Summary
In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.
Abstract
Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4–9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14–21 days of differentiation using a cocktail of 0.5 μg/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 μM indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.
Introduction
Adipose tissue is comprised of a heterogeneous mixture of cells, predominantly mature adipocytes and a stromal vascular fraction including fibroblasts, immune cells and adipose-derived stem cells (ADSCs)1,2,3. Primary ADSCs can be isolated directly from white adipose tissue and stimulated to differentiate into adipocytes, cartilage or bone cells4. ADSCs exhibit classical stem cell characteristics such as maintenance of multipotency in vitro and self-renewal; and are adherent to plastic in culture5,6. ADSCs are of important interest for the use in regenerative medicine due to their multipotency and ability to be easily harvested in large quantities using non-invasive techniques7. Adipogenic differentiation of ADSCs produces cells that functionally mimic mature adipocytes including lipid accumulation, insulin-stimulated glucose uptake, lipolysis, and adipokine secretion8. Their resemblance to mature adipocytes has led to the widespread use of ADSCs for physiological investigation of cellular characteristics and metabolic function of adipocytes. There is increasing evidence supporting the idea that the development of metabolic dysfunction and disorders originates at the cellular or tissue level9,10,11,12. Optimal ADSC differentiation is required for sufficient adipose tissue expansion, proper adipocyte function, and effective metabolic regulation13.
Protocols described in this manuscript are straightforward techniques utilizing standard laboratory equipment and basic reagents. The manuscript first describes the protocol for the isolation of primary ADSCs from fresh adipose tissue using mechanical and enzymatic digestion. Next, the protocol for proliferation and passaging of ADSCs in stromal medium is described. Lastly, the protocol for adipogenic differentiation of ADSCs is described. Following differentiation, these cells can be used for studies to better understand adipocyte metabolism and mechanisms of dysfunction. The protocols for confirmation of adipogenic differentiation and lipid droplet detection using Oil Red O and boron-dipyrromethene (BODIPY) staining are also described. The details of these protocols focused on primary ADSCs isolated from fresh omental adipose tissue of rhesus macaques. We and others have used this protocol to successfully isolate ADSCs from rhesus macaque subcutaneous and omental adipose tissues depots14,15. For the same amount of tissue used, we have observed that subcutaneous adipose tissue is more dense, tougher and yields less cells from digestion compared to omental adipose tissue. This protocol has also been used to isolate ADSCs from human adipose samples16.
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Protocol
All obtained tissues and procedures were approved by the Institutional Animal Care and Use Committee at the Louisiana State University Health Sciences Center and were performed in accordance with the guidelines of the National Institute of Health (NIH publication No. 85-12, revised 1996).
1. Preparation of buffers and solutions
- Prepare sterile 5-phosphate-buffered saline wash buffer (5-PBS) solution by adding 5% penicillin/streptomycin (pen/strep) and 0.25 μg/mL of fungal inhibitor to 1x PBS. Prepare sterile 2-PBS wash buffer (2-PBS) solution by adding 2% pen/strep and 0.25 μg/mL of fungal inhibitor in 1x PBS. Wash buffers can be stored at 4 °C for future use and must be used within 4 weeks.
NOTE: 5-PBS buffer is used for adipose tissue collection and initial washing to minimize possible contamination as tissue samples obtained at necropsy are collected in a non-sterile environment. - Prepare sterile collagenase buffer (CB) by combining 0.075% collagenase Type I (125 units/mg activity), 2% pen/strep, and 0.25 μg/mL of fungal inhibitor in Hank’s balanced salt solution (HBSS) with 1% Bovine Serum Albumin. CB must be used within 1 h.
- Prepare sterile ADSC growth medium using α-MEM buffer. Combine 100 mL of fetal bovine serum (FBS), 5 mL of 200 mM L-glutamine solution, 500 μL of 0.25 μg/mL of fungal inhibitor and 10 mL of pen/strep solution in 394 mL of α-MEM buffer. ADSC growth medium can be stored at 4 °C and must be used within 4 weeks.
NOTE: Heat inactivation of FBS is not necessary. - Prepare sterile ADSC differentiation medium by combining ADSC growth medium as prepared above and induction agents to achieve final concentration of 0.5 μg/mL dexamethasone, 0.5 mM isobutyl methylxanthine and 50 μM indomethacin.
2. ADSC isolation
- Tissue preparation and digestion
- Pipette ~10 mL of 5-PBS buffer into four 100 mm cell culture dishes.
- Transfer ~50 g of adipose sample to one of the 100 mm dishes containing 5-PBS. Wash the collected adipose tissue sample four times by transferring it sequentially across the four culture dishes containing 5-PBS.
- Transfer adipose sample to a clean 100 mm culture dish and thoroughly mince adipose tissue using scissors or two sterile scalpels. Mincing allows for increasing the surface area for efficient and complete enzymatic digestion.
- Transfer the minced adipose tissue to 50 mL plastic conical tube containing 13 mL of CB (1–3 cm section of tissue per 15 mL of CB).
- Rinse the culture dish with 2 mL of CB and transfer the medium to the 50 mL plastic conical tube.
NOTE: At this point, there should be a total of 15 mL of CB with tissue in a 50 mL plastic conical tube. - Pipette up and down several times using 25 mL serological pipette to facilitate mechanical tissue digestion.
- Incubate the 50 mL plastic conical tube containing tissue in CB on a rocker at medium speed at 37 °C for 30–60 min.
- ADSC plating for culture
- Add 10 mL of ADSC growth medium to the 50 mL tube containing tissue to neutralize enzyme activity. Pipette up and down several times using 25 mL serological pipette to separate any adipose tissue aggregates.
- Transfer the liquid portion to a new sterile 50 mL conical tube leaving the solids behind. Wash the original 50 mL tube 3 times with ~7 mL of 2-PBS buffer and transfer the liquid portion to the 50 mL tube.
- Centrifuge at 500 x g for 5 min to obtain a cell pellet. Carefully remove as much supernatant as possible using a serological pipette without disturbing the cell pellet. Do not decant.
- Resuspend the cell pellet in 1 mL of 1x red blood cell (RBC) lysis buffer and incubate at room temperature for 10 min. Add 5 mL of ADSC growth medium to the tube and centrifuge at 500 x g for 5 min, then carefully remove and discard the supernatant.
- Add 5 mL of ADSC growth medium and centrifuge at 500 x g for 5 min to wash cell pellet. Discard the supernatant.
- Resuspend the pellet in 2 mL of ADSC growth medium and filter through a 70 μm cell strainer into a new sterile 50 mL plastic conical tube.
- Rinse the cell strainer with an additional 2 mL of ADSC growth medium. Transfer 4 mL of the suspension containing ADSCs from the 50 mL conical tube to a sterile 100 mm culture dish.
- Wash the 50 mL conical tube 2 times with 3 mL of ADSC growth medium and transfer the liquid into the 100 mm culture dish containing ADSCs for a total of 10 mL of medium in culture dish.
- View cells under an inverted microscope at 10x magnification to check for floating cells in the media as shown in Figure 1A. Cells should be maintained in a CO2 incubator at 37 °C at 5% CO2 and 100% relative humidity.
- After 24 h, view cells under an inverted microscope to check for cell adherence. Aspirate ADSC growth medium from the plate and replace with 10 mL of fresh, warm (37 °C) media. Remove and replace media every 48 h until cells are 80%–90% confluent (Figure 1B).
NOTE: At least 10–20 % of cells will be adherent by 24 h. Check the cell culture for signs of contamination such as cell granularity, turbidity of the media, or growth of spores. Once cells are 80% confluent, they can be harvested for proliferation and cryopreservation or induced to differentiate. ADSCs can be expanded to 4–6 passages before losing their ability to efficiently proliferate or differentiate.
3. ADSC proliferation
- Cell detachment
- Remove ADSC growth medium using an aspirator/pipette. Rinse confluent ADSCs 2 times with 2 mL of sterile, room temperature PBS.
- Aspirate PBS and add 2 mL of 0.25 % trypsin-EDTA per 100 mm culture plate. Ensure that the entire surface area is covered with trypsin.
