Adipose Tissue-Derived Mesenchymal Stem Cells: Isolation, Expansion, and Characterization

  • Miriam Araña
  • Manuel Mazo
  • Pablo Aranda
  • Beatriz Pelacho
  • Felipe Prosper
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1036)

Abstract

Over the last decade, cell therapy has emerged as a potentially new approach for the treatment of cardiovascular diseases. Among the wide range of cell types and sources, adipose-derived mesenchymal stem cells have shown promise, mainly due to its plasticity and remarkable paracrine-secretion capacity, largely demonstrated at the in vitro and in vivo levels. Furthermore, its accessibility and abundance, the low morbidity of the surgical procedure, its easy isolation, culture, and long-term passaging capacity added to its immunomodulatory properties that could allow its allogeneic transplantation, making it one of the most attractive candidates for clinical application. In this chapter, we will focus on the methodology for the isolation, expansion, phenotypical characterization, differentiation, and storage of the adipose-derived stem cells.

Key words

Adipose tissue Stem cells Stromal vascular fraction ADSC Isolation Expansion Characterization Storage 

1 Introduction

The adipose tissue (AT) has traditionally been regarded as an energy-storing organ, composed of mature adipocytes, nurtured by an intermingling vasculature and containing fibroblasts and immune/hematopoietic cells. It was Zuk and coworkers however who first described the isolation of a population with multilineage differentiation capacity from human lipoaspirates [1], named as stromal vascular fraction (SVF) and composed by a mixture of phenotypes, including endothelial, smooth muscle, cardiac, immune, and stromal cells [1, 2, 3, 4]. Furthermore, if cultured under concrete conditions, SVF was homogenized, giving rise to the adipose-derived stem cells (ADSC), a pure mesenchymal cell population (see Fig. 1) able to give rise to mesodermal phenotypes such as osteoblasts, chondrocytes, adipocytes, and myogenic cells [5, 6, 7, 8]. Both SVF and ADSC differ in their phenotypic profile, their capacity of differentiation, and their secretome [9, 10, 11, 12], although both cases have shown, at the experimental level, a positive effect for the treatment of different pathologies at the experimental level (reviewed in [13]). Importantly, together with their therapeutic potential, adipose-derived stem cells present a number of advantages over other cell types. First, AT is easily accessible, requiring a minimally invasive and conventional surgery for its harvesting. Second, progenitor cells are present in a greater number [14] than in other tissues (e.g., bone marrow) and have a larger expansion potential [5, 15], making high-dosage treatments readily available. Third, the isolation process is relatively simple and does not require specific equipment, and fourth, cell culture and characterization do not imply the use of expensive reagents, making any prospective therapy more affordable. Thus, clinical application has been pursued, focusing not only on ADSC mesenchymal differentiation potential for bone or cartilage reconstruction surgery [16] but also on their implication on the healing process (seewww.clinicalTrials.gov, Identifier NCT00475410) and their immunomodulatory effects in pathologies like Crohn’s [17] and graft-versus-host disease (Identifier NCT01222039). Also, the putative beneficial effect of the ADSC at the paracrine level, stimulating different mechanisms such as cell survival, tissue revascularization, and tissue remodeling (reviewed in [18]), is being evaluated on heart and limb ischemia clinical trials (Identifier NCT00426868 and NCT01211028) and also in brain disease (Identifier NCT01453829).
Fig. 1

Adipose-derived stem cells (ADSC) isolation from adipose tissue. After mechanical and enzymatic digestion of an adipose tissue biopsy and following centrifugation to eliminate mature adipocytes [top layer ], the adipose stromal vascular fraction (SVF) (pellet) is plated and kept in culture during 3 weeks, deriving into a homogeneous mesenchymal cell population, the ADSC

In order to elucidate the real beneficial potential of the ADSC and compare the results of different clinical trials, it is indeed of capital importance a standardized isolation and expansion protocol together with an adequate ADSC characterization, since their phenotypic profile is not completely specific of ADSC and is shared by commonly contaminating cells as fibroblasts. Along this chapter we will describe the procedures for the isolation, expansion, storage, and characterization of mouse, rat, pig, and human AT-derived cells.

2 Materials

2.1 ADSC Isolation and Culture

  1. 1.

    Sterile dishes.

     
  2. 2.

