Name: An Efficient Method to Obtain Dedifferentiated Fat Cells (Video) | JoVE Journal
Uploaded: 2016-07-15T13:00:00.000+00:00
Duration: 6 min 11 s
Description: Learn an efficient cell culture method to generate high-quality dedifferentiated fat cells (DFAT) for regenerative medicine and tissue engineering applications. This protocol enhances cell proliferation and differentiation potential.

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5 minutes

This research was supported in part by Program for Creating Start-ups from Advanced Research and Technology (START Program) from the Japan Society for the Promotion of Science (ST261006IP, TM) and by Program for the Strategic Research Foundation at Private Universities (2014-2019) (S1411018, TM) from the Ministry of Education, Sports, Science and Technology.

Samples of human subcutaneous fat were obtained from patients undergoing surgery in the Departments of Plastic Surgery, Urology, Pediatric Surgery and Orthopedic Surgery of Nihon University Itabashi Hospital (Tokyo, Japan). The patients gave written informed consent, and the Ethics Committee of Nihon University School of Medicine approved the study.
1. Tissue Preparation
<ol>
	<li>Bring the tissue sample from the operating room to the laboratory.</li>
	<li>Wash 1-2 g of adipose tissue with 5 ml of PBS(-) at room temperature (RT) once, in a 50 ml tube and then place the sample in a 10 cm plastic dish.</li>
</ol>
2. Collagenase Digestion
<ol>
	<li>Remove the sample from the PBS(-), then add 10 ml of sterile 0.1% (w/v) collagenase solution (Collagenase type II) to the dish containing the tissue sample.</li>
	<li>Mince the tissue with surgical scissors for approximately 15 min, until it reaches a pureed consistency.</li>
	<li>Subsequently, digest the minced tissue in the 0.1% (w/v) collagenase solution at 37 &#176;C for 30 min (60 rpm) in a 50 ml tube with gentle agitation on a shaker.</li>
	<li>Filter the digested sample through a 100 &#181;m filter into a 50 ml tube. Next, pass 10 ml of DCM (see Table of Materials) supplemented with 2% FBS through the filter.</li>
</ol>
3. Cell Isolation
<ol>
	<li>Centrifuge the filtered cells at 100 x g for 1 min at RT.</li>
	<li>At this point, prepare 5 ml of DCM supplemented with 2% FBS in a 50 ml tube.</li>
	<li>Next, draw up the floating fat cells using a 1,000 &#181;l pipette and add the cells to a 50 ml tube containing medium.</li>
	<li>Subsequently, shake the 50 ml tube gently to mix the DCM medium with the cells.</li>
	<li>Then, centrifuge the tube at 100 x g for 1 min at RT.</li>
	<li>Discard the medium at the bottom of the tube using an 18 G needle and a 20 ml syringe.</li>
	<li>Next, add 10 ml of DCM supplemented with 2% FBS to the collected cells.</li>
	<li>Mix the DCM with the cells by shaking the 50 ml tube gently.</li>
	<li>Centrifuge the tube at 100 x g for 1 min at RT.</li>
</ol>
4. Plating Cells
<ol>
	<li>Add 41 ml of DCM or DMEM supplemented with 20% FBS to a 12.5 cm flask.</li>
	<li>Next, using a 200 &#181;l pipette add 40 &#181;l of fat cells to the flask.</li>
	<li>Then, fill up the flask with DCM or DMEM supplemented with 20% FBS. CRITICAL STEP: At this point, ensure that the cells are spread throughout the medium. Visually check the detached cells and afterwards keep the flask on the lid of a round petri dish to keep it flat.</li>
	<li>Finally, place the flask inside the incubator and leave it undisturbed in a 5% CO2 incubator at 37 &#176;C for 7 days.</li>
	<li>Following a week of incubation, invert the flask and check for DFAT cells. Next, assess DFAT cell generation proficiency as described10 since at this point fibroblast-like cells (DFAT cells) are located in regions distinct from fat cells.</li>
	<li>Add 5 ml of DCM or DMEM supplemented with 20% FBS to the flask following removal of the previous cell culture medium. Allow the DFAT cells to grow for another week after changing the medium.</li>
	<li>Using a cell counter, count the number of DFAT cells generated under various cell culture conditions (DCM vs. DMEM).</li>
</ol>

