This study was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant (371697-2011, AT). The authors want to acknowledge the help of bariatric surgeons Drs S. Biron, F-S. Hould, S. Lebel, O. Lescelleur, P. Marceau as well as Christine Racine and Caroline Gagnon from the IUCPQ Tissue Bank. We thank Mr Jacques Cadorette from the IUCPQ&#8217;s audiovisual services for video shooting and editing.

倫理声明：プロジェクトが前に患者の募集にIUCPQの研究倫理委員会によって承認されています。この記事/ビデオの目的のために、私たちは2人の患者から組織を得た：1）57キロのBMIが50.7キロ/ m 2であり、2）35歳の女性患者のBMIが62歳の男性患者/ m 2である 。実験は、両方の脂肪のコンパートメントで行うことができるが、このビデオの目的のために一種の脂肪区画に限られていた。ビデオの技術的側面は、患者1で行ったとFABP4免疫蛍光は、患者2からの脱分化細胞を用いて行った。 1.サンプル処理<ol><li>腹腔鏡肥満手術の時に大網および皮下脂肪区画から脂肪組織を収集するために外科医に依頼。 </li><li>すぐに室温で実験室に脂肪のサンプルを持ってきて、すぐに処理します。 </li><li>非無菌雰囲気の中で、研究室での消化を実行します。ザ·細胞は、最終的には培養室に移し、無菌条件下で培養される。汚染を避けるために、消化の前に蒸留し、濾過水をKRH緩衝液を調製し、濾過（0.22μmのフィルター）をたどる。フラスコとプレートの準備のための細胞培養フード中のエタノールの前に転送で十分にきれいなチューブ。 </li><li>事前加重ディッシュとレコード重量に脂肪組織を置きます。パラフィン包埋の前に少なくとも24時間、室温で10％ホルマリン緩衝液中の各組織サンプル（1cm未満2）の小片を固定する。免疫組織化学実験（技術図示せず）のために、この埋め込まれたサンプルを使用してください。 </li><li>全体の脂肪組織（ -技術図示していない例えば 、遺伝子発現）上のさらなる研究のために-80℃で保存する前に、液体窒素中で50 mlチューブとフラッシュ凍結に別の作品を配置します。 </li></ol> 2.コラゲナーゼ消化<ol><li> 50ミリリットルtuの中の残りの脂肪組織片を置き消化のためにも。 </li><li>消化管中の試料のグラム当たりのコラゲナーゼ（350 U / ml）を補充したKRH-WBを4mlを加える。 </li><li>はさみで脂肪組織をみじん切りに。 </li><li> 45分のインキュベーション（最大1時間）のために、シェーカーで37℃、90回転数の最大値を、みじん切り脂肪組織懸濁液を置きます。 </li></ol>脂肪細胞および前脂肪細胞の精製<ol><li>プラスチック製のビーカーにメッシュ400のナイロンを通して脂肪の少ないチャンクと半透明のソリューションを注ぐ。 </li><li>ピンセットで、ナイロンメッシュ上の細胞調製をこするとKRH-WBの5ミリリットルで洗浄する。 </li><li>微妙にその中にプラスチック管および管端に取り付けられた60ccのシリンジで50ミリリットルチューブに濾過し、細胞懸濁液を転送します。 </li><li>成熟脂肪細胞との懸濁液を、細胞が浮遊することによって、バッファの先頭に到達することを可能にする、約10分間放置する。 </li><li>ゆっくりと60ccのsyrinを使用して、チューブの底にバッファを吸引GE吸引。 </li><li>洗ってKRH-WBの20ミリリットルを追加します。 2追加の洗浄のためのステップ3.4から繰り返します。 </li><li>細胞の量に応じて、5または10 mlの最終容積に脂肪細胞の懸濁液をもたらすためにバッファを集める。セクション5のステップで追求しています。 </li><li>所望であれば、さらに、初代細胞培養の遠心分離によって60ccのシリンジ（3000回転、RT、5分）で集めバッファから間質 - 血管画分を回収し（技術が示されていない）。 </li></ol> 4.成熟脂肪細胞細胞数<ol><li>負荷の計数室（血球計）で穏やかに振盪脂肪細胞懸濁液10μl。四重に細胞数を実行します。 </li><li>孤立した成熟細胞の数を計算します。 </li></ol> T-25フラスコに5.成熟脂肪細胞脱分化<ol><li> DMEM / F12-20％ウシ血清でボリュームの3/4に25cm 2の組織培養フラスコを埋める。 </li><li>細胞数に応じて、フラスコに50万成熟細胞を注ぐ。 </li&gt; <li>培地で50ミリリットルチューブを使用して完全にフラスコを記入し、できるだけ多くの気泡を除去。 </li><li>フラスコ上の未換気キャップをねじ込みます。 </li><li>汚染を避けるためにインキュベーションの前にエタノールでフラスコを清掃してください。 </li><li>細胞接着が中断される場合があり文化の動きを避けるために、それに触れることなく、週に逆さまにフラスコをインキュベートする。 </li><li>反転した培養7日目に、フラスコを逆にする前に、静かにフラスコを操作し、突然の動きを避け、吸引によりフラスコ内のすべてのメディアを削除します。 </li><li> DMEM-F12-20％ウシ血清の12ミリリットルを追加し、標準的な技術を用いて細胞を養う。ろ過、ベントキャップフラスコに添加してもよい。 </li></ol> 6ウェルプレートに6成熟脂肪細胞脱分化<ol><li> 6ウェルプレートの各ウェルの底にカバースリップを置き</li><li>各カバースリップの上に½ &quot;プラスチックブッシングを追加します。 </li><li> 20％のウシ血清DMEM培地の8ミリリットルで井戸を埋める。 </li><li>各プラスチックブッシングにカバースリップを置く。 </li><li>スライドとスライドの下で細胞を注入するチューブ（ウェル当たり50,000細胞）との間にピペットチップを挿入します。 </li><li>週5％CO 2、37℃で標準的な細胞培養インキュベーター中でプレートをインキュベートする。 </li><li> 20％のウシ血清を補充した培地の各ウェルを含む2ミリリットル中に付着した細胞を用いた逆カバースリップと文化を追求する。 </li><li>免疫蛍光（技術が示されていない）を含むいくつかの目的のために脱分化を受けている細胞でカバーガラスを使用してください。 </li></ol>