- Incubate plate for ~7 min at 37 °C in 5% CO2 until ADSCs are detached. Remove plate from incubator and mechanically dislodge cells by forcefully pipetting the trypsin solution in the plate.
- Add 2 mL of ADSC growth medium to the dislodged cells and gently pipette to mix. Transfer cells to sterile 50 mL plastic conical tube. To ensure maximal cell recovery, rinse culture dish with another 2 mL of ADSC growth medium, and transfer medium to the conical tube containing detached cells.
- Centrifuge the 50 mL conical tube at 500 x g for 5 min at room temperature to obtain a cell pellet. Carefully decant supernatant without disturbing the cell pellet.
- Resuspend cell pellet in 5–6 mL of ADSC growth medium per 1 x 106 cells.
- Cell count and plating
- In a 1.5 mL microcentrifuge tube, dilute 10 μL of cell solution from above and 10 μL of 0.04% trypan blue solution (0.4% trypan blue diluted 1:10 in PBS) and count cells using a hemocytometer.
- Resuspend the desired number of ADSCs in growth medium. For 100 mm culture dish, plate ~3.0 x 105 cells in 10 mL ADSC growth media to allow for 80% confluence in 72 h or 100% confluency in 96 h.
- View the dish under an inverted microscope at 10x magnification prior to incubation to ensure presence of suspended cells. Maintain the cells at 37 °C at 5% CO2 and 100% relative humidity.
- Every 48 h aspirate the growth medium and replace with warm (37 °C) medium until cells are 80% to 90% confluent as shown in Figure 1B.
- Repeat steps from sections 3.1 and 3.2 for appropriate number of passages to obtain desired cell number.
NOTE: After passages 5 to 6, primary cells appear to undergo senescence; therefore, aim to obtain desired cell numbers by passage 4.
4. ADSC adipogenic differentiation
- Aspirate ADSC growth medium from adhered ADSCs that have reached 80% to 90% confluence.
- Quickly rinse ADSC cell layer with sterile, room temperature PBS.
- Add ADSC differentiation medium. For 100 mm culture dish, add 10 mL of ADSC differentiation medium.
- Replace medium every 3 days for approximately 14–21 days. Mature adipocytes are generally observed after 14 days of differentiation.
- Examine cells under an inverted light microscope at 40x magnification to confirm the presence of lipid droplet as shown in Figure 2A.
5. Adipocyte detection
NOTE: This section will describe staining protocols used for lipid droplet detection in differentiated adipocytes, however, immunofluorescent staining for CD105, a mesenchymal stem cell marker, can also be used for ADSC confirmation.
- Oil Red O staining
- Prepare 0.5% Oil Red O stock solution in isopropanol. For 20 mL of Oil Red O stock solution, dissolve 100 mg Oil Red O in 20 mL of isopropanol. Filter the solution through a sterile syringe filter with 0.2 μm membrane.
- Carefully aspirate the differentiation medium and wash cells 2 times by adding PBS along the sides of the well to not disturb the cell monolayer.
- Carefully aspirate PBS from cells and add enough neutral buffered formalin (NBF, 10%) to cover cell layer. Incubate at room temperature for 30–60 min.
- During the formalin fixation step, prepare Oil Red O working solution by diluting 3 parts of Oil Red O stock solution with 2 parts of distilled water. Mix the solution and filter through a sterile syringe filter with 0.2 μm membrane. Working solution is stable for up to 2 h.
- Carefully aspirate NBF and quickly wash (~ 15 s) the cell layer with distilled water. Add enough 60% isopropanol to cover the cell layer after aspirating water and incubate at room temperature for 5 min.
- Carefully aspirate isopropanol after 5 min incubation and add enough Oil Red O working solution to cover the cell layer and incubate at room temperature for 10–15 min.
- Carefully aspirate off the Oil Red O working solution and wash the cell layer several times with distilled water until the water becomes clear.
- Add PBS to the cell culture dish and examine cells under an inverted microscope for indication of differentiation and presence of lipid droplets (Figure 2B).
- BODIPY Staining
- Prepare 5 mM BODIPY stock solution by dissolving 1.3 g of BODIPY in 1 mL of DMSO. Prepare 2 μg/mL BODIPY working solution by diluting the stock solution 1:25,000 times in PBS.