<ol><li>Lanzoni, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concise+review%3A+clinical+programs+of+stem+cell+therapies+for+liver+and+pancreas%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Concise review: clinical programs of stem cell therapies for liver and pancreas.</a> Stem Cells. 31 (10), 2047-2060 (2013).</li>
<li>de Girolamo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem%2Fstromal+cells%3A+a+new+%22cells+as+drugs%22+paradigm.+Efficacy+and+critical+aspects+in+cell+therapy%5BTitle%5D)+AND+%22Curr.+Pharm.+Des%22%5BJournal%5D)">Mesenchymal stem/stromal cells: a new "cells as drugs" paradigm. Efficacy and critical aspects in cell therapy.</a> Curr. Pharm. Des. 19 (13), 2459-2473 (2013).</li>
<li>Lindroos, B., Suuronen, R., Miettinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+potential+of+adipose+stem+cells+in+regenerative+medicine%5BTitle%5D)+AND+%22Stem+Cell.+Rev%22%5BJournal%5D)">The potential of adipose stem cells in regenerative medicine.</a> Stem Cell. Rev. 7 (2), 269-291 (2011).</li>
<li>Yan, J., Tie, G., Xu, T. Y., Cecchini, K., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+as+a+treatment+for+peripheral+arterial+disease%3A+current+status+and+potential+impact+of+type+II+diabetes+on+their+therapeutic+efficacy%5BTitle%5D)+AND+%22Stem+Cell.+Rev%22%5BJournal%5D)">Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy.</a> Stem Cell. Rev. 9 (3), 360-372 (2013).</li>
<li>Ringden, O., Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stromal+cells+as+treatment+for+chronic+GVHD%5BTitle%5D)+AND+%22Bone+Marrow+Transplant%22%5BJournal%5D)">Mesenchymal stromal cells as treatment for chronic GVHD.</a> Bone Marrow Transplant. 46 (2), 163-164 (2011).</li>
<li>Matsumoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential%5BTitle%5D)+AND+%22J.+Cell.+Physiol%22%5BJournal%5D)">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J. Cell. Physiol. 215 (1), 210-222 (2008).</li>
<li>Lessard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human+adipose+stem+cells+through+dedifferentiation+of+mature+adipocytes+in+ceiling+cultures%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.</a> J. Vis. Exp. (97), (2015).</li>
<li>Lessard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+dedifferentiating+human+mature+adipocytes+from+the+visceral+and+subcutaneous+fat+compartments%3A+fibroblast-activation+protein+alpha+and+dipeptidyl+peptidase+4+as+major+components+of+matrix+remodeling%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.</a> PLoS One. 10 (3), 0122065(2015).</li>
<li>Peng, X., et al. Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPeng%2C+X+%22Biomed.+Res.+Int%22">Biomed. Res. Int</a>. 2015, 673651(2015).</li>
<li>Kono, S., Kazama, T., Kano, K., Harada, K., Uechi, M., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypic+and+functional+properties+of+feline+dedifferentiated+fat+cells+and+adipose-derived+stem+cells%5BTitle%5D)+AND+%22Vet.+J%22%5BJournal%5D)">Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells.</a> Vet. J. 199 (1), 88-96 (2014).</li>
<li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+pluripotent+stem+cells%3A+the+new+patient%5BTitle%5D)+AND+%22Nat.+Rev.+Mol.+Cell+Biol%22%5BJournal%5D)">Induced pluripotent stem cells: the new patient.</a> Nat. Rev. Mol. Cell Biol. 13 (11), 713-726 (2012).</li>
</ol>

The authors have nothing to disclose.