    Sterilized scissors or blades.

     
  3. 3.

    Collagenase type I.

     
  4. 4.

    0.2 μm filters.

     
  5. 5.

    ADSC basal medium: Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin.

     
  6. 6.

    100 and 40 μm filters.

     
  7. 7.

    Erythrocyte lysis buffer: 155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA.

     
  8. 8.

    Phosphate Buffer Saline (PBS).

     
  9. 9.

    Trypan blue.

     
  10. 10.

    Culture flasks 175 cm2.

     
  11. 11.

    0.05 % trypsin and 200 mg/l EDTA.

     
  12. 12.

    Freezing medium: DMEM, 50 % FBS, and 10 % dimethyl sulfoxide (DMSO).

     
  13. 13.

    Cryogenic vials.

     
  14. 14.

    Isopropanol.

     
  15. 15.

    Freezing container.

     
  16. 16.

    Conical tubes (50 and 15 ml).

     

2.2 Flow Cytometry

  1. 1.

    PBS.

     
  2. 2.

    Cytometry tubes.

     
  3. 3.
    Antibodies (see Table 1).
    Table 1

    Antibody list. Antibodies used for ADSC characterization by flow cytometry

    Antibody

    Specificity (species)

    Commercial reference

    CD29

    Pig

    BD 552369

    Human

    Immunotech 0791

    CD31

    Rat/pig

    BD 555027

    Mouse

    BD 551262

    Human

    BD 555446

    CD34

    Mouse

    BD 551387

    Human

    BD 345802

    CD44

    Rat

    BD 554869

    Human

    BD 555478

    Mouse

    BD 553134

    CD45

    Mouse

    BD 557235

    Rat

    BD 554881

    Human

    BD 557059

    CD73

    Rat

    BD 551124

    Human

    BD 550257

    CD90

    Human/pig

    BD 555596

    Rat

    BD 554898

    Mouse

    eBioscience 17090082

    CD105

    Human

    Ancell 326-050

    CD117

    Mouse

    BD 553356

    Human

    BD 332785

    HLA-A, HLA-B, HLA-C

    Human

    BD 555553

    HLA-DR, HLA-DP, HLA-DQ

    Human

    BD 555558

    MHC-I

    Mouse

    BD 553573

    MHC-II

    Mouse

    BD 553623

    RT1A

    Rat

    BD 559993

    RT1B

    Rat

    BD 554929

     
  4. 4.

    0.4 % paraformaldehyde (PFA).

     

2.3 Osteogenic Differentiation

  1. 1.

    Osteogenic differentiation medium: alpha-MEM supplemented with 0.2 mM ascorbic acid, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, 10 % FBS, and 1 % penicillin/streptomycin.

     
  2. 2.

    6-well cell culture plates.

     
  3. 3.

    10 % neutral buffered formalin solution.

     
  4. 4.

    Alizarin Red S solution.

     
  5. 5.

    Acetone.

     
  6. 6.

    Sodium citrate.

     
  7. 7.

    Alkaline staining: mix 6 mg of Fast Blue BB Salt with 1 ml of Naphtol AS-MX and 24 ml of distilled water.

     

2.4 Adipogenic Differentiation

  1. 1.

    Adipogenic differentiation medium: alpha-MEM supplemented with 50 μM indomethacin, 1 μM dexamethasone, 0.5 mM isobutyl-methylxanthine (IBMX), 10 % FBS, and 1 % penicillin/streptomycin.

     
  2. 2.

    6-well cell culture plates.

     
  3. 3.

    10 % neutral buffered formalin solution.

     
  4. 4.

    0.5 % Oil Red O in isopropanol.

     
  5. 5.

    Harris’ hematoxylin.

     

2.5 Chondrogenic Differentiation

  1. 1.

    Chondrogenic differentiation medium: Dulbecco’s Modified Eagle’s Medium High Glucose (4.5 g/l) (DMEM-HG) supplemented with 0.1 μM dexamethasone, 100 μg/ml sodium pyruvate, 50 μg/ml ascorbic acid, 1 % ITS + Premix Tissue Culture Supplement, 1 % penicillin/streptomycin, 500 ng/ml bone morphogenetic protein-6 (BMP-6), and 10 ng/ml transforming growth factor beta-1 (TGFβ-1).

