Name: a microphysiologic platform for human fat: sandwiched white adipose tissue
Uploaded: 2018-08-15T14:00:00
Description: Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

The authors would like to acknowledge the institutional support provided by LSU Health Sciences Center, which funded the project.

All tasks were performed in adherence to protocols #8759 and #9189, as approved by the IRB Office of LSUHSC-NO. All animal work was performed in adherence to protocol #3285 approved by the IACUC Office at LSUHSC-NO.
1. Seeding of Sandwiching Cell Sheets
NOTE: See Figure 1.
<ol>
	<li>Seed ADSCs at approximately 80% confluency in tissue culture plates (6 cm or 6-well plates). For each well of SWAT desired, seed 1 conventional tissue cul...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+National+Institutes+of+Health.+Microphysiological+Systems+Program+focuses+on+a+critical+challenge+in+the+drug+discovery+pipeline.">The National Institutes of Health. Microphysiological Systems Program focuses on a critical challenge in the drug discovery pipeline.</a> Stem Cell Research and Therapy. 4, (2013).</li><li>Wikswo, 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=The+relevance+and+potential+roles+of+microphysiological+systems+in+biology+and+medicine.">The relevance and potential roles of microphysiological systems in biology and medicine.</a> Experimental Biology and Medicine. 239 (9), 1061-1072 (2014).</li><li>. NIH-CASIS Coordinated Microphysiological Systems Program for Translational Research in Space (#RFA-TR-18-001) Available from: <a target="_blank" href="https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html">https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html</a> (2017)</li><li>Lau, F. H., Vogel, K., Luckett, J. P., Hunt, M., Meyer, A., Rogers, C. L., Tessler, O., Dupin, C. L., St Hilaire, H., Islam, K. N., Frazier, T., Gimble, J. M., Scahill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sandwiched+White+Adipose+Tissue:+A+Microphysiological+System+of+Primary+Human+Adipose+Tissue.">Sandwiched White Adipose Tissue: A Microphysiological System of Primary Human Adipose Tissue.</a> Tissue Engineering Part C: Methods. 24 (3), (2018).</li><li>Ebara, M., Hoffman, J. M., Stayton, P. S., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+microfluidic+channels+by+UV-mediated+graft+polymerization+of+non-fouling+and+'smart'+polymers.">Surface modification of microfluidic channels by UV-mediated graft polymerization of non-fouling and 'smart' polymers.</a> Radiation Physics and Chemistry. 76, 1409-1413 (2007).</li><li>Lin, J. B., Isenberg, B. C., Shen, Y., Schorsch, K., Sazonova, O. V., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-responsive+poly(N-isopropylacrylamide)+grafted+onto+microtextured+poly(dimethylsiloxane)+for+aligned+cell+sheet+engineering.">Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtextured poly(dimethylsiloxane) for aligned cell sheet engineering.</a> Colloids and Surfaces B: Biointerfaces. 99, 108-115 (2012).</li><li>Turner, W. S., Sandhu, N., McCloskey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering:+Construction+of+a+Multicellular+3D+Scaffold+for+the+Delivery+of+Layered+Cell+Sheets.">Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets.</a> Journal of Visualized Experiments. (92), e51044 (2014).</li><li>Bunnell, B., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+Isolation,+expansion+and+differentiation.">Adipose-derived stem cells: Isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li><li>Akiyama, Y., Kikuchi, A., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+poly(N-isopropylacrylamide)+grafted+layer+on+polystyrene+surfaces+for+cell+adhesion/detachment+control.">Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control.</a> Langmuir. 20 (13), 5506-5511 (2004).</li><li>Fried, S. K., Russell, C. D., Grauso, N. L., Brolin, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoprotein+lipase+regulation+by+insulin+and+glucocorticoid+in+subcutaneous+and+omental+adipose+tissues+of+obese+women+and+men.">Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men.</a> Journal of Clinical Investigation. 92 (5), 2191-2198 (1993).</li><li>Keipert, S., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brite/beige+fat+and+UCP1+-+is+it+thermogenesis?.">Brite/beige fat and UCP1 - is it thermogenesis?.</a> Biochimica et Biophysica Acta. 1837 (7), 1075-1082 (2014).</li></ol>

This protocol details the use of ADSCs to sandwich human white adipose tissue; human ADSC cell lines can be isolated via well-established protocols15. However, the system can be adapted for individualized research requirements (such as using 3T3L-1 cells to sandwich mouse WAT). This process involves handling primary human tissue. Standard safety precautions should be employed; handle human tissues as BSL-2 pathogens (e.g., HIV, HepC). Only handle tissue directly under a BSC. Wear all appr...