Mature adipocytes that undergo in vitro dedifferentiation, a process known as ceiling culture, may revert to a more primitive phenotype and gain proliferative abilities. These cells are referred to as dedifferentiated fat (DFAT) cells. The multilineage differentiation potential of DFAT cells was evaluated. Flow cytometry analysis and gene expression analysis revealed that DFAT cells were highly homogeneous in comparison to ASCs6. In fact, the cell-surface antigen profile of DFAT cells is very similar ...

Cell therapy and tissue engineering are hot topics in the field of regenerative medicine1-5. While these therapeutic modalities hold great promise, several technical issues currently hamper their clinical use. In this regard, as in the generation of iPS cells, all tissue engineering therapies must produce cells free of external gene transductions in order to maintain patient safety. Accordingly, we were the first group to successfully produce human DFAT cells6. Several other research groups have since adopted our method to generate DFAT cells of mammalian origin7-9, further highlighting the usefulness of our model.
Over the course of several studies, we have found that the quality of the cell culture environment may be modified by adjusting the content of the cell medium. This finding has led to an increase in the success rate of DFAT cell production and improved cell quality; both critical factors in efficiently generating cells for future clinical trials. In this regard, an improved DFAT culture medium (DCM, a medium similar to mesenchymal stem cell medium, which contains recombinant human insulin, serum albumin, L-glutamic acid, several fatty acids, and cholesterol) and a method for DFAT cell generation and proliferation was developed (more information about the contents of DCM is available upon request). Using this method high quality DFAT cells were generated with the ability to differentiate into several cell types including adipogenic, osteogenic, and chondrogenic cells. Altogether, this validated cell culture protocol enhances the quality of DFAT cells and may be quite useful for enhancing clinical applications of cell therapy and tissue engineering.

<table><tbody><tr><td>CSTI303-MSC medium&amp;nbsp;</td><td>CSTI</td><td>87-671</td><td>This medium is defined as DCM in the text</td></tr><tr><td>PBS(-)</td><td>Wako</td><td>166-23555</td><td>It does not contain Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>DMEM medium</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>172012</td><td></td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>C-6885</td><td></td></tr><tr><td>Scissors</td><td>Takasago Medical Industry Co., Ltd</td><td>TKZ-F2194-1</td><td></td></tr><tr><td>Shaker</td><td>TAITEC</td><td>Bioshaker V.BR-36</td><td></td></tr><tr><td>Falcon Cell Strainer 100 &amp;mu;m Yellow</td><td>CORNING LIFE SCIENCES&amp;nbsp;</td><td>DL 352360</td><td></td></tr><tr><td>Falcon 12.5 cm&amp;sup2; Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap</td><td>CORNING LIFE SCIENCES&amp;nbsp;</td><td>353107</td><td></td></tr><tr><td>18 G needle</td><td>NIPRO</td><td>02-002</td><td></td></tr><tr><td>20 ml Syringe&amp;nbsp;</td><td>NIPRO</td><td>08-753</td><td></td></tr><tr><td>Z Series Coulter Counter</td><td>BECKMAN COULTER</td><td>383550</td><td></td></tr></tbody></table>

an efficient method to obtain dedifferentiated fat cells

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

In this study, the method and toolkit for DFAT cell generation was improved (Figure 1). Our method allows us to generate DFAT cells using both DCM and DMEM medium containing 20% FBS (Figure 2A). As such, we compared the efficiency of DCM and DMEM in generating DFAT cells. In this regard, DCM enhanced DFAT cell proliferation by three times when compared to DMEM, regardless of the number of adipocytes (Figure 2A and B). Usi...

