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

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

1. 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 MgSO₄·7H₂O in 900 mL of water. Add 1.84 g of CaCl₂·2H₂O and adjust the volume to 1 L by adding water. Sterile filter through a 0.22 µm filter and store at 4 °C.
2. 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.
3. 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.
4. Adjust the pH of Medium 199 to 7.4.
5. Sterile filter both buffers and medium 199 through a 0.22 µm filter flask and warm to 37 °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 0.5−1 mL/well of Dulbecco's modified Eagle's medium/Ham'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 °C, 5% CO₂).

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.

1. 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.
2. 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.

3. Collagenase Treatment

1. 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 µm 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.
2. 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.
3. 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.
4. Digest the tissue at 37 °C in a shaking incubator with constant agitation at 150 rpm for 30−45 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.

4. Filtration of the Cell Suspension and Purification of the Mature Adipocytes

1. Place a funnel on top of a 1 L sterile flask and place a sterile 250 µm mesh filter inside the funnel. Pour the digested fat solution into the filter to remove the undigested tissue.
2. When all of the adipocyte suspension has passed through the filter, gently squeeze the mesh filter to increase the yield of adipocytes. Pour approximately 50−100 mL of wash buffer into the filter to rinse it and squeeze the filter again.
3. 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.
4. Let the suspension stand for 2−3 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).
5. 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.
6. Repeat the washing process in steps 4.3−4.5 three times to thoroughly wash the mature adipocytes and remove all of the collagenase.

5. Packing of the Mature Adipocytes

1. Open the nozzle and collect the purified mature adipocyte suspension into 50 mL conical tubes.
2. Lightly pack the mature adipocytes by spinning the tubes at 50 x g for 3 min.
3. Use a 18 G needle and a syringe to remove the remaining wash buffer below the adipocyte suspension.
4. 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.

6. Seeding of Mature Adipocytes

1. 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.
2. Pipette 30 µL 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.
3. 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.
4. 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.
5. 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).
6. 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.

7. Maintenance of Adipocytes and Harvesting for Analysis

1. 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.
2. To harvest RNA, remove the media as described in step 7.1, and add 500 µL 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 °C.