     
  2. 2.

    Conical tubes (15 ml).

     
  3. 3.

    10 % neutral buffered formalin solution.

     
  4. 4.

    Toluidine blue.

     
  5. 5.

    Acetic acid.

     
  6. 6.

    Ethanol.

     
  7. 7.

    Xylene.

     
  8. 8.

    DPX (BDH).

     

3 Methods

3.1 Ex Vivo Expansion of ADSC

Mammalian AT can be classified in three types, brown, beige, and white, of whom the last is the most abundant. In humans, white AT represents on average 16 % of body weight, distributed throughout the body but especially in the abdomen, buttocks, and abdominal zone. It appears however that not all of the fat accumulations are equivalent, and at least in mice, the potential of the cells can be different according to their location [19].

3.1.1 Cell Isolation

  1. 1.

    Place the AT sample (seeNote1 ) in a sterile dish and mince thoroughly (around 15 min) with scissors and blades until a mush is obtained (seeNote2).

     
  2. 2.

    Collect the minced tissue in a 50 ml tube and weigh it. Prepare 10 ml of collagenase I solution per 3 g tissue (for the collagenase I solution, dissolve 2 mg/ml collagenase type I in DMEM and filter through a 0.2 μm filter).

     
  3. 3.

    Incubate the minced tissue with the collagenase I solution during 1 h at 37 °C, gently shaking on a water bath (seeNote3).

     
  4. 4.

    After digestion, dilute the sample 1:1 in DMEM and 10 % FBS and sequentially filter through 100 and 40 μm filters.

     
  5. 5.

    Centrifuge the filtered sample at 600 × g for 7 min and discard the formed lipid layer on the top and the supernatant by collecting them with a pipette. Resuspend the pellet in 1 ml of basal medium and add 3 ml of erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 min at room temperature (RT).

     
  6. 6.

    Add 10 ml of PBS and centrifuge at 600 × g for 7 min.

     
  7. 7.

    Discard the supernatant and resuspend the pellet in 1 ml of basal medium.

     
  8. 8.

    Pick a small aliquot and count cells, checking viability through trypan blue dye exclusion (seeNote4).

     
  9. 9.

    Plate isolated cells (SVF) at a density of 15–30 × 103 cells/cm2 in basal medium (DMEM supplemented with 10 % FBS and 1 % antibiotics) (seeNotes5 and 6).

     
  10. 10.

    Incubate at 37 °C with 5.5 % CO2 (seeNote7).

     

3.1.2 Subculture of Cells

  1. 1.

    Remove the medium from the flasks and wash the cells with PBS. Add the appropriate volume of trypsin/EDTA (0.05 % trypsin; 200 mg/l EDTA) and incubate the flasks during 5 min at 37 °C to detach the cells (seeNote8).

     
  2. 2.

    Check complete detachment by gently tapping the side of the flask and observing cells under the microscope.

     
  3. 3.

    Inactivate trypsin adding pre-warmed basal medium (seeNote8).

     
  4. 4.

    Collect the cells with basal medium and plate them at 4 × 103 cells/cm2 (seeNote5).

     
  5. 5.

    Incubate the cells at 37 °C with 5.5 % CO2, 90–95 % humidity.

     

3.2 Long-Term Preservation

SVF or ADSC can be cryopreserved for later use. Optimal preservation is crucial for ensuring cell survival and proliferation after thawing. Cells can be aliquot and stored in liquid nitrogen at recommended density [20, 21].

3.2.1 Cryopreservation

  1. 1.

    Remove medium from flasks and wash cells with PBS. Add the appropriate volume of trypsin/EDTA (0.05 % trypsin; 200 mg/l EDTA) and incubate flasks during 5 min at 37 °C to detach cells (seeNote8).

     
  2. 2.

    Check cell detachment under the microscope and inactivate trypsin, adding basal medium (seeNote8).

     
  3. 3.

    Collect and centrifuge cells at 600 × g for 5 min.

     
  4. 4.

    Discard the supernatant and resuspend the cells at 1–10 × 106 cells/1 ml in freezing medium (40 % DMEM, 50 % FBS, 10 % DMSO).

     
  5. 5.

    Aliquot cells into previously labeled cryogenic vials (seeNote9).

     
  6. 6.

