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.

Declaración de Ética: El proyecto ha sido aprobado por el Comité Ético de Investigación de IUCPQ previo a la contratación del paciente. A los efectos de este artículo / video, obtuvimos tejidos de 2 pacientes: 1) un paciente varón de 62 años de edad con un IMC de 50,7 kg / m 2 y 2) una paciente de 35 años de edad con un IMC de 57 kg / m 2. Los experimentos se pueden realizar con las dos compartimentos de grasa, pero se han limitado a un compartimento de grasa para el propósito de este video. Aspectos técnicos del video se realizaron con el paciente 1 e inmunofluorescencia FABP4 se realizó con células indiferenciadas del paciente 2. Procesamiento 1. Muestra <ol><li> Pregunta a los cirujanos para recoger el tejido adiposo de los compartimentos de grasa omental y subcutáneo en el momento de la cirugía bariátrica laparoscópica. </li><li> Traer rápidamente las muestras de tejido adiposo al laboratorio a temperatura ambiente y procesar inmediatamente. </li><li> Realizar la digestión en el laboratorio, en un ambiente no estéril. Lacélulas eventualmente serán transferidos a la sala de cultivo y se cultivaron bajo condiciones estériles. Para evitar la contaminación, preparar tampón KRH con agua destilada y se filtró y el seguimiento de un (filtro 0.22μM) filtración antes de la digestión. Tubos Limpiar a fondo con etanol transferencia antes de la campana de cultivo celular para frasco y preparación plato. </li><li> Coloque el tejido adiposo en un plato pesado previamente y registrar el peso. Fijar un pequeño trozo de cada muestra de tejido (menos de 1 cm 2) en tampón de formalina al 10% a temperatura ambiente durante al menos 24 h antes de la inclusión en parafina. Utilice esta muestra embebido para experimentos de inmunohistoquímica (técnica no mostrado). </li><li> Coloque una pieza más en un tubo de 50 ml y el flash-congelación en nitrógeno líquido antes de almacenar a -80 ° C durante más estudios sobre los tejidos adiposos enteros (por ejemplo, la expresión de genes - técnica no se muestra). </li></ol> 2. digestión con colagenasa <ol><li> Coloque la pieza de tejido adiposo que permanece en un ml tu 50ser para la digestión. </li><li> Añadir 4 ml de KRH-WB suplementadas con colagenasa (350 U / ml) por gramo de la muestra en el tubo de digestión. </li><li> Picar el tejido adiposo con unas tijeras. </li><li> Coloque picada suspensión de tejido adiposo en una coctelera, 37 ° C, 90 rpm máximo, para una incubación de 45 minutos (máximo 1 hora). </li></ol> 3. Purificación de los adipocitos y preadipocitos <ol><li> Vierta la solución transparente con unos trozos de grasa a través de un nylon 400 mM de malla en un vaso de plástico. </li><li> Con pinzas, frotar la preparación de células en la malla de nylon y se lava con 5 ml de KRH-WB. </li><li> Transferir delicadamente la suspensión celular se filtró en un tubo de 50 ml con el tubo de plástico en ella y una jeringa de 60 cc unida a la extremidad del tubo. </li><li> Que la suspensión con adipocitos maduros reposar durante aproximadamente 10 min, permitiendo que las células alcanzan la parte superior de la memoria intermedia por flotación. </li><li> Lentamente aspirar el tampón en la parte inferior del tubo utilizando Syrin 60ccsucción ge. </li><li> Añadir 20 ml de KRH-WB para lavar. Repita desde el paso 3.4 para 2 lavados adicionales. </li><li> Recoger el tampón para llevar la suspensión de adipocitos a un volumen final de 5 o 10 ml, dependiendo de la cantidad de células. Continuar con los pasos en la sección 5. </li><li> Recuperar la fracción estromal-vascular de la memoria intermedia recogido con la jeringa de 60 cc por centrifugación (3000 rpm, RT, 5 min) para su posterior cultivo celular primario si se desea (técnica no se muestra). </li></ol> 4. adipocito maduro Cuenta de la célula <ol><li> Cargar 10 l de suspensión de los adipocitos sacudido suavemente en una cámara de recuento (hemocitómetro). Realizar el recuento de células por cuadruplicado. </li><li> Calcular el número de células maduras aisladas. </li></ol> 5. maduro Adipocito Desdiferenciación en T-25 Frasco <ol><li> Llene un 25 cm2 tejido cultivo en matraz a ¾ del volumen con DMEM / F12-20% de suero de ternera. </li><li> De acuerdo con el recuento de células, vierta 500.000 células maduras en el matraz. </li&gt; <li> Llene el frasco completamente utilizando un tubo de 50 ml con medio y eliminar tantas burbujas como sea posible. </li><li> Tornillo de la tapa sin ventilación en el matraz. </li><li> Limpiar el matraz con etanol antes de la incubación para evitar la contaminación. </li><li> Incubar el frasco boca abajo durante una semana sin tocarlo para evitar el movimiento de la cultura que puede perturbar la adhesión celular. </li><li> Antes de invertir el matraz a 7 días de cultivo invertida, manipular suavemente el matraz y eliminar todo el medio en el matraz por aspiración, evitando movimientos bruscos. </li><li> Añadir 12 ml de DMEM-F12-20 suero% ternera y cultivar células con técnicas estándar. A filtrada, tapa ventilada se puede añadir al matraz. </li></ol> 6. maduro Adipocito Desdiferenciación en una placa de 6 pocillos <ol><li> Coloque un cubreobjetos en la parte inferior de cada pocillo de una placa de 6 pocillos </li><li> Añadir un ½ &quot;casquillo de plástico en la parte superior de cada cubreobjetos. </li><li> Llenar los pocillos con 8 ml de 20% de medio de suero de ternero-DMEM. </li><li> Ponga un cubreobjetos en cada casquillo de plástico. </li><li> Inserte punta de la pipeta entre la corredera y el tubo para inyectar células bajo la corredera (50.000 células por pocillo). </li><li> Incubar las placas en una incubadora de cultivo celular estándar a 37 ° C con 5% de CO 2 durante una semana. </li><li> Cubreobjetos inversa con células unidas en cada pocillo que contiene 2 ml de medio suplementado con suero de ternera 20% y llevar a cabo la cultura. </li><li> Utilice cubreobjetos con células que experimentan desdiferenciación para varios propósitos incluyendo inmunofluorescencia (técnica no se muestra). </li></ol>

