[methods in molecular biology] somatic stem cells volume 879 || obtaining freshly isolated and...

269

Shree Ram Singh (ed.), Somatic Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 879,DOI 10.1007/978-1-61779-815-3_15, © Springer Science+Business Media, LLC 2012

Chapter 15

Obtaining Freshly Isolated and Cultured Mesenchymal Stem Cells from Human Adipose Tissue

Andrew C. Boquest and Philippe Collas

Abstract

The stromal compartment of adipose tissue harbors mesenchymal stem cells (MSCs) (also called stromal stem cells) that display extensive proliferative capacity and multilineage differentiation potential. Such cells offer a practical avenue of generating patient-matched tissue for use in regenerative medicine. It is rela-tively easy to isolate these cells from adipose tissue in large enough quantities (tens of millions) to allow for their clinical use in a native, uncultured form. Alternatively, MSCs from adipose tissue can be expanded and differentiated into the desired tissue type in vitro using straightforward cell culture techniques. In this chapter, we outline procedures for isolating large numbers of highly puri fi ed MSCs from human adipose tissue in their native, uncultured form and methods for their subsequent expansion and differentiation in vitro.

Key words: Adipose tissue , Mesenchyme , Mesenchymal stem cell , Stroma

The stromal compartment of mesenchymal tissues including bone marrow ( 1, 2 ) , muscle ( 3 ) , perichondrium ( 4 ) , and adipose ( 5– 7 ) contains a reservoir of stem cells with multilineage differentiation capacity. These cells, often referred to in the literature as “mesen-chymal stem cells” (MSCs), “stromal stem cells,” or “stromal precur-sor cells,” share many key characteristics including (1) the expression of speci fi c cell surface markers, (2) the ability to adhere to plastic to form fi broblastic-like colonies (often called CFU-F), (3) the capacity to proliferate extensively in vitro using relatively simple cell culture techniques, and (4) the ability to form several tissue types of the mesoderm lineage (e.g., bone, muscle, cartilage, and fat). Recent evidence also suggests that these cells possess the ability

1. Introduction

270 A.C. Boquest and P. Collas

to form nonmesodermal tissues including those of the ectoderm (e.g., neuronal-like cells) ( 7– 9 ) and endoderm (e.g., hepatocytes) ( 10 ) lineages. Furthermore, autologous MSCs can easily be sourced from the patient, which circumvents any complication related to tissue rejection posttransplantation. Given these alluring character-istics, MSCs offer a vast potential in the fi eld of regenerative medicine. Indeed, early clinical trials using adipose-derived MSCs show promising results of ef fi cacy in patients with a range of diseases including acute myocardial infarction, peripheral vascular disease, bony tissue defects including cranial bone loss, Crohn’s-related fi stula, and skin wounds ( 11 ) .

To date, the use of MSCs, whether for research or clinical use, has largely been limited to cultured cells. This is because MSCs are rare and therefore dif fi cult to isolate in large numbers in an uncul-tured form. For example, in human bone marrow, only 0.01–0.001% of nucleated cells form CFU-F ( 1 ) . Until recently, their ability to adhere to plastic and proliferate has been used as the con-ventional isolation method. In order to gain an in-depth under-standing of MSC biology and exploit their full therapeutic potential, it is imperative that a relatively simplistic nonculture isolation method for these cells is devised.

Since adipose tissue is easily obtainable by lipoaspiration in bulk quantities (liters), it represents the ideal choice of mesodermal tissue for isolating signi fi cant numbers of MSCs in their native, uncultured form. We have previously reported the isolation and extensive characterization of uncultured human MSCs from the stromal-vascular component of adipose tissue ( 9 ) . We found that cells with a CD45 − CD31 − CD34 + CD105 + surface marker pheno-type freshly isolated from adipose tissue form CFU-F, proliferate extensively, and can be differentiated towards several lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic under appropriate conducive conditions ( 9 ) . Further, from a prac-tical viewpoint, such cells can be freshly isolated in signi fi cant num-bers (tens of millions). Here, we describe methods for isolating MSCs from human adipose tissue as we perform it routinely in our laboratory.

1. Falcon 100 μ m cell strainers (Becton Dickinson). 2. Falcon 40 μ m cell strainers (Becton Dickinson). 3. MACS LD columns (Miltenyi). 4. MidiMACS separation unit (Miltenyi). 5. 50- and 15-mL Plastic conical tubes. 6. 25- and 162-cm 2 Cell culture fl asks.