    Place the cryogenic vials in a freezing container and transfer to a −80 °C freezer.

     
  7. 7.

    For long-term storage, after overnight freezing at −80 °C, move vials to a liquid nitrogen container.

     

3.2.2 Recovery of Cryopreserved Cells

  1. 1.

    Rapidly thaw the frozen vial in a pre-warm water bath. Maximum cell viability depends on the rapid thawing of frozen cells.

     
  2. 2.

    As soon as the cells are completely thawed, disinfect the outside of the vial with 70 % ethanol.

     
  3. 3.

    Transfer the cell suspension with a 1 ml pipette to a 15 ml conical tube containing approximately 10 ml of basal medium.

     
  4. 4.

    Mix the cell suspension by gently pipetting up and down.

     
  5. 5.

    Centrifuge the tubes at 600 × g for 5 min to pellet the cells and remove DMSO.

     
  6. 6.

    Discard the supernatant and resuspend the cell pellet in a suitable volume of basal medium (seeNote5).

     
  7. 7.

    Plate the resuspended cells at a density of 12 × 103 cells/cm2.

     

3.3 Cell Characterization

As stated above, the characterization of ADSC requires fulfilling both the phenotype and differentiation capacity criteria (see Fig. 3). As a heterogeneous population, freshly isolated SVF expresses hematopoietic markers (CD34 and CD45), mesenchymal markers (CD29, CD44, CD73, CD90, and CD105), endothelial cell markers (CD34 and CD31), and other stem cell markers (CD117). The subsequent culture and homogenization (taking usually 2–3 weeks) that gives rise to ADSC is reflected in their phenotypic profile, showing that stem cell, endothelial, or hematopoietic markers progressively disappear, obtaining a homogeneous population which only expresses mesenchymal cell markers [2, 6].

On the differentiation side, ADSC, similarly to bone marrow-derived mesenchymal stem cells (BM-MSC), have a multilineage differentiation capacity, giving rise to mesodermal lineages, including bone, fat, and cartilage [1]. However, although ADSC can be easily differentiated into the adipose lineage, it has been reported that they are less committed towards the osteogenic and chondrogenic lineages than BM-MSC [22, 23].

3.3.1 Flow Cytometry Analysis

The phenotypic analysis performed by flow cytometry with specific antibodies (see Table 1) must confirm the mentioned heterogeneity of the freshly isolated SVF and the homogeneity of the in vitro cultured ADSC (see Table 2) (seeNote10):
Table 2

Flow cytometry analysis

 

Mouse

Rat

Pig

Human

SVF (d0)

ADSC (d15)

SVF (d0)

ADSC (d21)

SVF (d0)

ADSC (d21)

SVF (d0)

ADSC (d21)

MHC-I

+++

++

+++

+++

n.d.

n.d.

++

+++

MHC-II

++

+

n.d.

n.d.

+

±

CD29

n.d.

n.d.

n.d.

n.d.

++

+++

++

+++

CD31

++

±

++

±

+

CD34

+

+

n.d.

n.d.

n.d.

n.d.

++

CD44

++

+++

++

+++

n.d.

n.d.

++

+++

CD45

++

+

+

n.d.

n.d.

+

CD73

n.d.

n.d.

+

+++

n.d.

n.d.

++

+++

CD90

+

++

+

+++

++

+++

++

+++

CD105

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

++

+++

CD117

n.d.

n.d.

n.d.

n.d.

+/−

Results of ADSC phenotype are shown as +++: ≥90 %, ++: 50–90 %, +: 5–50 %, ±: ≤5 %, −: 0 % positive cells. Data obtained from the average of 3–6 independent samples (d days in culture, n.d. not determined)

  1. 1.

    Remove medium from flasks, wash cells with PBS, and add the appropriate volume of trypsin/EDTA (0.05 trypsin; 200 mg/l EDTA). Incubate flasks during 5 min at 37 °C to detach cells (seeNote8).

     
  2. 2.

    Check cell detachment under the microscope and inactivate trypsin, adding basal medium (seeNote8).

     
  3. 3.

    Collect cells and centrifuge at 600 × g for 5 min.

     
  4. 4.

    Discard the supernatant. Add 1 ml of basal medium and count cells.

     
  5. 5.

    Resuspend 0.1–1 × 106 cells in 100 μl of PBS in a cytometry tube.