<ol>
	<li>Zhang, H. H., Kumar, S., Barnett, A. H., Eggo, M. 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" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dedifferentiated+adipocytes+show+similar+properties+to+bone+marrow-derived+mesenchymal+stem+cells.">Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.</a> Stem Cells. 30 (5), 965-974 (2012).</li><li>Perrini, S., 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=Differences+in+gene+expression+and+cytokine+release+profiles+highlight+the+heterogeneity+of+distinct+subsets+of+adipose+tissue-derived+stem+cells+in+the+subcutaneous+and+visceral+adipose+tissue+in+humans.">Differences in gene expression and cytokine release profiles highlight the heterogeneity of distinct subsets of adipose tissue-derived stem cells in the subcutaneous and visceral adipose tissue in humans.</a> PLoS One. 8 (3), e57892 (2013).</li><li>Kishimoto, N., 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=The+osteoblastic+differentiation+ability+of+human+dedifferentiated+fat+cells+is+higher+than+that+of+adipose+stem+cells+from+the+buccal+fat+pad.">The osteoblastic differentiation ability of human dedifferentiated fat cells is higher than that of adipose stem cells from the buccal fat pad.</a> Clin Oral Investig. , (2013).</li><li>Kou, L., 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=The+phenotype+and+tissue-specific+nature+of+multipotent+cells+derived+from+human+mature+adipocytes.">The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.</a> Biochem Biophys Res Commun. 444 (4), 543-548 (2014).</li><li>Jumabay, M., 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=Pluripotent+stem+cells+derived+from+mouse+and+human+white+mature+adipocytes.">Pluripotent stem cells derived from mouse and human white mature adipocytes.</a> Stem Cells Transl Med. 3 (2), 161-171 (2014).</li><li>Sugawara, A., Sato, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+dedifferentiated+fat+cells+for+periodontal+tissue+regeneration.">Application of dedifferentiated fat cells for periodontal tissue regeneration.</a> Hum Cell. 27 (1), 12-21 (2014).</li><li>Kikuta, S., 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=Osteogenic+effects+of+dedifferentiated+fat+cell+transplantation+in+rabbit+models+of+bone+defect+and+ovariectomy-induced+osteoporosis.">Osteogenic effects of dedifferentiated fat cell transplantation in rabbit models of bone defect and ovariectomy-induced osteoporosis.</a> Tissue Eng Part A. 19 (15-16), 1792-1802 (2013).</li><li>Obinata, D., 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=Transplantation+of+mature+adipocyte-derived+dedifferentiated+fat+(DFAT)+cells+improves+urethral+sphincter+contractility+in+a+rat+model.">Transplantation of mature adipocyte-derived dedifferentiated fat (DFAT) cells improves urethral sphincter contractility in a rat model.</a> Int J Urol. 18 (12), 827-834 (2011).</li><li>Jumabay, M., 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=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.

Desdiferenciación de adipocitos maduros con la técnica de cultivo de techo es un nuevo enfoque para la obtención de células madre adiposas de una pequeña muestra de tejido adiposo nativo. Basándonos en nuestra experiencia y la de otros 2, un gramo de tejido es suficiente para platear un matraz de 25 cm2 y para obtener una población de células DFAT para que la homogeneidad se ha demostrado por Poloni y colaboradores 3. Desdiferenciación adipocito parece posible con células de un...

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.

Los principales cambios morfológicos ocurren a adipocitos maduros durante la desdiferenciación (Figura 1). Como se muestra en la Figura 2, las células sometidas a la desdiferenciación se tiñeron con un anticuerpo anti-FABP4 para el análisis de fluorescencia. Las células con una morfología redonda expresaron la proteína FABP4 mientras que la mayoría de las células similares a fibroblastos no lo hizo. Después de desdiferenciación, células DFAT pueden cultivarse con procedimi...

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.

Generación de células madre adiposas Humano a través Desdiferenciación de adipocitos maduros en culturas de techo

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

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Research

JoVE Journal

Developmental Biology

11.6K vistas.  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.