrifugations in 10 milliliters of PBS, supplemented with 1% fetal bovine serum.
After the second centrifugation, resuspend the pellet in 5 milliliters of culture medium on ice, and collect the cells with a final centrifugation. Then, resuspend the cells in 5 milliliters of FACS buffer for counting.
To set up an NCADSC culture after their isolation by FACS according to standard protocols, place the cells at a 5 x 103 cells per cm2 concentration in each well, a 12-well culture plate in complete culture medium, at 37 degrees Celsius, and 5% carbon dioxide for 20 to 24 hours. The next day, wash the cells with 37 Celsius PBS, and feed the culture with fresh culture medium.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of the stromal vascular fraction (SVF)
Collect the periaortic arch adipose tissue (PAAT) of 5-6 mice into one 2 mL microcentrifuge tube containing 1 mL freshly prepared digestion medium and mince the tissue using surgical scissors in an Eppendorf tube at room temperature (RT).
<ol>
	<li>Transfer the mix into 50 mL tubes containing 9 mL of the digestion medium. Homogenize the tissues by pipetting up and down with a 1 mL pipette 10x.</li>
	<li>Incubate the tubes at 37 &#176;C with constant shaking at 100 rpm for 30&#8211;45 min and check every 5&#8211;10 min to prevent overdigestion. This is critical to improving cell viability and yield. 
	NOTE: Good tissue digestion will result in a homogenous, light yellow adipose tissue that is visible to the naked eye upon gently swirling the tube.</li>
	<li>Stop the digestion by adding 5 mL of HDMEM containing 10% FBS and 1% v/v PS at RT and mix well by pipetting.</li>
	<li>Centrifuge the cell suspension at 500 x g for 5 min at RT. The SVF will be visible as a brownish pellet. Carefully aspirate the floating adipocytes and decant the remaining supernatant without disturbing the SVF. Dissolve the SVF pellet in 10 mL of culture medium and filter through a 70 &#181;m cell strainer.</li>
	<li>Centrifuge the cell suspension at 500 x g for 5 min, remove the supernatant, and gently resuspend the pellet in 5 mL of erythrocyte lysis buffer in a 15 mL conical tube for 10 min at RT.</li>
	<li>Stop the reaction by adding 10 mL of 1x PBS containing 1% FBS. Centrifuge the cell suspension at 500 x g for 5 min at 4 &#176;C, remove the supernatant, and resuspend the pellet in 10 mL of 1x PBS containing 1% FBS.</li>
	<li>Centrifuge the cells again at 500 x g for 5 min at 4 &#176;C. Remove the supernatant and resuspend the pellet in 5 mL of culture medium in a 15 mL conical tube at 4 &#176;C.</li>
	<li>After a final round of centrifugation (500 x g for 5 min at 4 &#176;C), resuspend the pelleted cells in 5 mL of FACS buffer (PBS containing 10% FBS, 100 units/mL DNA I, and 1% v/v PS) on ice, and count the cells with a hemocytometer.</li>
</ol>
2. Isolation of NCADSCs by FACS
<ol>
	<li>Set up and optimize the cell sorter following the instruction manual. Select the 100 &#181;m nozzle, sterilize the collection tubes, install the required collection device, and set up the side streams.</li>
	<li>For sorting RFP+ cells, a 561 nm yellow/green laser and optical filter 579/16 are recommended. Perform the compensation using the negative control and the single-stained positive controls. See Figure 1A for the gating scheme.</li>
	<li>Filter the cells through a 40 &#181;m strainer, centrifuge at 500 x g for 5 min, and resuspend them in 2 mL of FACS buffer at a density of 0.5&#8211;1 x 107/mL. Transfer the cells to clearly labeled 5 mL round-bottom polystyrene tubes and load them into the sorter.</li>
	<li>Run the experimental sample tube at 4 &#176;C, turn on the deflection plates, and sort into a 15 mL conical tube precoated with RPMI containing 1% FBS and 1% v/v PS. 
	NOTE: Protect the samples from strong light to minimize RFP quenching.</li>
</ol>
3. Culture of NCADSCs
<ol>
	<li>Plate the sorted cells at a density of 5,000 cells/cm2 in a 12 well culture plate in complete culture medium and incubate at 37 &#176;C in a humid atmosphere with 5% CO2 for 20&#8211;24 h.</li>
	<li>Remove the culture medium, wash the cells with prewarmed (37 &#176;C) PBS to remove cell debris, and add fresh culture medium.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BSA</td>
			<td>VWR life sciences</td>
			<td>0332-100G</td>
			<td>50 mg/mL working concentration; lot #: 0536C008</td>
		</tr>
		<tr>
			<td>Collagenase, Type I</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythrocyte Lysis Buffer</td>
			<td>Invitrogen</td>
			<td>00-4333</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Corning</td>
			<td>R35-076-CV</td>
			<td>50 mg/mL working concentration; lot #: R2040212FBS</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>HDMEM</td>
			<td>Gelifesciences</td>
			<td>SH30243.01</td>
			<td>Lot #: AD20813268</td>
		</tr>
		<tr>
			<td>PBS (Phosphate buffered saline)</td>
			<td>ABCONE (China)</td>
			<td>P41970</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Qi, Y., et al. <a href="https://app.jove.com/v/60691">Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue.</a> J. Vis. Exp. (2020)
This video demonstrates a technique for isolating and culturing fluorescently labeled neural crest-derived adipose-derived stem cells (ADSCs) from murine periaortic arch adipose tissue. Following isolation, the NCADSCs are detected, sorted using fluorescence-activated cell sorting, and subsequently cultured on a plate, promoting cell proliferation.

<img alt="Figure 1" src="/files/ftp_upload/22550/22550_Figure1.jpg" /> 
Figure 1: Adipogenic differentiation of NCADSCs isolated from PAAT. (A) General gating scheme for characterizing and sorting NCADSCs (RFP) populations. (B) Fluorescence microscope images show that the NCADSCs adhered and expanded after 96 h seeding on a 12 well culture plate. (C&#8211;G) Representative images showing that oil red O stained NCADSC...

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of the stromal vascular ...

Take harvested transgenic mouse periaortic adipose tissues containing fluorescent protein-expressing adipose-derived stem cells descended from neural crest cells, or NCCs.&#160;&#160;
Add digestion media and mince the tissues.
Transfer the minced tissue with the digestion media to a tube. Mechanically dissociate the fragments.&#160;
Incubate. Collagenase in the media digests the tissue&#39;s extracellular matrix, releasing stem cells, adipocytes, and fibroblasts.
Add serum-containing media to stop collagenase activity. Centrifuge and discard the supernatant containing adipocytes.
Resuspend the cells in media, and filter to remove aggregates.
Centrifuge the filtrate and remove the supernatant. Resuspend the cells in erythrocyte lysis buffer to lyse erythrocytes.
Add serum-containing buffer to stop the lysis. Centrifuge and discard the supernatant.
Resuspend the cells in buffer.
Perform fluorescence-activated cell sorting to isolate fluorescent stem cells.
The isolated stem cells can be used for further experiments.

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Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

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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.

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Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue

Transkrypcja

Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue