[methods in molecular biology] basic cell culture protocols volume 946 || isolation, enumeration,...

315

Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946,DOI 10.1007/978-1-62703-128-8_20, © Springer Science+Business Media, LLC 2013

Chapter 20

Isolation, Enumeration, and Expansion of Human Mesenchymal Stem Cells in Culture

Ravenska Wagey and Brenton Short

Abstract

Human bone marrow (BM) contains a population of non-hematopoietic stem cells also termed stromal cells, mesenchymal cells or multipotent mesenchymal stromal cells (MSCs). These cells have unique stem cell-like properties including their ability to self-renew, differentiate into multiple tissue types, and modu-late immune cell responses through paracrine effects. These properties have positioned mesenchymal cells as biological agents in clinical trials for various diseases since the 1990s. Mesenchymal cells have been iso-lated from various tissues and cultured using various media and methods resulting in a lack of standardiza-tion in culture methods for these cells. Consequently, cells cultured in different laboratories exhibit different characteristics of MSC-like cells. This chapter outlines protocols for optimal isolation, enumera-tion, and expansion of human MSCs from BM in fetal bovine serum (FBS)-containing medium, as well as in xeno-free medium.

Key words: FBS-containing medium , Xeno-free medium , Human mesenchymal stem cells , CFU-F , Expansion , Bone marrow mononuclear cells

The non-hematopoietic stem cells in the bone marrow (BM), also termed multipotent marrow stromal cells or mesenchymal stem cells (MSCs), are a heterogeneous population of plastic-adherent, fi broblast-like cells, which in culture are able to self-renew and dif-ferentiate into bone, adipose, and cartilage tissue ( 1– 3 ) . Recently, cells with characteristics of MSCs have been isolated from various tissues including adipose tissue, cord blood, skeletal muscle, amni-otic fl uid, fetal liver and lung, synovium, and the circulatory system ( 4– 10 ) . Accumulating evidence indicates a perivascular location for these cells, leading to the suggestion that MSCs are pericytes which

1. Introduction

316 R. Wagey and B. Short

exist in close proximity to endothelial cells in capillaries and microvessels in multiple organs ( 11 ) .

Friedenstein and colleagues in the 1960s conducted the initial studies to characterize single cell suspensions of BM stromal cells. His work revealed the osteogenic potential of non-hematopoietic cells in the BM. These plastic-adherent, fi broblast like cells are able to give rise to colonies of cells termed colony forming unit- fi broblasts (CFU-F) ( 12, 13 ) . The CFU-F assay is now widely used as an in vitro assay to quantify the frequency of MSCs from a speci fi c donor tissue. There appears to be a strong correlation between age and proliferative potential, with decreasing progeni-tor cell proliferation and number associated with increasing age ( 14 ) . Since abnormal function of stromal precursor cells has been implicated in several diseases, the CFU-F assay provides the means to examine the quality and quantity of MSC progenitors from speci fi c tissues and donors prior to cell expansion.

Studies have shown that the clonogenic precursor cells initiat-ing these colonies are capable of differentiating into multiple mes-enchymal lineages including adipose tissue, cartilage, bone, and muscle ( 15– 18 ) . More recently, it has been reported that MSCs are also capable of giving rise to non-mesodermal cell types including neural cells ( 19 ) and cardiac muscle ( 20 ) .

The multi-differentiation potential of MSCs, as well as their ef fi cacy in the modulation of other cell types (paracrine effect), have generated considerable interest in utilizing MSCs for various cell-based therapies and as vehicles for gene therapy in the fi eld of tissue engineering and regenerative medicine. Accordingly, MSC have already been used in clinical trials including the treatment of childhood Osteogenesis Imperfecta (OI) ( 21 ) , in the facilitation of hematopoietic reconstitution following HSC transplantation ( 22 ) and in the prevention and treatment of graft versus host disease (GvHD) ( 23 ) . This chapter describes the methods for isolation and culture of human MSCs in FBS-containing medium.