2. Materials

2.1. Laboratory Equipment

27115 Obtaining Freshly Isolated and Cultured Mesenchymal Stem Cells…

7. Empty 500-mL sterile medium bottles. 8. 0-, 25-, and 10-mL Disposable plastic pipettes. 9. Swing-out centrifuge with buckets for 50- and 15-mL tubes. 10. Fluorescence microscope fi tted for FITC viewing. 11. Cell incubator set at 100% humidity, 37°C, and 5% CO 2 in air.

1. Lipoaspirate, commonly obtained from a clinic. 2. Column buffer: phosphate buffered saline (PBS, pH 7.2), 0.5%

fetal bovine serum (FBS), 2 mM EDTA. 3. Hanks Balanced Salt Solution (HBSS, without phenol red). 4. FBS. Aliquot and store frozen. Thaw at 4°C. 5. Collagenase A type I (Sigma-Aldrich). 6. Antibiotics: penicillin–streptomycin mix. 7. Fungizone. 8. Red blood cell lysis buffer: 2.06 g/L Tris Base, pH 7.2,

7.49 g/L NH 4 Cl. Sterile fi lter. Can be kept at room tempera-ture for 4 weeks.

9. DNase I: 1,000× stock (10 mg/mL) in HBSS. Aliquot and freeze.

10. Bench media: HBSS with 2% FBS and antibiotics. 11. DMEM:F12 medium. 12. 0.25% Trypsin-EDTA solution. 13. MACS anti-human CD45 magnetic microbead-conjugated

mouse monoclonal antibody (Miltenyi). 14. Anti-human CD31 FITC-conjugated mouse monoclonal anti-

body (Serotec). 15. MACS anti-FITC Microbeads (Miltenyi).

We have found that MSCs can be isolated from lipoaspirate col-lected from several regions of the body including hip, thigh, and abdominal regions. It is desirable that at least 300 mL of lipoaspi-rate is collected from the patient to isolate uncultured MSCs in signi fi cant numbers (10 7 -range). Using the technique described later, we routinely isolate up to 10 7 adipose stromal stem cells with greater than 95% purity from 300 mL of lipoaspirate. However, yields can vary widely between patients. The actual adipose tissue volume used for digestion after the washing steps is usually about two thirds of the collected lipoaspirate volume.

2.2. Reagents

3. Methods

3.1. Collection and Storage of Lipoaspirate


It is necessary to wash the lipoaspirate extensively to remove the majority of the erythrocytes and leukocytes. The following proce-dures should be performed under aseptic conditions, ideally in a laminar air fl ow cabinet.

1. Place a maximum of 300 mL of lipoaspirate into a used sterile medium bottle (see Note 1).

2. Allow the adipose tissue to settle above the blood fraction. 3. Remove the blood using a sterile 25-mL pipette. 4. Add an equivalent volume of HBSS with antibiotics and fungi-

zone and fi rmly tighten the lid. 5. Shake vigorously for 5–10 s. 6. Place the bottle on the bench and allow the adipose tissue to

fl oat above the HBSS. This will take 1–5 min depending on the sample.

7. Carefully remove the HBSS using a 50-mL pipette. 8. Repeat the above washing procedure (steps 4–7) three times. 9. Medium from the fi nal wash should be clear. If it is still red,

wash again by repeating steps 4–7.

Dispersion of adipose tissue is achieved by collagenase digestion. Collagenase has the advantage over other tissue digestive enzymes in that it can ef fi ciently disperse adipose tissue while maintaining high cell viability.

1. Make up collagenase solution just prior to digestion. The fi nal volume required is half that of the washed adipose tissue vol-ume. Add powdered collagenase to HBSS at a fi nal concentra-tion of 0.2%. Dissolve the required amount of collagenase into 40 mL of HBSS and fi lter sterilize into the remaining working volume. Add DNase I to a fi nal concentration of 10 μ g/mL antibiotics and fungizone.

2. Add the washed adipose tissue to large cell culture fl asks (100 mL/162 cm 2 fl ask).

3. Add collagenase solution. 4. Resuspend the adipose tissue by shaking the fl asks vigorously

for 5–10 s. 5. Incubate at 37°C on a shaker for 1–2 h, manually shaking the

fl asks vigorously for 5–10 s every 15 min. 6. Prepare 200 mL of washing medium consisting of HBSS con-

taining 2% FBS, antibiotics, and fungizone. 7. On completion of the digestion period, the digested adipose

tissue should have a “soup-like” consistency, containing few, if any, clumps of undigested tissue.

8. Directly add FBS to a fi nal concentration of 10% to cease col-lagenase activity.

3.2. Separation of the Stromal Vascular Fraction

3.2.1. Lipoaspirate Washing

3.2.2. Collagenase Digestion


After digestion, the ability of lipid- fi lled adipocytes to fl oat is used to separate them from the stromal-vascular fraction (SVF).

1. Dispense the collagenase-digested tissue into 50-mL tubes. Avoid dispensing undigested tissue. Centrifuge at room tem-perature at 400 × g for 10 min.

2. After centrifugation, use a 50-mL pipette to aspirate the fl oating adipocytes, lipids, and the digestion medium. Leave the SVF pellet in the tube.

We have found that the SVF predominantly contains erythrocytes, leukocytes, endothelial cells, and MSCs. Erythrocytes are removed fi rst using the red blood cell lysis buffer.

1. Resuspend thoroughly each SVF pellet in 20 mL of cell lysis buffer at room temperature.

2. Incubate at room temperature for 10 min. 3. Centrifuge at 300 × g for 10 min and aspirate the cell lysis

buffer.

It is essential to obtain a cell suspension free from undigested tissue and cell clumps to effectively separate MSCs from other cell types using antibody-conjugated magnetic beads. To achieve this, gross undigested tissue is removed using gravity. Cell strainers are then used to separate small tissue clumps.

1. Resuspend SVF pellets thoroughly in 2 mL of washing medium using a 1-mL pipette.

2. Pipet the cells up and down several times to reduce clumping. 3. Pool the pellets into two 50-mL tubes. 4. Allow undigested tissue clumps to settle by gravity for

~1 min. 5. Aspirate and pass the suspended cells through 100 μ m cell

strainers. 6. Pass the fi ltered cells through 40 μ m cell strainers. 7. Perform a cell count. 8. Centrifuge at 300 × g for 10 min using a low brake setting. 9. If the desired outcome is simply to obtain cultured MSCs, pro-

ceed to Subheading 3.4 .

MSCs are separated from remaining cells using magnetic cell sorting. Unwanted endothelial (CD31 + ) and leukocytes (CD45 + ) are mag-netically labeled and eliminated from the cell suspension when applied to a column under a magnetic fi eld. Magnetically labeled

3.2.3. Separation of the Stromal Vascular Fraction

3.3. Separation of MSCs from the Stromal Vascular Fraction

3.3.1. Removal of Erythrocytes

3.3.2. Removal of Cell Clumps and Remaining Undigested Tissue

3.3.3. Separation of MSCs from Endothelial Cells and Leukocytes by Magnetic Cell Sorting


cells are retained in the column, whereas unlabeled stem cells with a CD45 − CD31 − phenotype pass through the column and are collected. To this end, CD31 + and CD45 + cells are labeled with FITC-conjugated anti-CD31 and anti-CD45 antibodies. The stained cells are magnetically labeled by the addition of anti-FITC-conjugated magnetic microbeads. This approach presents the advantage that cell purity after separation can be assessed by fl ow cytometry or fl uorescence microscopy. For the following steps, use cold buffer and work on ice to reduce cell clumping.

1. Resuspend and pool the sedimented pellets in 10 mL of col-umn buffer.

2. Remove all remaining cell clumps by passing the suspension through a 40- μ m cell strainer.

3. Perform a cell count. 4. Transfer cells to a 15-mL tube and centrifuge at 300 × g for

10 min at 4°C using a low brake setting. 5. Resuspend the cell pellet in column buffer and label with anti-

CD31 FITC-conjugated and anti-CD45 FITC-conjugated antibodies according to the manufacturer’s recommendations (see Note 2). We resuspend cells in 100 μ L of column buffer and add 10 μ L of each antibody/10 7 cells.

6. Mix well and incubate for 15 min in the dark at 4°C (resus-pend the cells after 7 min of incubation).

7. Wash the cells to remove unbound antibody by adding 2 mL of column buffer/10 7 cells. Centrifuge at 300 × g for 10 min at 4°C using a low brake setting.