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

The overall goal of this novel cell culture method is to generate dedifferentiated fat cells with greater efficiency. Our method may help researchers working in the field of regenerative medicine answer key questions about the several processes involved in producing adipogenic, osteogenic, and chondrogenic cells. Since the primary advantage of our cell culture protocol is that it enhances the quality of DEFAT cells, it may be applied in the near future to improve current applications in cell therapy and tissue engineering.After bringing the patient's subcutaneous fat sample to the lab from the operating room, weigh the tissue and place a one to two gram sample of adipose tissue into a 15 milliliter tube. Then wash the sample with five milliliters of PBS and then transfer the sample to a 10 centimeter plastic dish. Remove the PBS.Then add 10 milliliters of sterile 0.1%weight per volume collagenase to the dish containing the sample. Next, use surgical scissors to mince the tissue until it reaches a pureed consistency. Transfer the minced tissue and collagenase solution to a 15 milliliter centrifuge tube and digest at 37 degrees Celsius for 30 minutes, with shaking at 60 RPM.Following the digestion, filter the digested sample through a 100 micron filter into a fresh 15 milliliter tube. Then, pass 10 milliliters of DEFAT culture medium supplemented with 2%FBS through the filter. To isolate the fat cells from the cell suspension, first centrifuge the filtered cells at 100 times g for one minute at room temperature.During the centrifugation, prepare five milliliters of DCM supplemented with 2%FBS in a 15 milliliter tube. Next, use a 1000 microliter pipette to draw up the floating fat cells from the centrifuged tube and add the cells to a 15 milliliter tube containing medium. Shake the tube gently to mix the DCM medium with the cells.Then, centrifuge the tube at 100 times g for one minute at room temperature. After centrifuging, use an 18 gauge needle and a 20 milliliter syringe to remove the medium at the bottom of the tube. Then add 10 milliliters of DCM supplemented with 2%FBS to the cells and mix by shaking the tube gently.Centrifuge the cells again at 100 times g for one minute at room temperature. To begin plating, add 41 milliliters of DCM or DMEM supplemented with 20%FBS to a 12.5 centimeter flask. Then use a 200 microliter pipette to draw up 40 microliters of fat cells and add them to the flask.Then fill the flask with DCM or DMEM supplemented with 20%FBS. It is critical that the cells are spread throughout the medium. Place the flask on the lid of a round Petri dish to keep it flat.Finally, place the flask inside an incubator set to 5%carbon dioxide and 37 degrees Celsius and leave it undisturbed for seven days. After a week of incubation, invert the flask and check for dedifferentiated fat cells. Next, aspirate the media and then add five milliliters of DCM or DMEM supplemented with 20%FBS to the flask.Allow the DEFAT cells to grow for another week after changing the medium. After a week, use a cell counter to count the number of DEFAT cells generated under various cell culture conditions. This image shows DEFAT cell colonies following 14 days of culture in DMEM.Here the DEFAT cells are shown after 14 days of culture in DCM. In each case, the scale bar represents 200 microns. DEFAT cell proliferation was quantified using a cell counter, and the efficiency of DCM and DMEM in generating DEFAT cells, was compared.As seen here, enhanced cell proliferation is observed with the use of DCM. Here, human DEFAT cells, generated using DCM, were cultured for three weeks in the appropriate induction media to evaluate the redifferentiation and differentiation abilities of DEFAT cells. The cells were positive for osteogenic capacity by alkaline phosphatase stain and by staining with Alizarin Red S.Finally, adipogenic capacity was shown with an Oil Red O stain.It's quite very important to ensure that collagenase with rudiment and fat cell isolation are both performed with the utmost of care in order to successfully generate high quality DEFAT cells. Through these efforts, our validated cell culture method aims to facilitate both current and future research efforts in the fields of developmental biology and regenerative medicine.

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Learn an efficient cell culture method to generate high-quality dedifferentiated fat cells (DFAT) for regenerative medicine and tissue engineering applications. This protocol enhances cell proliferation and differentiation potential.

An Efficient Method to Obtain Dedifferentiated Fat Cells

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