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ceiling+culture+of+mature+human+adipocytes:+use+in+studies+of+adipocyte+functions.">Ceiling culture of mature human adipocytes: use in studies of adipocyte functions.</a> J Endocrinol. 164 (2), 119-128 (2000).</li><li>Matsumoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential.">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J Cell Physiol. 215 (1), 210-222 (2008).</li><li>Poloni, A., et al. <a target="_blank" 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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+fat+cells+convert+to+cardiomyocyte+phenotype+and+repair+infarcted+cardiac+tissue+in+rats.">Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats.</a> J Mol Cell Cardiol. 47 (5), 565-575 (2009).</li><li>Ohta, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+adipocyte-derived+cells,+dedifferentiated+fat+cells+(DFAT),+promoted+functional+recovery+from+spinal+cord+injury-induced+motor+dysfunction+in+rats.">Mature adipocyte-derived cells, dedifferentiated fat cells (DFAT), promoted functional recovery from spinal cord injury-induced motor dysfunction in rats.</a> Cell Transplant. 17 (8), 877-886 (2008).</li><li>Moreno-Navarrete, J. M. F. -. r., Symonds, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Adipose Tissue Biology. , 17-38 (2012).</li><li>Poulos, S. P., Dodson, M. V., Hausman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+models+for+differentiation:+preadipocytes+and+adipocytes.">Cell line models for differentiation: preadipocytes and adipocytes.</a> Exp Biol Med (Maywood. 235 (10), 1185-1193 (2010).</li><li>Casteilla, L., Penicaud, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity).">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity).</a> Methods Mol Biol. 456, 23-38 (2008).</li><li>Tchernof, A., Despres, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+human+visceral+obesity:+an+update.">Pathophysiology of human visceral obesity: an update.</a> Physiol Rev. 93 (1), 359-404 (2013).</li><li>Rodbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+Isolated+Fat+Cells.+I.+Effects+of+Hormones+on+Glucose+Metabolism+and+Lipolysis.">Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis.</a> J Biol Chem. 239, 375-380 (1964).</li><li>Watson, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Markers+and+Functional+Attributes+of+Human+Adipose-Derived+Stem+Cells+and+Dedifferentiated+Adipocyte+Cells+from+Subcutaneous+Fat+of+an+Obese+Diabetic+Donor.">Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor.</a> Adv Wound Care. 3 (3), 219-228 (2014).</li></ol>