     
  6. 6.

    Add 10 μl of each antibody (see Table 1) and incubate during 15 min at RT. The incubation must be done in the dark.

     
  7. 7.

    Add 2 ml of PBS and vortex the sample.

     
  8. 8.

    Centrifuge at 600 × g for 5 min.

     
  9. 9.

    Remove the supernatant and resuspend the pellet in 400 μl of 0.4 % PFA.

     
  10. 10.

    Store the cell suspensions immediately at 4 °C in the dark until analysis (seeNote11).

     

3.3.2 ADSC Differentiation

Aside from marker expression, ADSC must meet the differentiation criteria for a proper characterization (seeNote12).

Osteogenic Differentiation (SeeNote13)
  1. 1.

    Trypsinize cells and plate 5 × 103 cells/cm2 in ADSC medium.

     
  2. 2.

    One day after, change medium to osteogenic differentiation medium.

     
  3. 3.

    Change medium every 3 days.

     
  4. 4.

    Maintain cells for 3 weeks in culture with the osteogenic differentiation medium.

     
  5. 5.

    After 3 weeks of differentiation, cells can be fixed and stained with Alizarin Red S or alkaline phosphatase.

     
Alizarin Red S Staining
  1. 1.

    Aspirate the medium from each well.

     
  2. 2.

    Fix cells with 10 % formalin during 30 min at RT.

     
  3. 3.

    Carefully aspirate the formalin and rinse first with PBS and distilled water.

     
  4. 4.

    Stain with 2 % Alizarin Red S solution pH 4.5 in distilled water for 5 min.

     
  5. 5.

    Wash three times with distilled water to remove remaining staining solution.

     
  6. 6.

    Visualize stained samples using a microscope.

     
Alkaline Phosphatase Test
  1. 1.

    Wash cells with PBS and then fix cells with 60 % acetone in 3 mM sodium citrate during 30 s, then wash with distilled water.

     
  2. 2.

    Stain samples with alkaline staining (see Subheading 2.3) for 30 min at RT and dark.

     
  3. 3.

    Wash three times with distilled water to remove remaining staining solution.

     
  4. 4.

    Visualize stained samples using a microscope.

     
Adipogenic Differentiation (SeeNote14)
  1. 1.

    Culture ADSC cells in basal medium until 95–100 % confluence is reached.

     
  2. 2.

    Wash cells with PBS and add the adipogenic differentiation medium.

     
  3. 3.

    Change medium every 3 days.

     
  4. 4.

    Maintain for 3 weeks in culture in differentiation medium.

     
  5. 5.

    After 3 weeks of differentiation, cells can be fixed and stained with Oil Red O.

     
Oil Red O Staining
  1. 1.

    Aspirate the medium from each well.

     
  2. 2.

    Fix cells with 10 % formalin during 30 min, then wash with PBS and distilled water.

     
  3. 3.

    Stain with 60 % Oil Red O solution in distilled water (from a stock solution of 0.5 % Oil Red O in isopropanol) 20 min.

     
  4. 4.

    Wash three times with PBS to remove remaining Oil Red O solution.

     
  5. 5.

    Counterstain samples with Harris’ hematoxylin diluted 1:2 in distilled water.

     
  6. 6.

    Visualize stained samples using a microscope.

     
Chondrogenic Differentiation (SeeNote 15)
  1. 1.

    Trypsinize and count cells.

     
  2. 2.

    Centrifuge ADSC cells (2 × 105 cells) at 450 × g for 5 min to form a pelleted micromass. Discard the supernatant.

     
  3. 3.

    Culture pellets in 15 ml conical tubes with chondrogenic medium.

     
  4. 4.

    Change medium every 3 days, avoiding removing pellets.

     
  5. 5.

    Maintain for 3 weeks in culture in chondrogenic differentiation medium.

     
  6. 6.

    Collect pellets and use them for toluidine blue staining.

     
Toluidine Blue Staining
  1. 1.

    Fix pellets with 10 % formalin during 30 min.

     
  2. 2.

    Transfer the pellets to 70 % ethanol in H2O.

     
  3. 3.

    Dehydrate the samples and embed the pellets with paraffin, following the routine histological procedures (see elsewhere).

     
  4. 4.

