We thank Xiao-Rong Peng and Stefan Hallen for providing resources and optimizing the adipocyte isolation, Martin Uhrbom for technical assistance, and Daniel Olausson and Malin L&#246;nn for coordinating and providing the human adipose.

Anonymous samples of adipose tissue were collected from the abdominal region of female patients undergoing elective surgery at Sahlgrenska University Hospital in Gothenburg, Sweden. All study subjects received written and oral information before giving written informed consent for the use of the tissue. The studies were approved by The Regional Ethical Review Board in Gothenburg, Sweden.
NOTE: An overview of the method is provided in Figure 1.
1. Preparation of Buffers, Tissue Culture Media, and Culture Plates
<ol>
	<li>Prepare a 5x Krebbs Ringer (KR) stock by dissolving 35.1 g of NaCl, 1.75 g of KCl, 0.82 g of KH2PO4, and 1.48 g of MgSO4&#183;7H2O in 900 mL of water. Add 1.84 g of CaCl2&#183;2H2O and adjust the volume to 1 L by adding water. Sterile filter through a 0.22 &#181;m filter and store at 4 &#176;C.</li>
	<li>From the 5x KR stock, prepare 1 L of buffer containing 1x KR, 25 mM HEPES, 2 mM glucose, and 2% bovine serum albumin (BSA) (referred to hereafter as wash buffer). Adjust the pH to 7.4.</li>
	<li>Prepare 500 mL of collagenase buffer containing 1x HBSS + CaCl2 + MgCl2, 2% BSA, and 450 mL of water (referred to hereafter as digestion buffer). 
	NOTE: The collagenase should not be added to the digestion buffer until after the adipose tissue has been weighed in step 2.2.</li>
	<li>Adjust the pH of Medium 199 to 7.4.</li>
	<li>Sterile filter both buffers and medium 199 through a 0.22 &#181;m filter flask and warm to 37 &#176;C. 
	NOTE: To save time, the tissue culture media can be prepared and added to plates before processing the adipose tissue. For a 24-well plate format (Figure 1A), use 0.5&#8722;1 mL/well of Dulbecco&#39;s modified Eagle&#39;s medium/Ham&#39;s nutrient mixture F12 (DMEM/F12), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (penn/strep) and 20 nM insulin. Small molecules and other stable pharmacological agents can also be added at this time to the media in the desired layout. Place the plates in a tissue culture incubator (37 &#176;C, 5% CO2).</li>
</ol>
2. Dissection of Human Subcutaneous Adipose Tissue
NOTE: Work within a biological safety cabinet and use sterile technique throughout the isolation process, as well as exclusive use of autoclaved and sterile equipment.
<ol>
	<li>Place the human adipose tissue in a 15 cm petri dish and add a small volume of medium 199 to keep it moisturized during the dissection.</li>
	<li>Working with pieces of adipose approximately the size of a golf ball, grasp large fibrotic vessels with tweezers and gently release the adipocytes by scraping the adipose with the back of closed scissors. Discard the large pieces of fibrotic tissue. Weigh the trimmed fat.</li>
</ol>
3. Collagenase Treatment
<ol>
	<li>Add collagenase to the digestion buffer (see step 1.3) at a concentration of 1 mg/mL. Sterile filter the solution using a 0.22 &#181;m sterile filter. 
	NOTE: Three mL of digestion buffer per gram of fat is recommended (i.e., for 100 g of fat, prepare 300 mL of digestion buffer with 300 mg of type 2 collagenase). 
	CAUTION: Type 2 Collagenase is hazardous to eyes, skin and may cause respiratory irritation. Wear gloves, eye protection, and work in a ventilated hood when handling the collagenase.</li>
	<li>Move approximately 10 g of adipose tissue to a 15 cm petri dish and mince the fat carefully using a pair of curved scissors until it becomes a smooth homogenous mixture and there are no large pieces of adipose left. Repeat the process until all of the fat has been processed. 
	NOTE: Each round should take approximately 2 min and the adipose pieces should be small enough so that they can be pipetted using a wide-bore pipette tip. This step is crucial to yielding high quality adipocytes. If the pieces are too large, digestion times will have to be extended, compromising cell viabilty.</li>
	<li>Transfer the minced fat into 50 mL conical tubes using a spoon. Add 10 mL of minced tissue and 30 mL of digestion buffer to each tube. Scale down to appropriate volumes if less than 10 mL of minced fat is available.</li>
	<li>Digest the tissue at 37 &#176;C in a shaking incubator with constant agitation at 150 rpm for 30&#8722;45 min. After 30 min, check on the process every 5 min to avoid over-digestion. 
	NOTE: The digestion is complete when the adipose solution is homogenous without any large pieces and has an apricot color.</li>
</ol>
4. Filtration of the Cell Suspension and Purification of the Mature Adipocytes
<ol>