The authors declare no conflict of interest.

天井培養技術と成熟脂肪細胞の脱分化は、天然の脂肪組織の小サンプルから脂肪幹細胞を得るための新たなアプローチである。我々の経験など2のそれに基づいて、組織の1グラムを25cm 2のフラスコにプレートし、均質性がPoloni及び共同研究者3によって実証されているDFAT細胞の集団を得るために十分である。脂肪細胞の脱分化は、独立して自分の年齢、性別、その他の...

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture1. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent2. Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells2. Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells5. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture2.
DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox23. They also express markers specific to each of the three germ layers7. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature3. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease10-12. 
In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation13. However, these cells originate from mouse embryo tissue13. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology14. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent's abdominal cavity prevents from extrapolating directly to humans15. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential16. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes. 
Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples17, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine serum albumine</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma</td>
			<td>A4036</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A0278</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Type I collagenase</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS-004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, no phenol red</td>
			<td>Gibco-Life Technologies</td>
			<td>11039-021</td>
			<td>Add to medium : 20% calf serum, gentamicin (50&#181;g/ml) and fungizone (2.5&#181;g/ml)</td>
		</tr>
		<tr>
			<td>Calf Serum, iron supplemented, from formula-fed calves</td>
			<td>Sigma</td>
			<td>C8056-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 In plastic bushing</td>
			<td>Iberville</td>
			<td>2704-CP</td>
			<td>SKU:1000120918 (Home Depot)</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Linde</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin soluton, neutral buffered, 10%</td>
			<td>SIGMA</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60cc syringes</td>
			<td>BD Syringe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit stock buffer (KRH)</td>
			<td></td>
			<td></td>
			<td>Prepare stock buffer as following: 25mM HEPES pH7.6, 125mM NaCl, 3.73mM KCl, 5mM CaCl2.2H2O, 2.5mM MgCl2.6H2O, 1mM K2HPO4. Adjust pH to 7.4.</td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit-Working Buffer (KRH-WB)</td>
			<td></td>
			<td></td>
			<td>Add the following components freshly to KRH buffer: 4% bovine serum albumin, 5mM glucose, 0.1&#181;M adenosine, 560 &#181;M ascorbic acid</td>
		</tr>
		<tr>
			<td>KRH-WB supplemented with Type I collagenase</td>
			<td></td>
			<td></td>
			<td>Add 350U/ml of Type I collagenase</td>
		</tr>
		<tr>
			<td>T25 unvented cap tissue culture flask</td>
			<td>Sarsted or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>BD Falcon or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22x22</td>
			<td>Fisherbrand</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

主な形態学的変化は、脱分化（ 図1）の間に脂肪細胞を成熟起こる。 図2に示すように、脱分化を受けている細胞は、蛍光分析のために抗FABP4抗体で染色した。線維芽細胞様細胞の大部分はしなかったのに対し、丸い形態を有する細胞は、FABP4タンパク質を発現した。脱分化した後、DFAT細胞は数回の継代のための標準的な手順で培養することができる。我々は、ヒト大...

Watch this Scientific Journal Video about Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures at JoVE.com

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

天井の培養における成熟脂肪細胞の脱分化を経てヒト脂肪幹細胞の生成

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

generation-human-adipose-stem-cells-through-dedifferentiation-mature

Research

JoVE Journal

Developmental Biology

11.6K ビュー.  IUCPQ Research Center. Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.