8. Aspirate the supernatant completely and resuspend the cell pel-let in 90 μ L of column buffer/10 7 cells. Add 10 μ L of MACS anti-FITC magnetic microbeads/10 7 cells.

9. Mix well and incubate for 15 min at 4°C (resuspend the cells after 7 min of incubation).

10. Wash the cells to remove unbound beads by adding 2 mL of column buffer/10 7 cells. Centrifuge at 300 × g for 10 min at 4°C using a low brake setting.

11. Aspirate the supernatant completely and resuspend the cell pel-let in 500 μ L of column buffer.

12. For magnetic cell separation, use the MACS LD column speci fi cally designed for depletion of unwanted cells. Place a MACS LD column onto the MidiMACS separation unit or onto a compatible unit.

13. Prepare the column by washing with 2 mL of column buffer. 14. Apply the cell suspension to the column and collect the fl ow-

through unlabeled cells in a 15-mL tube.


15. Wash the unlabeled cells through the column by twice adding 1 mL of column buffer. Collect the total ef fl uent.

16. Check for stem cell purity as described in Subheading 3.3.4 . 17. If higher purity is required, centrifuge the collected cells at

300 × g for 10 min at 4°C using a low brake setting and repeat steps 11–16.

18. Perform a cell count. 19. Centrifuge at 300 × g for 10 min at 4°C using a low brake

setting. 20. Use the cells as required or freeze the cells according to stan-

dard protocols.

Success of obtaining uncultured MSC samples of high purity varies between donors for unclear reasons. It is therefore recommended that MSC purity is determined after each isolation. Since both endothelial cells and leukocytes are immunolabeled with FITC-conjugated antibodies, a fl uorescence-based assay can be used to assess contaminating cells. We fi nd that fl uorescence microscopy is suf fi cient to evaluate purity. A more accurate assessment can be made, however, by using fl ow cytometry.

1. Place 5 μ L of the collected cell fraction onto a glass slide. 2. View under white light. MSCs have an evenly round pheno-

type, approximately 10 μ m in diameter, whereas endothelial cells have an irregular shape (Fig. 1a ).

3. Observe the samples under epi fl uorescence. 4. Determine the percentage of fl uorescent cells under fi ve fi elds

of view (Fig. 1b , c). The average represents an approximate percentage of non-MSCs in the sample.

3.3.4. Assessment of MSC Purity

Fig. 1. Mesenchymal stem cells (MSCs) puri fi ed from lipoaspirate and identi fi cation of contaminating CD45 + and CD31 + cells. ( A ) MSCs isolated from collagenase-digested lipoaspirate. These cells have an even round phenotype. ( B , C ) Identi fi cation of contaminating CD45 + and CD31 + cells among MSCs isolated from adipose tissue. Cells recovered from liposuction material are labeled using anti-CD31 FITC- and anti-CD45 FITC-conjugated antibodies prior to puri fi cation of CD45 − and CD31 − cells by negative selection. Unwanted endothelial cells (CD31 + ) and leukocytes (CD45 + ) are eliminated from the cell suspension. As a quality control assessment, fl uorescence microscopy examination ( B ) of the fl ow-through cells enables identi fi cation of a low proportion of contaminating CD45 + CD31 + cells ( arrows ). Bars, 20 μ m.


Culture can also be used to further validate the successful isolation of MSCs from adipose tissue using the above procedure. MSCs, when cultured, adhere to plastic and acquire a fi broblastic-like morphology. It may take several days before all adherent cells change their morphology. We usually fi nd that approximately 50% of cells isolated as earlier will plate under the correct culture condi-tions. However, plating ef fi ciency can vary substantially between donors.

1. Resuspend 10 5 freshly isolated MSCs in 2 mL of DMEM:F12 medium containing 50% FBS, antibiotics, and fungizone (see Note 3).

2. Add the cell suspension to a 25-cm 2 cell culture fl ask. 3. Smear the cell suspension across the entire surface by rocking

the fl ask. 4. Incubate in a humidi fi ed incubator at 37°C, 5% CO 2 . 5. Observe the cells daily using an inverted phase contrast

microscope. 6. It usually takes several days before those cells which form a

fi broblastic morphology start dividing. 7. Estimate the percentage of cells which adhere to the plastic

surface (Fig. 2a ) and form a fi broblastic-like morphology after 7 days of culture (Fig. 2b ).