	<li>Place a funnel on top of a 1 L sterile flask and place a sterile 250 &#181;m mesh filter inside the funnel. Pour the digested fat solution into the filter to remove the undigested tissue.</li>
	<li>When all of the adipocyte suspension has passed through the filter, gently squeeze the mesh filter to increase the yield of adipocytes. Pour approximately 50&#8722;100 mL of wash buffer into the filter to rinse it and squeeze the filter again.</li>
	<li>Pour the isolated adipocyte suspension from the flask into a separation funnel and add wash buffer until the funnel is almost completely filled. Gently invert the funnel a few times to mix the adipocyte suspension with the buffer.</li>
	<li>Let the suspension stand for 2&#8722;3 min until there is a distinct separation of 2 layers (Figure 1B), with a top yellow layer containing the mature adipocytes and free lipid and a bottom layer containing the buffer and the adipose stroma vascular fraction (SVF).</li>
	<li>Open the nozzle on the separation funnel, and slowly elute the bottom solution into a sterile flask (cells from the SVF can be pelleted and collected after centrifugation at 200 x g for 7 min). Keep the top layer with the mature adipocytes in the separation funnel.</li>
	<li>Repeat the washing process in steps 4.3&#8722;4.5 three times to thoroughly wash the mature adipocytes and remove all of the collagenase.</li>
</ol>
5. Packing of the Mature Adipocytes
<ol>
	<li>Open the nozzle and collect the purified mature adipocyte suspension into 50 mL conical tubes.</li>
	<li>Lightly pack the mature adipocytes by spinning the tubes at 50 x g for 3 min.</li>
	<li>Use a 18 G needle and a syringe to remove the remaining wash buffer below the adipocyte suspension.</li>
	<li>Remove the free lipid layer (oil from the small portion of adipocytes that broke during the isolation procedure) floating on top of the mature adipocytes by using a pipette. 
	NOTE: To reduce the risk that the adipocytes will drip off the membranes in step 6.5, it is important that the lipid layer and all wash buffer have been removed when the mature adipocytes are seeded onto membranes. For this reason collect the adipocytes in 3 tubes. The samples that are collected last will have the most carryover lipid and will therefore be of the lowest quality. However, with careful pipetting these samples can be used.</li>
</ol>
6. Seeding of Mature Adipocytes
<ol>
	<li>Open the package containing the permeable membrane inserts (Table of Materials) and take out the membrane component. Flip it upside down and place on a sterile surface so the membranes face the ceiling.</li>
	<li>Pipette 30 &#181;L of packed mature adipocytes onto each of the membranes (Figure 1C). Avoid touching the membrane with the tip. Use wide-bore pipette tips to seed the cells, or use scissors to cut off a small piece of a pipette tip to make it wider.</li>
	<li>Gently invert the 50 mL tubes with packed adipocytes several times throughout the seeding process to ensure an even distribution of adipocytes with different sizes.</li>
	<li>Bring the prepared multiwell plates containing the media from the incubator to the biosafety cabinet and remove the lids. Pick up the membranes seeded with adipocytes and grasp it from the bottom so that it can be inverted in step 6.5.</li>
	<li>In one smooth movement invert the membranes with the adipocytes on top so that the seeded adipocytes are now facing down (Figure 1D). Lower the plate with adipocytes into the wells containing media (Figure 1E).</li>
	<li>Put the lid on the plate and carefully transfer the plate into a tissue culture incubator. Avoid rapid movement of the plate since adipocytes can easily be dislodged initially. 
	NOTE: It takes a few days for the cells to adhere more firmly to the membrane.</li>
</ol>
7. Maintenance of Adipocytes and Harvesting for Analysis
<ol>
	<li>Change the media at least every 7 days. Remove and add media via the cutout hole in each membrane insert. To remove the media, use a syringe and a needle, an aspirating wand, or a pipette with a p200 tip. To add media, use a pipette and slowly pipette new media into the wells along the side of the wall to avoid disturbing the adipocytes. 
	NOTE: Adipocytes have been cultured for 2 weeks with one media change after 7 days. However, different adipose origins and experimental questions may benefit from increased media changes.</li>
	<li>To harvest RNA, remove the media as described in step 7.1, and add 500 &#181;L of lysis buffer (Table of Materials) directly into the wells to lyse the cells. To fix the cells for imaging, add formaldehyde directly to the well at a final concentration of 1%. 
	NOTE: The cells can then be stored at 4 &#176;C.</li>
</ol>