- Carefully aspirate the differentiation medium and wash cells 2 times by adding PBS along the sides of the well to not disturb the cell monolayer.
- Carefully aspirate PBS and add enough BODIPY working solution to cover the cell layer and incubate at 37 °C for 30 min.
NOTE: From this point, protect cells from light by covering plate with foil. - Carefully aspirate the BODIPY working solution and wash the cell layer 3 times with PBS.
- Fix cells by adding 4% paraformaldehyde and incubating at room temperature for 15 min.
- Carefully aspirate the fixation buffer and wash cells 2 times by adding PBS along the sides of the well to not disturb the cell monolayer.
- Add PBS to cell culture dish and examine cells under an inverted fluorescent microscope for evidence of differentiation and presence of lipid droplet (Figure 2C). Image cells using a FITC/EGFP/Bodipy Fl/Fluo3/DiO Chroma filter set. Excitation wavelength is at 480 nm with a band width of 40 nm and emission wavelength is at 535 nm with a bandwidth of 50 nm. A similar or appropriate filter set, and microscope can be used.
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Representative Results
The ADSCs isolated from rhesus macaque adipose tissue samples were seeded on culture plates and is shown in Figure 1. On the day of plating, cells are non-adherent and float in the culture dish as shown in Figure 1A. Within 72 h, ADSCs will become 80% confluent and are ready for adipocyte differentiation (Figure 1B). ADSCs exhibit strong adipogenic characteristics after chemical induction. After 14 days of differentiation, mature adipocytes can be observed via light microscopy (Figure 2A). To confirm ADSC adipogenic differentiation various stains may be used to visualize lipid droplets of the differentiated mature adipocytes. On day 14 of differentiation, cells were stained and fixed with Oil Red O (Figure 2B) and BODIPY (Figure 2C) for lipid droplet visualization. The black arrow represents a differentiated adipocyte (Figure 2B). These data indicate that following this protocol, ADSCs were generated from rhesus macaque adipose tissue and differentiated into mature adipocytes.
Figure 1: Light micrographs of ADSCs on the day of plating and at 80% cell confluency. (A) Representative light micrograph of stromal vascular cells on the day of plating following ADSC isolation from fresh rhesus macaque adipose tissue (10x magnification). (B) Representative light micrograph of ADSCs at 80% confluency (20x magnification). Please click here to view a larger version of this figure.
Figure 2: Micrographs of ADSCs at day 14 of differentiation. (A) Representative light micrograph obtained on day 14 of ADSC differentiation (40x magnification). (B) Representative micrograph of Oil Red O staining at day 14 of ADSC differentiation (20x magnification). (C) Representative micrograph of boron-dipyrromethene (BODIPY) staining at day 14 of ADSC differentiation (20x magnification). Please click here to view a larger version of this figure.
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Discussion
ADSC isolation, proliferation and differentiation protocols are straight-forward and reproducible, but they require careful technique to ensure adequate isolation, healthy expansion, and efficient differentiation. A sterile working environment is critical for all cell culture experiments. Bacteria or fungi may be introduced into cell cultures through contaminated tools, media or work environment. Fungal contamination is indicated by spore growth in the culture, while bacterial contamination is indicated by the presence of turbidity of the media. We suggest disinfecting the biosafety cabinet and all pipettes, bottles and tools before use, turning on the laminar air flow at least 15 minutes before using the biosafety cabinet and disinfecting the cabinet and all pipettes after use to reduce risk for contamination. Also, we suggest autoclaving nonfilter pipette tips for vacuum aspiration and flushing the aspirator with sterile water followed by 70% ethanol after use. A culture dish with media only can be placed in the humidified incubator at 37 °C and 5 % CO2 for a few days to check sterility of media. Contaminated cultures should be bleached for 30 minutes and discarded. The cell culture incubator should also be disinfected.