    Cut the samples in 5 μm sections using a microtome.

     
  5. 5.

    Deparaffinize the sections 15 min at 60 °C and immerse twice on xylene (for 5 and 15 min).

     
  6. 6.

    Rehydrate the sections using decreasing alcohol series (100, 96, 80, 70 %, 2 min each), followed by a rinse with tap water for 5 min.

     
  7. 7.

    Rinse the sections with deionized water for 1 min.

     
  8. 8.

    Stain samples with 1 % toluidine blue reagent and 1 % acetic acid (1:4) for 10 min.

     
  9. 9.

    Rinse the stained sections with distilled water.

     
  10. 10.

    Dehydrate in graded ethanol (96 and 100 %, 2 min each) followed by two steps in xylene for 15 min, and mount with DPX.

     
  11. 11.

    Visualize samples using a microscope.

     

4 Notes

  1. 1.

    It is important to collect samples in a sterile container and process them maintaining sterile conditions and within the first 24 h to obtain the highest cell yield. The AT samples are processed in a Class II biological laminar flow hood, and the personnel processing the samples must wear a lab coat, gloves, and surgical mask.

     
  2. 2.

    Lipoaspirate samples are directly incubated with the collagenase solution; mincing is not required. Nevertheless, an appropriate mincing of the sample is mandatory for a proper enzymatic digestion. In our experience, a careful and thorough mincing facilitates filtration and renders a higher cell yield.

     
  3. 3.

    For rat or mouse adipose tissue processing, incubate the samples with the collagenase solution during 30 min.

     
  4. 4.

    Approximately, 0.5–1 million cells per gram of processed adipose tissue are obtained.

     
  5. 5.

    For a 175 cm2 culture flask, add 18–20 ml of basal medium.

     
  6. 6.
    As shown in Table 3 there are several similar media formulations available for the cultivation of ADSC, with the most frequent basic components being DMEM or alpha-MEM supplemented with 10 % FBS. Also, bFGF is used to enhance the proliferation rate [24]. Animal serum is also added to support optimal cell growth as it provides crucial cues for the adequate cell growth. For clinical application, however, animal components must be avoided. As a consequence, a very low human serum expansion medium and a completely serum-free medium have been developed [25]. Another option is the replacement of bovine serum by autologous serum [26] in order to prevent undesirable animal-derived viral transmissions or immunologic reactions. A recently used serum substitute for culture of ADSC is platelet lysate obtained by subjecting platelets to several cycles of freezing-thawing and collecting the supernatants. Recent studies have compared the ADSC and bone marrow MSC yield obtained with platelet lysate in comparison with other serum-containing media [27, 28].
    Table 3

    Different cell medias used for ADSC expansion

    Basic medium

    Serum

    Supplements

    References

    DMEM

    10 % FBS

    [6, 7]

    DMEM-LG

    10 % FBS

    [29]

    DMEM F12

    10 % FBS

    [22, 30]

    α-MEM

    10 % FBS

    [26]

    α-MEM

    10 % FBS

    1 ng/ml bFGF

    [24]

     
  7. 7.
    ADSC are cultured in plastic flasks at 37 °C with a 95 % humidified atmosphere of 5.5 % CO2. ADSC form fibroblast-like loose colonies composed of spindle-shaped cells which are visible under microscope 1–2 days after their isolation. The colonies grow and cells proliferate, reaching confluence in approximately 3–4 days. ADSC proliferate at high density and are subcultured when they reach 80–90 % confluence (see Fig. 2); it is important to note that the ADSC growth is not inhibited by cell-to-cell contact. For subculturing, cells are washed to eliminate old medium and serum and detached with trypsin/EDTA. ADSC are replated at 4 × 103 cells/cm2, requiring 3 weeks for proper homogenization. Media must be changed every 3 days.
    Fig. 2

    Adipose-derived stem cell (ADSC) culture. Representative photographs of rat adipose tissue-derived cells at d0, d2, d4, d7, d14, and d21 of cell culture. Scale bars: 50 μm

     
  8. 8.

    For a 175 cm2 culture flask, wash with 10 ml of PBS, trypsinize with 2 ml trypsin/EDTA, and inactivate the trypsin with 6 ml of basal medium (DMEM supplemented with 10 % FBS and 1 % antibiotics).

     
  9. 9.