There are also several publications on culturing MSCs in serum-free formulations including the use of platelet lysates in the medium ( 24– 26 ) . The medium formulation, as well as the culture method for isolation and expansion of MSCs, can determine the characteristics of the cultured cells (e.g., Multipotent Adult Progenitor Cells—MAPC, Marrow-isolated adult multilineage inducible cells—MIAMI, and Mesenchymal Stem Cells—MSCs) ( 3, 27, 28 ) . Hence, MSCs cultured in a xeno-free medium can have different morphology and growth characteristics compared to cells cultured in serum-containing medium. The protocol for expansion of MSCs in the xeno-free formulation described here, called MesenCult™ -XF, has been optimized for culturing cells in this particular medium with the selected substrate and dissociation enzyme. The xeno-free formulation, MesenCult™-XF contains components selective for growth of MSCs while suppressing

31720 Culture of Human Mesenchymal Stem Cells

growth of hematopoietic cells at early passage. Hence, superior clonogenic growth, higher purity of MSCs at lower passage, and greater expansion of cells can be obtained in MesenCult™-XF compared to FBS-containing medium. In this chapter we also describe the protocol to isolate, enumerate, and expand human MSCs in a xeno-free medium formulation.

1. 14 mL polypropylene tubes. 2. 50 mL conical tubes. 3. Recommended tissue culture plates/ fl asks: 6-well plates

(Corning Catalog #3516) or T-25 cm 2 Tissue culture fl asks (Falcon Catalog #353109).

4. Para fi lm®.

1. Dulbecco PBS without Ca 2+ and Mg 2+ . 2. PBS + 2% FBS. 3. Methanol, ACS Grade. 4. Giemsa Stain Solution. 5. Ficoll-Paque™ PLUS. 6. 3% Acetic Acid with Methylene Blue. 7. Trypan Blue. 8. 0.25% Trypsin-EDTA. 9. Complete MesenCult™ Medium consisting of MesenCult™

MSC Basal Medium (Human; StemCell Catalog #05401) and Mesenchymal Stem Cell Stimulatory Supplements (Human; StemCell Catalog #05402). To prepare the medium, thaw the supplements at room temperature or 2–8°C overnight. Add the entire contents of the supplements to the basal medium and mix thoroughly. Complete MesenCult™ Medium (Human) is stored at 2–8°C and should be prepared in volumes that can be used within 1 month (see Note 1).

10. L -Glutamine 200 mM. 11. Distilled water (autoclaved). 12. Iscove’s MDM (IMDM). 13. Fresh Bone marrow (25 mL) or frozen cultured BM-MSCs. 14. Human Serum Albumin (HSA; quality cell culture-tested and

veri fi ed nontoxic for MSCs) for preparation of 10% HSA in Iscove’s MDM.

15. 0.5 M EDTA (Ethylenediaminetetraacetic acid).

2. Materials

2.1. Tissue Culture

2.2. Reagents


16. Cryostor. 17. Minimum Essential Medium Eagle (MEM), Alpha Modi fi cation

(see Note 2). 18. MesenCult™-ACF Enzymatic Dissociation Solution (StemCell

Cat #05427). 19. MesenCult™-ACF Enzyme Inhibition Solution (StemCell Cat

#05428). 20. MesenCult™-XF Basal Medium (StemCell Catalog #05421). 21. MesenCult™-XF Supplement (5×; StemCell Catalog #05422). 22. MesenCult™-XF Attachment Substrate (StemCell Catalog

#05425). 23. 0.2 μ m fi lter.

Isolation and establishment of human MSC cultures should be performed in Level II Biosafety hoods using sterile techniques.

When working with fresh bone marrow (BM) samples, the cells need to be processed to remove the red blood cells or to enrich for the desired cells prior to culture. If the BM is freshly collected from the posterior iliac crest or sternum of the BM donor, heparin (5000 U) should be added as an anticoagulant following aspiration of the BM. When purchasing fresh BM, heparin is already added. MSCs are obtained from fresh BM samples by isolating mononu-clear cells using density gradient separation as detailed below.

1. Purchase/obtain 25 mL of fresh bone marrow. 2. Dilute heparinized unprocessed BM cells at a 1:2 ratio in

PBS + 2% FBS (i.e., 25 mL BM and 50 mL of PBS + 2% FBS for a total volume of 75 mL)

3. In three 50 mL conical tubes (BD Catalog # 352070), pipette 17 mL Ficoll-Paque™ PLUS (StemCell Catalog #07907/07957) into each tube. Carefully layer 25 mL diluted BM on top of the Ficoll-Paque™ PLUS in each tube using a smaller diameter pipette (e.g., 10 mL instead of 25 mL pipette). Slightly tilt the 50 mL conical tube containing Ficoll-Paque™ and slowly release (dropwise) BM from a 10 mL pipette on top of the Ficoll layer.

4. Centrifuge at room temperature (15–25°C) for 30 min at 300 × g in a benchtop centrifuge with the brake off .

5. Remove and discard the upper plasma layer with a pipette without disturbing the plasma: Ficoll-Paque™ PLUS interface.

3. Methods

3.1. Culture, Expansion, and Enumeration of MSC in FBS-Containing Medium

3.1.1. Isolation of MSCs from Unprocessed Human Bone Marrow


Carefully pipette and retain the mononuclear cells located at the interface layer and place in a new 50 mL conical tube. Suspend the mononuclear cells in 40 mL cold (2–8°C) PBS + 2% FBS Buffer. Mix gently by pipetting the cell suspension.

6. Centrifuge the cells at 300 × g (~1,200 rpm) for 10 min at room temperature in a benchtop centrifuge with the brake on . Aspirate the supernatant and suspend the cells in 1–2 mL Complete MesenCult™ Medium (Human)

7. Dilute an aliquot of cells (e.g., 10 μ L) 1/50 to 1/100 in 3% Acetic Acid with Methylene Blue and count the total number of nucleated cells using a hemacytometer.

1. Primary BM mononuclear cells should be plated at densities between 2.0 and 12 × 10 4 cells/cm 2 in Complete MesenCult™ Medium (Human). For example, dilute the cells (after the total nucleated cell count) to a stock cell concentration of 3 × 10 6 cells/mL in Complete MesenCult™ Medium (Human). Plate three different cell densities by adding 1.0, 0.67, and 0.33 mL of the cell stock to separate T-25 cm 2 culture-treated dishes fi lled with Complete MesenCult™ Medium (Human) to a total volume of 8–10 mL. This will yield fi nal cell concentrations of 3 × 10 6 cells, 2 × 10 6 cells and 1 × 10 6 cells per fl ask. For 6-well plates, cells should be plated at densities between 3 × 10 5 cells/well and 1 × 10 6 cells/well.

2. Place the T-25 cm 2 tissue culture fl asks or 6-well plates into a 37°C humidi fi ed incubator with 5% CO 2 in air and >95% humidity for 10–14 days. Maximum colony size and numbers are typically observed at 14 days (see Note 3).

1. Remove the medium from the CFU-F cultures using a 10 mL pipette and discard it into the biohazardous waste. The adher-ent colonies will remain attached to the plate. This staining procedure can be done on a benchtop since sterility is not required.

2. Gently wash the culture dishes or fl asks twice by adding PBS (e.g., 2 mL/well of a 6-well plate or 5 mL/T25 cm 2 fl ask) to the CFU-F culture. The wash is to remove any remaining medium. Discard the PBS from the two washes into the waste.

3. Add 5 mL of methanol, using a 5 mL pipette, to each culture dish or fl ask and incubate for 5 min at room temperature to fi x the cells.

4. Remove the methanol using a 10 mL pipette and discard into the biohazardous waste. Let the culture dishes or fl asks air dry at room temperature for about 5–10 min.

5. Add 5 mL of Giemsa Staining Solution to each culture dish or fl ask and leave for 5–10 min.

3.1.2. Plating Cells for the CFU-F Assay

3.1.3. Staining of CFU-F-Derived Colonies


6. Remove the Giemsa Staining Solution using a 10 mL pipette, discard the Giemsa solution and rinse the culture dishes or fl asks under a low running tap. Swirl and discard the water. A minimum of four rinses are required until water remains clear. Allow the tissue culture dishes or fl asks to dry at room temperature for about 1–2 h.

CFU-F colonies from human cells in FBS-containing medium are typically between 1 and 8 mm in diameter and should be scored both macroscopically and microscopically (for con fi rmation of col-ony numbers). Photographs of representative CFU-F-derived col-onies are shown in Fig. 1 . It is important to note that some colonies do not take up enough stain to be easily visible macroscopically, and therefore it is important to verify the number of colonies counted by scoring colonies microscopically. We recommend tak-ing a felt-tip pen and marking each CFU-F on the bottom of the well when counted. This prevents counting colonies more than once.