Adipose tissue is the primary organ of obesity, which carries direct annual medical costs between $147 billion and $210 billion in the U.S.1. The accumulation of adipose tissue also contributes to other leading causes of death such as heart disease, type II diabetes, and certain types of cancer2. In vitro culture models are essential for metabolic studies and drug development, but current research models of adipose tissue have major deficiencies. Adipocytes are fragile, buoyant, and terminally differentiated cells that will not adhere to cell culture plastics, and therefore cannot be cultured using conventional cell culture methods. Since the 1970s, several methods have been used in attempts to overcome these barriers, including the use of glass coverslips, ceiling culture, suspension culture, and extracellular matrices3,4,5,6,7. However, these methods have been marked by cell death and dedifferentiation, and they are typically used for no more than a two-week study period. Moreover, these models do not attempt recapitulate the native adipose microenvironment as they do not maintain the intact ECM, the interactions between adipocytes and stromal support cells, nor the contractile forces cells exert on each other in in vivo WAT.
In the absence of a gold-standard primary adipose culture method, adipose research has relied primarily on differentiated pre-adipocytes (diffAds). DiffAds are multilocular, adherent, and metabolically active. By contrast, primary white adipocytes are unilocular, nonadherent, and demonstrate relatively low metabolism. The failure of current adipose culture models to recapitulate the physiology of healthy mature adipose tissue is likely a major factor in the absence of FDA-approved medications that directly target adipocytes. In fact, the lack of physiologic in vitro organ models is a major problem across most organs and disease.
In its position paper announcing the creation of its Microphysiological Systems (MPS) program, the National Institutes of Health (NIH) reported that the 2013 success rate across all human pharmaceutical clinical trials was only 18% for phase II and 50% for phase III clinical trials8.The MPS program is designed to directly address the inability of in vitro monoculture to model human physiology. The NIH defines MPSs as culture systems comprised of human primary or stem cells in multicellular 3D constructs that recapitulate organ functioning. Unlike reductionist models of homogeneous, immortalized cell cultures, MPSs should accurately model cell-cell, drug-cell, drug-drug, and organ-drug interactions9. Unlike short-term primary culture methods, NIH standards dictate MPS sustainability over 4 weeks in culture8. Further details of the MPS program can be found at the NIH&#39;s RFAs (#RFA-TR-18-001)10.
We have developed a simple, novel, adaptable, and inexpensive adipose MPS termed &#34;sandwiched white adipose tissue&#34; (SWAT)11. We overcome the natural buoyancy of adipocytes by &#34;sandwiching&#34; minced primary adipose tissue between sheets of adipose-derived stromal cells (ADSCs) (Figure 1). The resulting 3D construct recapitulates the cell-cell contact and the native adipose microenvironment by surrounding mature adipocytes with a natural adipocyte support cell population. SWAT has been validated by demonstrating 8-week viability, response to exogenous signaling, adipokine secretion, and engraftment into an animal model.

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">10x HBSS</td>
			<td style="width:92px;">Thermofisher</td>
			<td style="width:115px;">14185052</td>
			<td style="width:393px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma-aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine</td>
			<td>Sigma-aldrich</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermofisher</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 Media</td>
			<td>Thermofisher</td>
			<td>11043023</td>
			<td>Phenol red-free&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250&#181;m Mesh Filter</td>
			<td>Pierce</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2&#181;m Syringe Filter</td>
			<td>Celltreat</td>
			<td>229747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5mL Luer-Lok syringes&#160;</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Metal Washers</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3g&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Incubated Orbital Shaker</td>
			<td>VWR</td>
			<td>10020-988</td>
			<td style="width:393px;">Samples should be pitched at 45&#176; angle to facilitate collagenase digestion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Block</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to 37-40&#176;C and placed under Biosafety Cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to ~75&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specialized Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;">Upcell Dishes 6cm of 6-multiwell</td>
			<td>Nunc</td>
			<td>174902&#160; or 174901</td>
			<td style="width:393px;">These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: &#60;6cm for 6cm dish, &#60;3.5cm for 6-well plate; approximate mass: 6.7g for 6cm dish, 5.1g for 6-well plate</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Plastic Plunger Apparatus</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics</td>
		</tr>
	</tbody>
</table>

a microphysiologic platform for human fat: sandwiched white adipose tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

Viability of SWAT was initially assessed by serial brightfield imaging of individual WAT clusters (n = 12) over approximately 7.6 weeks. Clusters remained secured in place on the monolayer throughout this time. Slight morphological changes were observed with individual adipocytes warping slightly or shifting positions. However, adipocytes neither become multilocular over time, indicating a lack of dedifferentiation, nor did they exhibit any visible signs of cell death such as cellular ble...

Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

This breakthrough method allows us, for the first time, to keep fat alive and healthy ex vivo for several consecutive weeks for the study of adipose biology. The main advantage of this technique is that the tissue remains stable over eight weeks in the culture while recapitulating the adipose microenvironment including the surrounding native adipose stromal support cells. Begin by seeding ASC at an approximately 80%confluency in tissue culture plates.For each well of SWAT desired, seed one conventional tissue culture well and one well of corresponding size on a PNIPAAm coated tissue culture plastic plate. Maintain the ASC in complete culture medium at 37 degrees Celsius and 5%CO2 for six to eight days, changing the medium every two days. Twenty-four hours before the SWAT seeding, wrap the rim of the plunger disk of each plunger for the SWAT seeding apparatus at least two times with lab tape to prevent leakage of the gelatin solution.In an ethanol-disinfected biosafety cabinet, spray down a 15 milliliter conical tube rack containing empty, uncapped 15 milliliter conical tubes, as well as each tape wrapped plunger with 70%ethanol, placing each plunger into an empty tube as it is disinfected. Spray approximately 6.3 gram metal washers and place them on the rack along with a pair of hooked forceps. Then close the cabinet sash and turn on the UV light to facilitate overnight drying and sterilization of the seeding apparatus materials.When the cells reach 100%confluency and demonstrate a striated pattern, add 0.75 grams of gelatin powder to 10 milliliters of HBSS. And balance the pH of the solution with 100 microliters of sodium hydroxide. Add the gelatin to a 75-degree Celsius water bath and shake the tube vigorously every five minutes until the powder dissolves into solution.Strain the homogenous gelatin solution through a syringe filter and apply the gelatin to the plungers. The taped edges of the plungers will serve as a mold while the gelatin solidifies. When the gelatin has solidified unwrap the tape and use the hooked forceps to remove the gelatin from the outer edge of the plunger, taking care that the remaining gelatin in the center of the plunger is completely level, to maximize the contact with the ASC sheet.Once the gelatin has been removed from all of the plungers, gently apply each plunger to a PNIPAAm coated ASC plate using the metal washers to weigh down the plungers. After a 1.5-hour incubation at room temperature, transfer the plates to an ice-water bath for another 1.5 hours to complete the dissociation of the cell sheet from the PNIPAAm coated plate surface. Then, clean the bottom of the PNIPAAm coated plates to remove any non-sterile water.Upon receipt of the human adipose tissue, add cold cell culture medium to one 1.5 milliliter microcentrifuge tube per culture to be seeded. And wash large segments of the adipose tissue three times in sterile PBS, removing as much PBS as possible between washes. After the last wash, use forceps and a sterile razor to coarsely mince the tissue, removing as much vasculature and fascia as possible until the solution takes on thick liquid consistency with as few visible individual segments of white adipose tissue as possible.Once a proper tissue consistency has been reached, use a modified P1000 micro-pipette tip to transfer the appropriate volume of minced tissue to each previously prepared 1.5 milliliter tube and briefly mix the minced tissue with the medium within the tubes. Next, replace the medium in each ASC culture with one volume of white adipose tissue solution, and gently transfer the gelatin plungers to the white adipose tissue mixture on the base ASC plates. Examine the monolayers in the PNIPAAm coated plates under a microscope to confirm cellular detachment and move the plates to the surface of a 37 to 40 degree Celsius heat block in the biosafety cabinet.Then add two to three milliliters of warmed culture medium to incubate the cells and to facilitate gelatin melting. After 30 minutes gently remove the plungers from the plate surface, cover the cultures with the appropriate plate lids and place the plates in the cell culture incubator. When the gelatin has completely liquified, aspirate and replace the cell culture medium with the appropriate volume of phenol red free M199 medium supplemented with insulin, dexamethasone, and antibiotics.Over 7.5 weeks of culture, the SWAT clusters remain secured in place on the monolayer with only slight morphological changes observed in individual adipocytes. Clear Nile red staining of unilocular adipocyte clusters at two weeks of culture demonstrates that the cells are indeed adipocytes with intact membranes, rather than artifact, cysts, or dead adipocytes. Propidium iodide staining of 53-day-old SWAT and Oil Red O staining of 51-day-old SWAT cultures confirm that the adipocytes maintain viability even at advanced time points.Immunocytochemistry demonstrates the appropriate expected protein expression and localization of mature adipocyte markers within the SWAT and quantitative PCR indicates the expression of key adipocyte genes at the transcriptional level. The evaluation of basil endocrine function indicates basil leptin secretion from the SWAT cultures as detected by ELISA, and glycerol release is also able to be induced in response to chemical stimulation. Xenotransplantation of SWAT-cultured adipocytes reveals clearly visible and readily recovered fat deposits 10 days after transplant that stain for human but not mouse adipocyte markers.Once mastered, this technique can be completed in about five hours if it is performed properly. It's important to remember to process the primary tissue quickly to minimize cell death. Following this procedure, other methods like real time quantitative PCR, immunostaining, or functional assays can be performed to answer additional questions about how adipocytes respond to various drugs or toxins.After watching this video you should have a good understanding of how to perform SWAT culture as a stable and reliable platform for your adipose research. Don't forget that working with primary human tissue can be hazardous, and that precautions such as wearing the proper PPE and handling the tissue under biosafety cabinets should always be taken while performing this procedure.

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