Generation of MSC lines is required to evaluate their differentia-tion capacity and proliferative ability and to expand cell numbers for possible clinical application. We have kept lines of stem cells generated as described earlier for longer than 6 months in culture without induction of senescence (see Note 4).

1. After at least 7 days of culture, replace the medium with DMEM:F12 containing antibiotics, 20% FBS, and no fungizone.

3.4. Culture of MSCs

3.4.1. Validation of MSC Isolation and Viability by Cell Culture

3.4.2. Generation of an MSC Line

Fig. 2. Morphology of isolated and seeded MSCs derived from adipose tissue. ( A ) Cells adhere to the plastic surface and ( B ) after 7 days of culture, acquire a fi broblastic-like morphology. Bars, 20 μ m. ( C ) Monoclonal culture derived from a single adipose tissue-derived stem cell. Bar, 50 μ m.


2. Subculture cells using standard methods of trypsinization using 0.25% trypsin–EDTA solution after a further week of culture to form a cell line (Fig. 2c ).

3. Subculture the cells weekly with a splitting ratio of 1:3. 4. To evaluate the “stemness” of adipose stem cell lines estab-

lished, we recommend differentiating the cells towards the adipogenic (Fig. 3a ) and osteogenic (Fig. 3b ) mesodermal lineages according to Zuk et al. ( 5 ) .

1. If overnight storage of lipoaspirate is required, we recommend storage at room temperature. We have found that overnight storage at 4°C hardens the lipid component of the aspirate, dramatically reducing MSC isolation ef fi ciency.

2. If the desired outcome is to obtain a combination of MSCs and endothelial cells (e.g., for a particular clinical application), omit the anti-CD31 antibody.

3. We initially plate freshly isolated stem cells in medium contain-ing 50% FBS (which is substantially higher than what is nor-mally used for cell culture; being 10–20%) in a minimal volume suf fi cient to “smear” the cell suspension across the surface of a cell culture fl ask. We fi nd that this technique maximizes cell contact with the plastic surface and promotes plating.

4. We fi nd that the time taken in culture for MSCs to enter a senescent phase varies substantially between donors for reasons yet to be determined.

4. Notes

Fig. 3. In vitro differentiation of cultured human adipose tissue-derived stem cells towards ( A ) the adipogenic pathway and ( B ) the osteogenic pathway according to Zuk et al. ( 5 ) . ( A ) To visualize intracellular lipid droplets, cells are fi xed and stained with oil-red O. ( B ) Mineralization of the extracellular matrix is visualized by staining with Alizarin red. Bars, 50 μ m.


References

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2. Gronthos S, Zannettino AC, Hay SJ, Shi S, Graves SE, Kortesidis A, Simmons PJ (2003) Molecular and cellular characterisation of highly puri fi ed stromal stem cells derived from human bone marrow. J Cell Sci 116:1827–1835

3. Howell JC, Lee WH, Morrison P, Zhong J, Yoder MC, Srour EF (2003) Pluripotent stem cells identi fi ed in multiple murine tissues. Ann N Y Acad Sci 996:158–173

4. Arai F, Ohneda O, Miyamoto T, Zhang XQ, Suda T (2002) Mesenchymal stem cells in per-ichondrium express activated leukocyte cell adhesion molecule and participate in bone mar-row formation. J Exp Med 195:1549–1563

5. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based ther-apies. Tissue Eng 7:211–228

6. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH (2002) Human

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8. Safford KM, Hicok KC, Safford SD, Halvorsen YD, Wilkison WO, Gimble JM, Rice HE (2002) Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 294:371–379

9. Woodbury D, Reynolds K, Black IB (2002) Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and meso-dermal genes prior to neurogenesis. J Neurosci Res 69:908–917

10. Snykers S, De Kock J, Rogiers V, Vanhaecke T (2009) In vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art. Stem Cells 27:577–605

11. Hong SJ, Traktuev DO, March KL (2010) Therapeutic potential of adipose-derived stem cells in vascular growth and tissue repair. Curr Opin Organ Transplant 15:86–91

[methods in molecular biology] somatic stem cells volume 879 || obtaining freshly isolated and...

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