Ensure that there is a linear relationship between the cell num-bers plated and the resulting colony numbers, by con fi rming that there are twice as many colonies when 2 × 10 6 cells are plated as compared to 1.0 × 10 6 cells. Likewise, there should be twice as many colonies when 1.0 × 10 6 cells are plated as compared to 0.5 × 10 6 cells. Ideally there should be 10–40 colonies per T-25 cm 2 fl ask. Linearity may not be observed outside of this range as the cells would have been under- or over-plated (see Note 4).

3.1.4. Enumeration of CFU-F-Derived Colonies

Fig. 1. Photographs of representative CFU-F-derived colonies cultured in MesenCult™ Proliferation medium. A large colony ( Left ) and a medium size colony ( Right ).


Con fl uent mesenchymal cell cultures can be produced when cells from BM are plated at relatively high densities on tissue-culture-treated fl asks or dishes in Complete MesenCult™ Medium (Human). Mesenchymal cell numbers can then be expanded by splitting the cells when they become 70–80% con fl uent. If cells remain in a highly con fl uent state (>90%) for a signi fi cant time (days) it may reduce their longevity and their potential to differentiate. Optimal and over-con fl uent densities for passaging are shown in Fig. 2a, b , respec-tively. Culture-expanded mesenchymal cells can be used for a number of applications including plasticity studies, assessment of differentia-tion or expansion potential, and the evaluation of phenotype.

1. Plate primary cells at 1.0–5.0 × 10 5 cells/cm 2 in complete MesenCult™ Medium (Human) in tissue-culture-treated dishes or fl asks. The recommended cell numbers for a T-25 cm 2 fl ask in 10 mL Complete MesenCult™ Medium (Human) are 2.5 × 10 6 –1.0 × 10 7 BM mononuclear cells. For frozen marrow stromal cells (P 1 ) plate between 1.25 × 10 5 cells to 2.5 × 10 5 cells/T-25 cm 2 fl ask.

2. Observe mesenchymal cell cultures under a microscope to ensure that the cells are at an adequate stage for passaging (~80% con fl uence). This should take approximately 7–14 days for primary BM cells but less time (3–6 days) for culture-expanded cells. If the medium in the fl ask or dish appears acidic (more yellow in color than orange/red) prior to reaching 80% con fl uence, a half-medium change can be done by removing one half of the acidic medium and replacing it with fresh Complete MesenCult™ Medium (Human) pre-warmed to 37°C (see Note 5).

3. If the cells are ready to be passaged, remove the Complete MesenCult™ Medium (Human) leaving the adherent

3.1.5. Expansion and Passaging of Cultured Mesenchymal Cells

Fig. 2. Optimal density (80% con fl uency) for passaging cells ( Left ) and over-con fl uent density (100% con fl uency) for pas-saging ( Right )


mesenchymal cells behind. Wash the cells with PBS (add PBS to the culture to cover the entire culture, swirl the dish/ fl ask in PBS to remove residual FBS-containing medium and remove PBS with a pipette).

4. For cells in a T-25 cm 2 fl ask, add 3–5 mL Trypsin-EDTA to cover the cells and incubate at 37°C for 3–5 min.

5. Check under the microscope to ensure that the mesenchymal cells have detached. If a small percentage of cells are still attached, gently tap the fl ask on a bench surface to detach the remaining cells. Add 1 mL FBS or 5 mL of Complete MesenCult™ Medium (Human) to neutralize the action of the trypsin.

6. Collect the trypsinized cells into a 14 mL tube and centrifuge the cells at 300 × g for 8 min at room temperature with the brake on. Remove the supernatant and suspend the cell pellet in 1–2 mL of Complete MesenCult™ Medium (Human).

7. Perform a cell count using Trypan Blue dye exclusion by diluting an aliquot of cells (e.g., 10 μ L) 1/3 to 1/10 with Trypan Blue. Replate the cells at 4–10 × 10 4 cells/cm 2 . Alternatively, the cells can be divided into new tissue-culture-treated fl asks at a recom-mended dilution of 1/4 (e.g., one T-25 cm 2 tissue-culture-treated fl ask containing 80% con fl uent mesenchymal cells can be passaged into four T-25 cm 2 tissue-culture-treated fl asks).

Mesenchymal cells can be frozen at any passage. Studies in our laboratory have shown that cryopreserved cells from passage num-bers 2–7 maintain their phenotype and differentiation potential. Before beginning have all reagents COLD (2–8°C) and label ster-ile cryovials using an indelible marker.

1. Make up 20% Dimethyl Sulfoxide (DMSO) in Fetal Bovine Serum and fi lter-sterilize using a 0.2 μ m fi lter. Keep on ice.

2. Harvest the cells from the tissue culture surface as described in Subheading 3.1.5 , steps 3–5. Centrifuge the cells at 300 × g , 25°C for 7 min. Remove the supernatant and suspend the cells in FBS to give a maximum concentration of 2 × 10 6 cells/mL. Place this cell suspension on ice.

3. Slowly add 20% DMSO in FBS dropwise to the cells. Mix cells gently with 20% DMSO in FBS at a ratio of 1:1 (the fi nal cell suspension will be 90% FBS/10% DMSO). Transfer 1 mL of cells in freezing medium to each cryovial. The fi nal cell con-centration will be ~1 × 10 6 cells per vial.

4. Place the cryovials immediately into a thawed 70% isopropanol freezing container and place the container in a −135°C freezer overnight. On the next day, remove frozen vials from the freez-ing container and store at −135°C (or colder) or in liquid nitrogen (see Note 6).

3.1.6. Freezing Mesenchymal Cells


1. Thaw the cells quickly in a 37°C water bath or a beaker of warm water in a tissue culture hood. Wipe the cryovial with 70% ethanol.

2. Gently transfer the cells into a 50 mL centrifuge tube by pipetting the cells from the cryovial into the centrifuge tube (see Note 7).

3. Slowly add 15 mL IMDM containing 2% FBS drop-wise while holding the tube and gently swirling. Then fi ll tube to 50 mL with IMDM containing 2% FBS. Gently invert the tube to mix.

4. Centrifuge the cells at 300 × g for 8 min. 5. Discard the supernatant and “ fl ick” the tube gently to suspend

the pellet. 6. Suspend the cells at the desired concentration in Complete

MesenCult™ Medium (Human).

1. Thaw MesenCult™-XF Supplement (5×; StemCell Catalog #05422) overnight at 2–8°C. Add the entire MesenCult™-XF Supplement (100 mL) to one bottle (400 mL) of MesenCult™-XF Basal Medium (StemCell Catalog #05421). Add L -Glutamine to a fi nal concentration of 2 mM. Cells cul-tured in MesenCult™-XF medium will not grow without addi-tion of 2 mM L -glutamine. This is now referred to as Complete MesenCult™-XF Medium. Complete MesenCult™-XF Medium can be stored at 2–8°C for no more than 5 days (see Note 8).

2. Tissue culture fl asks need coating with MesenCult™-XF Attachment Substrate (StemCell Catalog #05425) to support cell adherence. It is recommended to coat plates 1 day prior to usage (i.e., coat overnight at 2–8°C), but if time is limited, they can be coated for 2 h at 15–25°C (room temperature) prior to use.

3. Coating plates for the CFU-F assay: Dilute MesenCult™-XF Attachment Substrate 1/40 in sterile PBS without Ca 2+ and Mg 2+ (StemCell Catalog #37350). Gently mix by inverting the tube twice. Prepare an amount slightly more than required, to account for pipetting variability. For example, to coat one 6-well plate, dilute 167 μ L MesenCult™-XF Attachment Substrate in 4.8 mL PBS and add 800 μ L per well.

4. To coat plates for cell expansion from primary tissue: Dilute MesenCult™-XF Attachment Substrate 1:20 in sterile PBS without Ca 2+ and Mg 2+ and gently mix by inverting the tube twice. Prepare an amount slightly more than required, to account for pipetting variability. For example, to coat one T-75 cm 2 fl ask, dilute 250 μ L MesenCult™-XF Attachment Substrate in 5 mL PBS.

3.1.7. Thawing Mesenchymal Cells

3.2. Culture, Expansion and Enumeration of MSC in Xeno-Free Medium

3.2.1. Preparation of Reagents and Coating of Plates


5. To coat plates for cell expansion from previously cultured cells: Dilute MesenCult™-XF Attachment Substrate 1:28 in sterile PBS without Ca 2+ and Mg 2+ . Gently mix by inverting the tube twice. Prepare an amount slightly more than required, to account for pipetting variability. For example, to coat one T-75 cm 2 fl ask, dilute 185 μ L MesenCult™-XF Attachment Substrate in 5 mL PBS. Substrate volumes to use and sug-gested plates or fl asks are indicated in Tables 1 .

6. Wrap plates with Para fi lm®, sealing the junction between the base and lid, and incubate at 2–8°C (in the refrigerator) over-night or for 2 h at 15–25°C (room temperature). For fl asks, seal the vent on the cap with Para fi lm® and incubate as described.

7. If plates/ fl asks were incubated overnight at 2–8°C, bring to room temperature (approximately 20 min) prior to washing.

Table 1 Volume of attachment substrate for coating fl asks

Size Volume of attachment substrate Suggested plates/ fl asks

6-Well plate 800 μ L/well Corning Catalog #3516

T-25 cm 2 fl ask 2 mL/ fl ask BD Falcon Catalog #353109

T-75 cm 2 fl ask 5 mL/ fl ask BD Falcon™ Catalog #353136

Table 2 Plating densities for expansion of primary BM mononuclear cells in MesenCult™-XF

Tissue culture vessel

Volume of medium Surface area

Suggested plating densities

6-Well plate 2.5 mL/well 9.5 cm 2 /well 3 × 10 5 cells/well

4.5 × 10 5 cells/well


T-25 cm 2 10 mL/ fl ask 25 cm 2 / fl ask 8.0 × 10 5 cells/ fl ask

10 × 10 5 cells/ fl ask

12.5 × 10 5 cells/ fl ask





Gently pipette off, and discard, remaining MesenCult™-XF Attachment Substrate without touching the newly coated surface.

8. Wash plates/ fl asks once with sterile distilled water by slowly pipetting water down the side of the well/ fl ask, being careful not to scrape the newly coated surface. Swirl gently to rinse the entire surface and then carefully aspirate off water.

9. Allow to dry for at least 15 min at 15–25°C prior to use.

Prior to initiating this protocol prepare 500 mL Isolation Buffer (PBS + 0.5% HSA + 2 mM EDTA) by adding 25 mL HSA (10% stock solution in sterile dH 2 O) and 2 mL EDTA (0.5 M stock solution) to 473 mL of 1× PBS. Once made, the Isolation Buffer can be stored at 2–8°C for 1 month. If any of the components are not sterile (i.e., EDTA), be sure to fi lter sterilize the individual components or the complete buffer with a 0.2 μ m fi lter.

1. Count the total number of nucleated cells in the BM sample by taking 10 μ L BM and diluting it 1/40 to 1/100 with 3% Acetic Acid with Methylene Blue (StemCell Catalog #07060). Count cells using a hemacytometer (see Note 9).

2. Warm 50 mL Isolation Buffer at room temperature for 20 min prior to use. Dilute BM approximately 1:3 with room tempera-ture Isolation Buffer (e.g., dilute 25 mL BM with 50 mL Isolation Buffer for a total volume of 75 mL).

3. Pipette 17 mL Ficoll-Paque™ PLUS into each of three 50 mL conical tubes. Carefully layer 25 mL diluted BM on top of the Ficoll-Paque™ PLUS in each tube.

4. Centrifuge at room temperature (15–25°C) for 30 min at 300 × g in a benchtop centrifuge with the brake off.

5. Remove and discard the upper plasma layer without disturbing the plasma:Ficoll-Paque™ PLUS interface. Carefully remove and retain the mononuclear cells located at the interface layer and place in a new 50 mL conical tube. Suspend the mononu-clear cells in 40 mL cold (2–8°C) Isolation Buffer. Mix gently by pipetting.

6. Centrifuge the cells at 300 × g for 10 min at room temperature in a benchtop centrifuge with the brake on. Remove the super-natant and suspend cells in 1–2 mL cold Isolation Buffer.

7. Dilute an aliquot of cells (i.e., 10 μ L) 1/50 in 3% Acetic Acid with Methylene Blue and count the total number of nucleated cells using a hemacytometer.

8. Dilute cells in Complete MesenCult™-XF Medium at a fi nal concentration of 1 × 10 6 cells/mL.

3.2.2. Preparation of a Mononuclear Cell Suspension from Fresh Human Bone Marrow


The cell source for setting up the CFU-F assay can be either mono-nuclear cells from a fresh BM sample or culture-expanded mesen-chymal cells. It is recommended not to use previously frozen BM mononuclear cells, as freezing mononuclear cells may affect the viability of mesenchymal progenitor cells which are present at low frequency in the BM. CFU-F assays must be performed using tissue-culture-treated plates that have been coated with MesenCult™-XF Attachment Substrate (StemCell Catalog #05425) as described in Subheading 3.2.1 .

1. Using fresh BM-derived mononuclear cells processed accord-ing to Subheading 3.2.2 seed cells at three different densities (between 1.5 and 5 × 10 4 cells/cm 2 ) in Complete MesenCult™-XF Medium. For example: In a 6-well plate add 150, 250 and 500 μ L cells (stock: 1 × 10 6 cells/mL) for a con-centration of 1.5 × 10 5 cells/well, 2.5 × 10 5 cells/well and 5.0 × 10 5 cells/well in 2 mL MesenCult™-XF Medium.

2. When using culture-expanded mesenchymal stem cells, seed 25–250 cells per well of a 6-well plate at fi ve different densities in Complete MesenCult™-XF Medium (see Note 10)

3. Place the cultures in a 37°C incubator with 5% CO 2 in air and 95% humidity for 9–12 days. After day 7, monitor the growth of colony-forming cells daily, to prevent overgrowth. Cultures should be stained before adjacent colonies become too large and merge (see Note 10)

4. Gently remove MesenCult™-XF Medium from CFU-F cul-tures with a 5 mL or 10 mL pipette and discard. Adherent CFU-F colonies will remain attached.

5. Gently wash colonies once with 2 mL PBS per well of a 6-well plate to remove any residual culture medium. Fix cultures with methanol and stain with Giemsa as described in Subheading 3.1.3 .

6. Refer to Subheading 3.1.4 for details on CFU-F enumeration.

1. When initially plating bone marrow mononuclear cells in MesenCult™-XF Medium for expansion, plate between 3.0 and 7.0 × 10 4 cells/cm 2 in Complete MesenCult™-XF Medium into tissue-culture-treated plates/ fl asks that have been coated with MesenCult™-XF Attachment Substrate (StemCell Catalog #05425), as described in Subheading 2.1 . Suggested plating densities are outlined in Table 2 (see Note 11)

2. Place the cultures in a 37°C incubator with 5% CO 2 in air and 95% humidity for 9–13 days.

3. Observe primary MSCs under a microscope 7 days post-plat-ing to determine if they are ready for passaging or if the medium is acidic and a half-medium change needs to be performed.

3.2.3. Plating, Staining, and Enumerating Cells in the CFU-F Assay

3.2.4. Expansion and Passage of Cultured Mesenchymal Stem Cells


Cells should be passaged when cultures are 80% con fl uence in MesenCult™-XF medium (see Note 12).

4. If the cells are ready to be passaged, warm the MesenCult™-ACF Enzymatic Dissociation Solution (Cat #05427) and the MesenCult™-ACF Enzyme Inhibition Solution (Cat #05428) to room temperature. Do not incubate at 37°C (see Note 13)

5. To passage cells, slowly remove the medium from the cultures with a 5 mL or 10 mL pipette. The adherent cells will remain attached to the culture dish.

6. Wash cells with 2 mL sterile Ca/Mg 2+ free PBS per well of a 6-well plate to remove residual culture medium. Remove PBS with a 5 mL or 10 mL pipette.

7. Add 1 mL MesenCult™-ACF Enzymatic Dissociation Solution to each well of a 6-well plate, 3 mL to a T-25 cm 2 fl ask, or 6 mL to a T-75 cm 2 fl ask. Incubate at 37°C for 2–5 min.

8. After 2 min, observe cells under the microscope to ensure that all cells have detached. Gently tap plate/ fl ask to detach remain-ing cells. If cells remain adherent, return to incubator for 1 min further and again observe cells microscopically to assess cell detachment. Do not incubate for longer than 6 min.

9. Add 1 mL MesenCult™-ACF Enzyme Inhibition Solution to each well of a 6-well plate, 3 mL to a T-25 cm 2 fl ask or 6 mL to a T-75 cm 2 fl ask.

10. For cells cultured in a 6-well plate pipette the 2 mL cell sus-pension into a 14 mL polystyrene tube and wash each well with 3 mL MEM Alpha to recover remaining cells. Add MEM Alpha to bring the total volume to 8 mL. Duplicate wells seeded at the same density may be pooled into one tube if desired. See Note 14 for the protocols to deal with cells cul-tured in other sizes of culture fl asks.

11. Centrifuge cells at 300 × g for 8 min at room temperature with the brake on.

12. Remove the supernatant and suspend the cell pellet in 0.5–1 mL Complete MesenCult™-XF Medium.

13. Dilute an aliquot of cells (i.e., 10 μ L) 1/3 to 1/10 with Trypan Blue and perform a viable cell count using a hemacytometer.