A major difference between the protocols within this manuscript and those previously published is the use of BSA in our ADSC collagenase digestion buffer which allows for isolation of mature adipocytes and ADSCs. Additionally, our protocol utilizes ADSC growth medium supplemented with 20% FBS compared to 10% FBS as suggested by most protocols. We have noticed that rhesus macaque primary ADSCs proliferate and differentiate better with 20% FBS. ADSC differentiation in vitro is induced by lineage-specific induction agents. The induction agents used in this protocol are widely accepted for in vitro adipogenic differentiation of ADSCs. However, unlike many previously published protocols, the adipogenic differentiation cocktail does not include insulin or PPARγ agonists. We and others have used this protocol to successfully differentiate both rhesus macaque and human ADSCs14,15,16.
In this protocol, ADSC adipogenic differentiation is confirmed by lipid droplet detection using Oil Red O and BODIPY staining techniques. Though these stains are well recognized in the field, there are other methods commonly used including flow cytometry for detection of CD105 to identify ADSCs, or markers of endothelial and immune cells to establish purity of ADSCs17.
Use of ADSCs provides a valuable tool for regenerative medicine and metabolic research. ADSC isolation and proliferation produces a large number of stem cells that can be used for several downstream applications and experimental techniques including but not limited to those described in this protocol. A major intended use for differentiated adipocytes in this protocol includes the use of metabolic assays such as insulin-stimulated glucose uptake, lipogenesis, and stimulated lipolysis18. In addition, ADSCs can be differentiated into osteogenic and chondrogenic lineage and used for downstream analysis including for tissue engineering7.
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Disclosures
None.
Acknowledgments
The authors would like to thank Curtis Vande Stouwe for his technical assistance. The research underlying development of the protocols was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (5P60AA009803-25, 5T32AA007577-20 and 1F31AA028459-01).
Materials
| Name | Company | Catalog Number | Comments |
| 0.4 % trypan blue | Thermo-Fisher | 15250061 | |
| 1.5-ml microcentrifuge tube | Dot Scientific | 707-FTG | |
| 100 % isopropanol | Sigma-Aldrich | PX1838-P | |
| 100-mm cell culture dish | Corning | 430167 | |
| 3-Isobutyl-1-methylxanthine | Sigma-Aldrich | I7018 | |
| 50-mL plastic conical tube | Fisher Scientific | 50-465-232 | |
| 70-µm cell strainer | Corning | CLS431751 | |
| a-MEM | Thermo-Fisher | 12561056 | |
| Aluminum foil | Reynolds Wrap | ||
| BODIPY | Thermo-Fisher | D3922 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | 05470 | |
| Centrifuge | Eppendorf | 5810 R | |
| Collagenase, Type I | Thermo-Fisher | 17100017 | |
| Dexamethasone-Water Soluble | Sigma-Aldrich | D2915 | |
| Dimethyl sulfoxide, DMSO | Sigma-Aldrich | D2650 | |
| Distilled water | Thermo-Fisher | 15230162 | |
| Fetal Bovine Serum, USDA-approved | Sigma-Aldrich | F0926 | |
| Fungizone/Amphotericin B (250 ug/mL) | Thermo-Fisher | 15290018 | |
| Hanks' Balanced Salt Solution (HBSS) | Thermo-Fisher | 14175095 | |
| Hemacytometer with cover slip | Sigma-Aldrich | Z359629 | |
| Indomethacin | Sigma-Aldrich | I7378 | |
| Inverted light microscope | Nikon | DIAPHOT-TMD | |
| L-glutamine (200 mM) | Thermo-Fisher | 25030081 | |
| Laboratory rocker, 0.5 to 1.0 Hz | Reliable Scientific | Model 55 Rocking | |
| Neutral buffered formalin (10 %) | Pharmco | 8BUFF-FORM | |
| Oil Red O | Sigma-Aldrich | O0625 | |
| Paraformeldehyde | Sigma-Aldrich | P6148 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo-Fisher | 15140122 | |
| Phosphate buffered saline (PBS), pH 7.4 | Thermo-Fisher | 10010023 | |
| Red blood cell (RBC) lysis buffer | Qiagen | 158904 | |
| Serological pipettes, 2 to 25 mL | Costar Stripettes | ||
| Standard humidified cell culture incubator, 37 °C, 5 % CO2 | Sanyo | MCO-17AIC | |
| Trypsin-EDTA (0.25%) | Thermo-Fisher | 25200056 |
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