<ol>
	<li>Guilherme, A., Virbasius, J. V., Puri, V., Czech, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+dysfunctions+linking+obesity+to+insulin+resistance+and+type+2+diabetes.">Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes.</a> Nature Reviews Molecular Cell Biology. 9 (5), 367-377 (2008).</li><li>Rosen, E. D., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+we+talk+about+when+we+talk+about+fat.">What we talk about when we talk about fat.</a> Cell. 156 (1-2), 20-44 (2014).</li><li>Lotta, L. 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=Integrative+genomic+analysis+implicates+limited+peripheral+adipose+storage+capacity+in+the+pathogenesis+of+human+insulin+resistance.">Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of human insulin resistance.</a> Nature Genetics. 49 (1), 17-26 (2017).</li><li>Gustafson, B., Hedjazifar, S., Gogg, S., Hammarstedt, A., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+and+impaired+adipogenesis.">Insulin resistance and impaired adipogenesis.</a> Trends in Endocrinology and Metabolism. 26 (4), 193-200 (2015).</li><li>Gesta, 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=Culture+of+human+adipose+tissue+explants+leads+to+profound+alteration+of+adipocyte+gene+expression.">Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression.</a> Hormone and Metabolic Research. 35 (3), 158-163 (2003).</li><li>Fain, J. N., Cheema, P., Madan, A. K., Tichansky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dexamethasone+and+the+inflammatory+response+in+explants+of+human+omental+adipose+tissue.">Dexamethasone and the inflammatory response in explants of human omental adipose tissue.</a> Molecular and Cellular Endocrinology. 315 (1-2), 292-298 (2010).</li><li>Lessard, J., 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=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.">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), e0122065 (2015).</li><li>Asada, 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=Ceiling+culture-derived+proliferative+adipocytes+retain+high+adipogenic+potential+suitable+for+use+as+a+vehicle+for+gene+transduction+therapy.">Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy.</a> American Journal of Physiology - Cell Physiology. 301 (1), C181-C185 (2011).</li><li>Shen, J. F., Sugawara, A., Yamashita, J., Ogura, H., 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=Dedifferentiated+fat+cells:+an+alternative+source+of+adult+multipotent+cells+from+the+adipose+tissues.">Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues.</a> International Journal of Oral Science. 3 (3), 117-124 (2011).</li><li>Harms, M. J., 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+Human+White+Adipocytes+Cultured+under+Membranes+Maintain+Identity,+Function,+and+Can+Transdifferentiate+into+Brown-like+Adipocytes.">Mature Human White Adipocytes Cultured under Membranes Maintain Identity, Function, and Can Transdifferentiate into Brown-like Adipocytes.</a> Cell Reports. 27 (1), 213-225 (2019).</li><li>Wang, Q. A., Scherer, P. E., Gupta, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methodologies+for+the+study+of+adipose+biology:+insights+gained+and+opportunities+ahead.">Improved methodologies for the study of adipose biology: insights gained and opportunities ahead.</a> Journal of Lipid Research. 55 (4), 605-624 (2014).</li><li>Gregoire, F. M., Smas, C. M., Sul, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+adipocyte+differentiation.">Understanding adipocyte differentiation.</a> Physiological Reviews. 78 (3), 783-809 (1998).</li><li>Ruiz-Ojeda, F. J., Ruperez, A. I., Gomez-Llorente, C., Gil, A., Aguilera, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Models+and+Their+Application+for+Studying+Adipogenic+Differentiation+in+Relation+to+Obesity:+A+Review.">Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review.</a> International Journal of Molecular Sciences. 17 (7), 1040 (2016).</li><li>McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+shape,+cytoskeletal+tension,+and+RhoA+regulate+stem+cell+lineage+commitment.">Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.</a> Developmental Cell. 6 (4), 483-495 (2004).</li><li>Lackey, D. E., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+metabolism+by+the+innate+immune+system.">Regulation of metabolism by the innate immune system.</a> Nature Reviews Endocrinology. 12 (1), 15-28 (2016).</li><li>Saltiel, A. R., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+linking+obesity+and+metabolic+disease.">Inflammatory mechanisms linking obesity and metabolic disease.</a> The Journal of clinical investigation. 127 (1), 1-4 (2017).</li><li>Lee, Y. S., Wollam, J., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Integrated+View+of+Immunometabolism.">An Integrated View of Immunometabolism.</a> Cell. 172 (1-2), 22-40 (2018).</li></ol>

