Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

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- to 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 ug/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.

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, we describe ADSC differentiation using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

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) ^1–3. Primary ADSCs can be isolated directly from white adipose tissue and stimulated to differentiate into adipocytes, cartilage or bone cells ⁴. ADSCs exhibit classical stem cell characteristics such as maintenance of multipotency in vitro and self-renewal; and are adherent to plastic in culture^5,6. ADSCs are of important interest for 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 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 level^9–12. Optimal ADSC differentiation is required for sufficient adipose tissue expansion, proper adipocyte function, and effective metabolic regulation¹³.

The protocols described in this manuscript are straightforward techniques utilizing standard laboratory equipment and basic reagents. The manuscript first describes the protocol for 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 depots^14,15. For the same amount of tissue used, we’ve 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.

PROTOCOL:

Note: 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

1.1.

Prepare sterile 5-phosphate-buffered saline wash buffer (5-PBS) solution using 5 % penicillin/streptomycin (pen/strep) in 1X PBS and 0.25 μg/mL of Fungizone. Prepare sterile 2-PBS wash buffer (2-PBS) solution using 2 % pen/strep in 1X PBS and 0.25 μg/mL of Fungizone. 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.

1.2.

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 Fungizone in Hank’s balanced salt solution (HBSS) with 1 % Bovine Serum Albumin. CB must be used within 1 hour.

1.3.

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 Fungizone 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.

1.4.

Prepare sterile ADSC differentiation medium by combining ADSC growth medium as prepared above and induction agents to achieve final concentration of 0.5 ug/mL dexamethasone, 0.5 mM isobutyl methylxanthine and 50 μM indomethacin.

2. ADSC Isolation

2.1. Tissue preparation and digestion

2.1.1.

Pipette ^~10 mL 5-PBS buffer into four 100-mm cell culture dishes.

2.1.2.

Transfer ^~50 g 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.

2.1.3.

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.

2.1.4.

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).

2.1.5.

Rinse 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.

2.1.6.

Pipet up and down several times using 25 mL serological pipette to facilitate mechanical tissue digestion.

2.1.7.

Incubate the 50-mL plastic conical tube containing tissue in CB on a rocker at medium speed at 37 °C for 30 – 60 min.

2.2. ADSC plating for culture

2.2.1.

Add 10 mL of ADSC Growth Medium to the 50-mL tube containing tissue to neutralize enzyme activity. Pipet up and down several times using 25-mL serological pipette to separate any adipose tissue aggregates.

2.2.2.

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.

2.2.3.

Centrifuge at 500 × 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.

2.2.4.

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 × g for 5 min, then carefully remove and discard the supernatant.

2.2.5.

Add 5 mL of ADSC growth medium and centrifuge at 500 × g for 5 min to wash cell pellet. Discard supernatant.

2.2.6.

Resuspend 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.

2.2.7.

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.

2.2.8.

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.

2.2.9.

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.

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).

2.2.10.

After 24 hours, 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 hours until cells are 80 % - 90 % confluent (Figure 1B).

Note: At least 10–20 % of cells will be adherent by 24 hours. 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

3.1. Cell detachment

3.1.1.

Remove ADSC growth medium using an aspirator/pipette. Rinse confluent ADSCs 2 times with 2 mL of sterile, room temperature PBS.

3.1.2.

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.

3.1.3.

Incubate plate for ^~7 minutes 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.

3.1.4.

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.

3.1.5.

Centrifuge the 50-mL conical tube at 500 × g for 5 minutes at room temperature to obtain a cell pellet. Carefully decant supernatant without disturbing the cell pellet.

3.1.6.

Resuspend cell pellet in 5 to 6 mL of ADSC growth medium per 1 × 10⁶ cells.

3.2. Cell count and plating

3.2.1.

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.

3.2.2.

Resuspend desired number of ADSCs in growth medium. For 100-mm culture dish, plate ^~3.0 × 10⁵ cells in 10 mL ADSC growth media to allow for 80 % confluence in 72 hours or 100 % confluency in 96 hours.

3.2.3.

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.

3.2.4.

Every 48 hours aspirate the growth medium and replace with warm (37 °C) medium until cells are 80 % to 90 % confluent as shown in Figure 1B.

3.2.5.

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

4.1.

Aspirate ADSC growth medium from adhered ADSCs that have reached 80 % to 90 % confluence.

4.2.

Quickly rinse ADSC cell layer with sterile, room temperature PBS.

4.3.

Add ADSC differentiation medium. For 100-mm culture dish, add 10 mL of ADSC differentiation medium.

4.4.

Replace medium every 3 days for approximately 14–21 days. Mature adipocytes are generally observed after 14 days of differentiation.

4.5.

Examine cells under an inverted light microscope at 40X magnification to confirm presence of lipid droplet as shown in Figure 2A.

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).

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.

5.1. Oil Red O Staining

5.1.1.

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.

5.1.2.

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.

5.1.3.

Carefully aspirate PBS from cells and add enough neutral buffered formalin (NBF, 10 %) to cover cell layer. Incubate at room temperature for 30 – 60 minutes.

5.1.4.

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 solution and filter through a sterile syringe filter with 0.2-μm membrane. Working solution is stable for up to 2 hours.

5.1.5.

Carefully aspirate NBF and quickly wash (^~ 15 s) the cell layer with distilled water. Carefully aspirate the water and add enough 60 % isopropanol to cover the cell layer and incubate at room temperature for 5 minutes.

5.1.6.

Carefully aspirate the isopropanol and add enough Oil Red O working solution to cover the cell layer and incubate at room temperature for 10 – 15 minutes.

5.1.7.

Carefully aspirate off the Oil Red O working solution and wash the cell layer several times with distilled water until the water becomes clear.

5.1.8.

Add PBS to the cell culture dish and examine cells under an inverted microscope for evidence of differentiation and presence of lipid droplets (Figure 2B).

5.2. BODIPY Staining

5.2.1.

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.

5.2.2.

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.

5.2.3.

Carefully aspirate PBS and add enough BODIPY working solution to cover the cell layer and incubate at 37 °C for 30 minutes.

Note: From this point, protect cells from light by covering plate with foil.

5.2.4.

Carefully aspirate the BODIPY working solution and wash the cell layer 3 times with PBS.

5.2.5.

Fix cells by adding 4 % paraformaldehyde and incubating at room temperature for 15 minutes.

5.2.6.

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.

5.2.7.

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). Cells were imaged 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.

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 hours, 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.

DISCUSSION:

Critical steps in the protocol:

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.

Modifications and troubleshooting:

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 ADSCs^14–16.

Limitation of the technique:

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.

Future applications:

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.