    It is recommended to label the cryogenic vials with the number of the experiment, cryopreserved cells, and cell passage; also, it is important to add the cryopreservation date.

     
  10. 10.

    Although some of the markers depicted on Table 1 are different among species, this does not reflect differences among those populations but lack of specific antibodies. Researcher is highly encouraged to try cross-reactivity of existing antibodies or newly developed ones. However, it is important to keep in mind the above requirements for each population, which must be met for a proper characterization.

     
  11. 11.

    For best results, analyze the cells on the flow cytometer as soon as possible.

     
  12. 12.
    As multipotent progenitors, ADSC give rise to osteoblasts, chondrocytes, and adipocytes. At least two of these lineages must be achieved for a proven multipotency. Cell differentiation is triggered by culturing ADSC for 3 weeks with specific induction media, as specified below. As depicted on Table 4, media supplements may vary slightly depending on the chosen protocol.
    Table 4

    Factors for ADSC differentiation

    Differentiation

    Differentiation factors

    References

    Osteogenic

    1,25-Dihydroxycholecalciferol, β-glycerophosphate, ascorbic acid, dexamethasone

    [22, 23, 29, 31]

    Adipogenic

    Insulin, IBMX, dexamethasone, indomethacin

    [23, 32, 33]

    Chondrogenic

    TGF-β2, TGF-β3, dexamethasone, insulin, transferrin, ITS, sodium-l-ascorbate, sodium pyruvate, ascorbate-2-phosphate

    [22, 23, 29, 33]

    BMP bone morphogenetic protein, IBMX 3-isobutyl-1-methylxanthine, TGF-β transforming growth factor-β

     
  13. 13.
    This assay is performed in 6-well cell culture plates. Add 3 ml medium/each well. After 3 weeks of culture with the osteogenic medium, verify the differentiation towards osteoblasts by Alizarin Red S staining and alkaline phosphatase tests. Alizarin Red S stains calcified depositions characteristic of osteogenic differentiation, whereas alkaline phosphatase tests the activity of this enzyme (see Fig. 3b).
    Fig. 3

    ADSC characterization. (a) ADSC phenotype characterization. Representative example of antigen expression of human ADSC by flow cytometry analysis. The dotted line corresponds to isotype control IgG and the red line to the specific antibody. Expression of each antigen is also indicated as percentage. (b) ADSC differentiation potential. ADSC differentiates in vitro towards osteocytes (Alizarin Red and alkaline phosphatase stainings), adipocytes (Oil Red O staining), and chondrocytes (toluidine blue staining). Fibroblasts’ staining is shown as negative control. Scale bars: 250 μm (AC, EH  ), 100 μm (d  )

     
  14. 14.

    This assay is performed in 6-well cell culture plates. Add 3 ml medium/each well. After the differentiation is completed (3 weeks), Oil Red O staining can be performed to visualize lipid deposit and ascertain if lineage conversion has been successful (see Fig. 3b).

     
  15. 15.

    This assay is performed in 15 ml polypropylene centrifuge tubes to obtain cell aggregates. Cartilage produces an extracellular matrix rich in collagen type II, type X, or aggrecans. The chondrogenic differentiation can thus be assessed by staining the samples with toluidine blue, whereby cartilaginous extracellular matrix stains purple and undifferentiated tissue stains blue (see Fig. 3b).

     

Notes

Acknowledgement

This work was supported in part by funds from the ISCIII (RD06/0014, PI10/01621, CP09/00333), MINECO (PLE2009-0116), FP7 Program (INELPY), and the “UTE project CIMA.”

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Miriam Araña
    • 1
  • Manuel Mazo
    • 2
  • Pablo Aranda
    • 1
  • Beatriz Pelacho
    • 1
  • Felipe Prosper
    • 3
  1. 1.Laboratory of Cell Therapy, Division of CancerFoundation for Applied Medical Research, Clínica Universidad de Navarra, University of NavarraPamplonaSpain
  2. 2.Hematology and Area of Cell TherapyClínica Universidad de Navarra, University of NavarraPamplonaSpain
  3. 3.Laboratory of Cell Therapy, Division of CancerFoundation for Applied Medical Research, Hematology and Area of Cell Therapy, Clínica Universidad de Navarra, University of NavarraPamplonaSpain

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