The authors have nothing to disclose.

Membrane mature adipocyte aggregate culture (MAAC) is a new method for the long-term culture of freshly isolated mature adipocytes. In setting up MAAC there are a few critical steps in the protocol that greatly impact the yield, quality, and viability of the mature adipocytes. Much effort should be put into mincing the fat in step 3.2 since this step directly influences the amount of time the adipocytes are exposed to the collagenase. If the pieces of adipose are too large, the digestion time will have to be extended which negatively impacts the viability of the cells. Conversely, if the tissue is processed too finely with scissors, viability can be impacted as well. For successful culture of mature adipocytes as MAAC, one should give special attention to the following steps: for successful seeding of the adipocytes onto the membranes, it is crucial that free lipid and carryover wash buffer is removed from the mature adipocytes in steps 5.3 and 5.4. Remaining lipid or wash buffer will reduce the surface tension of the adipocytes and increase the risk of adipocytes dripping-off the membranes when they are flipped. When the adipocytes are seeded and in media, the cells remain in contact with the membrane primarily through buoyancy, thus a slow and gentle technique is recommended when changing media to not lose cells. Remove media from the bottom of the wells as described in step 7.1 and add media by slowly pipetting media down the sides of the walls. Lastly, a time saving suggestion is to prepare the plates with media and treatments before the adipocyte isolation process. Particularly for complex experimental designs, pre-preparing the plates can save much time and ensures that the adipocytes are placed in media with their treatments as soon as they are isolated.
One benefit of using MAAC compared to differentiating preadipocytes is that the MAAC media used is very simple and does not require an unphysiological hormone cocktail. Here we have cultured MAAC in glucose-rich media (DMEM/F12), 10% FBS, 1% penn/strep, and 20 nM insulin. Importantly, we have found that insulin is absolutely required for the rosiglitazone/pioglitazone driven induction of UCP110. Insulin, however, is not required to maintain the cells&#39; adipogenic phenotype. Thus, depending upon the experimental question, insulin can be included or omitted.
The procedure detailed above has been optimized for the isolation and culture of human adipocytes. However, mouse, and possibly other organism&#39;s adipocytes, can also be cultured as MAAC. If mouse adipocytes are to be cultured as MAAC there are additional considerations and precautions that should be kept in mind. We have found that mature adipocytes from mice are much more fragile than those from humans. As a result, the digestion time should be shortened to an absolute minimum to increase cell viability. We also found that adipocytes from young mice (8-week-old and younger) provided the most robust and reproducible results. Lastly, mouse MAAC can be cultured for up to one week, however given that their adipogenic phenotype appears less stable than humans (which can be cultured through at least two weeks) we recommend culturing mouse MAAC for the minimum required time to address experimental questions.
Since the MAAC model is based on using permeable membranes, one advantage of this technique is the possibility of co-culturing mature adipocytes with other cell types. We have previously demonstrated the ability of mature adipocytes and macrophages to crosstalk through the use of MAAC10. This opens up opportunities to further explore the linkage between obesity, insulin resistance, and immune responses15,16,17. Future experiments could incorporate other cell types such as hepatocytes, preadipocytes, endothelial cells, or pancreatic cells to further increase the complexity and the translational relevance of the MAAC model and investigate crosstalk between multiple cell types.
Even though MAAC has been shown to be superior at maintaining functionality and identity of the adipocytes relative to other adipocyte in vitro models, it also has limitations that need to be considered. In comparison to using adipocytes differentiated from precursor cells, MAAC is a more laborious and time-consuming model. Plates with membranes are also more expensive compared to regular cell culture plates. Importantly, mature adipocytes need to be freshly isolated each time upon seeding and cannot be expanded or frozen into stocks like precursor cells. Thus, this requires having access to fresh white adipose tissue samples, but also adds a level of complexity stemming from donor to donor variations.
Here we have presented a detailed protocol for isolating human mature adipocytes and setting up MAAC. We have demonstrated that adipocytes cultured as MAAC remains viable through two weeks, their adipogenic gene signature is preserved, and they respond to diverse pharmacological agonism. Using MAAC allows for the study of cross-talk betweeen adipocytes and other cell types, and the assessment of long term phenotypic changes of mature adipocytes in response to different stimuli.

The worldwide increase in obesity and obesity-related co-morbidities necessitates the development of new therapeutics. White adipose tissue (WAT) is an important regulator of whole-body metabolism, energy homeostasis, and is a central player in the development of insulin resistance and type 2 diabetes (T2D)1,2. During chronic excess calorie consumption, adipocytes enlarge to handle the surplus of energy. However, adipocyte lipid storage capacity can become exceeded, resulting in an elevation of circulating levels of fatty acids and increased storage in peripheral non-adipose tissues and leading to lipotoxicity3,4.
The lack of adipocyte in vitro models with high translational relevance is a key challenge in the development of new treatments for obesity and T2D. The ex vivo explant model, where small pieces of adipose tissue are cultured, is associated with rapid alterations in adipogenic gene expression driven by hypoxia and inflammation5,6. Ceiling cultures (CCs) where mature adipocytes float and adhere to the top of media-filled flasks, rapidly dedifferentiate into fibroblast-like cells lacking lipid7,8,9,10. The most commonly used model is adipocytes differentiated in vitro from committed precursors. The differentiated cells are, however, morphologically distinct from mature adipocytes in vivo since they are much smaller in size and lack a unilocular lipid droplet. Other limitations with this model include the unphysiological need of a chemical cocktail to drive differentiation, as well as variability in differentiation efficiency which can be affected by a number of factors11,12,13,14.
We have recently developed membrane mature adipocyte aggregate culture (MAAC), a method for long term culture of freshly isolated mature adipocytes, where adipocytes are cultured under permeable membranes10. Unbiased analysis of RNA sequencing data has shown that relative to adipose tissue explants and in vitro-differentiated adipocytes, MAAC is most similar to freshly isolated adipocytes. MAAC can be used to culture mature adipocytes isolated from both subcutaneous and visceral adipose tissue, as well as adipocytes from both obese and lean subjects. This methodology permits the study of long-term adipocyte phenotypic changes and facilitates co-culture of adipocytes with other cell types. Here we provide a detailed protocol for the isolation of mature adipocytes from human adipose tissue and how to set up the MAAC system.

&#160; &#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclaved scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved spoons</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer RLT</td>
			<td>QIAGEN</td>
			<td>79216</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>CaCl2*2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes, 50 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter flask 0.2 &#181;m pore size, 75 mm</td>
			<td>Thermo Scientific</td>
			<td>156-4020</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Filter flask 0.2 &#181;m pore size, 75 mm</td>
			<td>Thermo Scientific</td>
			<td>158-0020</td>
			<td>1000 mL</td>
		</tr>
		<tr>
			<td>HBSS+CaCl2+MgCl2</td>
			<td>Gibco</td>
			<td>14065-49</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin (Actrapid Penfill)</td>
			<td>Novo Nordisk A/S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Merck</td>
			<td>104873</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Gibco</td>
			<td>10012-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh filter (250 &#181;M)</td>
			<td>Sintab AB</td>
			<td>6111-025043</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4*7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles, 18G, 1.20x40 mm</td>
			<td>Sterican</td>
			<td>613-2948</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencillin-Streptomycin (Penn/Strep)</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, 150x21 mm</td>
			<td>Thermo Scientific</td>
			<td>168381</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR master mix</td>
			<td>Applied Biosystems</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantstudio 7 Flex Real-Time PCR machine</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini kits</td>
			<td>QIAGEN</td>
			<td>74106</td>
			<td></td>
		</tr>
		<tr>
			<td>Separation funnel</td>
			<td>VWR</td>
			<td>527-0008</td>
			<td>For large scale preparation</td>
		</tr>
		<tr>
			<td>Separation funnel</td>
			<td>VWR</td>
			<td>527-0005</td>
			<td>For small scale preparation</td>
		</tr>
		<tr>
			<td>Shaking incubator (37 &#176;C)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes, 5 mL</td>
			<td>Omnifix</td>
			<td>612-2892</td>
			<td>100 st</td>
		</tr>
		<tr>
			<td>Tissue culture incubator (37 &#176;C, 5% CO2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transwells, 24-well (6,5 mm)</td>
			<td>Costar</td>
			<td>3397</td>
			<td>Permeable membrane inserts</td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Invitrogen</td>
			<td>10296010</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Type 2 Collagenase</td>
			<td>Worthington</td>
			<td>LS004177</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of human mature adipocytes using membrane mature adipocyte aggregate cultures (maac)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Mature adipocytes cultured as MAAC preserves their function, phenotype, and can be used to study adipocyte responses to various pharmacological treatments. After 1 week of culture MAAC isolated from subcutaneous adipose tissue maintain the characteristic unilocular lipid droplet found only in mature adipocytes (Figure 2A). MAAC was cultured for 1 week while treated with either the PPAR&#947; agonists rosiglitazone (Rosi) and pioglitazone (Pio), or the glucocorticoid receptor (GR) agonist dexamethasone (Dex) to determine if different nuclear hormone receptor (NHR) agonists drive predicted changes in downstream target genes in MAAC. Rosi and Pio increased the expression of the PPAR&#947; responsive genes FABP4 and LPL by 4 and 2&#8722;3 fold, respectively, whereas dexamethasone had no effect (Figure 2B). Similarly, dexamethasone robustly drove the gene expression of the GR target genes APOD and FKBP5 by 13- and 55-fold respectively, while the PPAR&#947; agonists had no significant effects. We have previously demonstrated that freshly isolated human mature white adipocytes can transdifferentiate into a brown-like phenotype in MAAC when treated with Rosi10. A 7 day treatment with Rosi or Pio robustly induced the gene expression of the brown fat-specific gene UCP1 by 44-65 fold, as well as increased the expression of the brown fat marker PDK4 12-18 fold (Figure 2C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60485/60485fig1a.jpg" /> 
Figure 1: Visual diagram of MAAC setup. (A) Preparation of medium. (B) Isolation and packing of mature adipocytes. (C) Seeding mature adipocytes onto membranes. (D) Inverting membranes while keeping adipocytes attached. (E) Lowering the membranes into the medium and changing the medium. This figure has been modified from Harms et al.10. <a href="https://www.jove.com/files/ftp_upload/60485/60485fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60485/60485fig2a.jpg" /> 
Figure 2: MAAC maintains unilocular appearance through one week and respond to diverse pharmacological agonism. (A) Representative 4x and 10x bright field images of MAAC after one week of culture. Adipocytes that have an average diameter of 100 &#181;m with large unilocular lipid droplets are easily discernable. (B) mRNA levels of PPAR&#947; target genes and glucocorticoid receptor (GR) target genes in MAAC after 7 days of treatment with vehicle (Vehc), rosiglitazone (Rosi), pioglitazone (Pio), or dexamethasone (Dex). Rosi, Pio and Dex were all used at a final concentration of 10 &#181;M. (C) mRNA levels of brown fat-enriched genes in MAAC after 7 days of treatment with vehicle (Vehc), rosiglitazone (Rosi), pioglitazone (Pio), or dexamethasone (Dex). Rosi, Pio and Dex were all used at a final concentration of 10 &#181;M. For all gene expression data, TATA-binding protein (TBP) was used as an internal normalization control. Statistics were calculated using one-way ANOVA with Tukey&#39;s multiple comparisons test. (mean &#177; SD, n = 3, *p &#60; 0.05; **p &#60; 0.01; ***p &#60; 0.001). <a href="https://www.jove.com/files/ftp_upload/60485/60485fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

&#160; &#160;

Watch this Scientific Journal Video about Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC at JoVE.com

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

isolation-culture-human-mature-adipocytes-using-membrane-mature

Universidad San Sebastian

Research

JoVE Journal

Biology

18.8K Views.  AstraZeneca. Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Video: Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...