14. Suspend cells in Complete MesenCult™-XF Medium for plat-ing into new tissue-culture-treated plates/ fl asks that have been coated with MesenCult™-XF Attachment Substrate, as described in Subheading 3.2.1 . The recommended plating density for passaged cells is between 1.5 and 4.0 × 10 3 cells/cm 2 . The optimal plating densities for each tissue culture vessel are indicated in Table 3 .


15. Culture the cells in a 37°C incubator with 5% CO 2 in air and 95% humidity until they reach 80% con fl uence. When cells reach 80% con fl uence and are ready to be passaged, repeat steps 4–16 of Subheading 3.2.4 . A half-medium change is only necessary if the medium appears acidic (yellowish in color) prior to reaching 80% con fl uence (see Note 15).

1. If less than 500 mL will be required in a month, smaller vol-umes can be prepared. Prepare Complete MesenCult™ Medium (Human) by diluting Mesenchymal Stem Cell Stimulatory Supplements (Human) 1/10 with MesenCult™ MSC Basal Medium. For example, prepare 100 mL of Complete MesenCult™ Medium (Human) by adding 10 mL of Mesenchymal Stem Cell Stimulatory Supplements (Human) to 90 mL of MesenCult™ MSC Basal Medium (Human).The complete medium is stable at 2–8°C for 1 month. Repeated freezing and thawing is not recommended as it may lead to suboptimal performance (i.e. lower cell expansion).

2. Suggested Basal media for culturing MSCs include MesenCult™ MSC Basal medium-Human-(StemCell Cat. #05401), alpha MEM, or McCoy 5A medium. The supplement consists of pre-tested FBS. Each FBS lot should be pretested for optimal for-mation of CFU-F and long-term expansion of human MSCs.

3. CFU-F assays can also be set up using culture-expanded BM cells to assess the cloning ef fi ciency of the cells. The plating density for cultured cells ranges from 10 to 150 cells/cm 2 .

4. Notes

Table 3 Plating densities for expansion of cultured cells in MesenCult™-XF

Tissue culture vessel

Volume of medium Surface area

Suggested plating densities

6-Well plate 2.5 mL/well 9.5 cm 2 /well 1.5 × 10 4 cells/well




T-75 cm 2 15 mL/ fl ask 75 cm 2 / fl ask 15 × 10 4 cells/ fl ask

25 × 10 4 cells/ fl ask


4. Each bone marrow sample is unique and thus the number of CFU-F may vary depending on a number of factors including age, presence of disease and previous treatments given to the patient. To achieve optimal CFU-F number, size, and mor-phology it is recommended to prescreen FBS batches. There is no need to supplement the prescreened FBS with any growth factors.

5. The proliferative ability of each BM sample is donor-dependent and can be affected by a number of factors including age, dis-ease, or whether the sample comes from a transplant recipient. Therefore not all BM samples may be con fl uent in a week and a half-medium change may help cells to proliferate in some samples. It is important to observe the culture on a regular basis. Cells cultured at high density (>90%) and at high passage number (>P6) tend to loose their telomerase length and have reduced DNA methylation leading to reduced proliferation and differentiation potentials.

6. In order to ensure high cell viability upon freezing and thaw-ing do not let cells sit in freezing medium at room tempera-ture. Keep the cells on ice and transfer within 5 min to the freezing container.

7. Do not vortex cells at anytime. 8. MesenCult™-XF Supplement can be aliquoted into smaller

working volumes and stored at −20°C until required for use. Repeated thawing and freezing is not recommended. Complete MesenCult™-XF Medium should be prepared in volumes that can be used within 5 days. Prepare an amount suitable for your needs by diluting MesenCult™-XF Supplement 1/5 ( fi nal dilution) in MesenCult™-XF Basal Medium (i.e., 20 mL MesenCult™-XF Supplement + 80 mL MesenCult™-XF Basal Medium).

9. Acetic acid 3% with Methylene Blue will lyse red blood cells and white blood cell membranes. The remaining white blood cell nuclei will stain lightly with Methylene Blue.

10. Plating different cell densities will ensure that the resulting numbers of colonies can be scored. The proliferative potential of CFU-F from various bone marrow samples is widely vari-able. If too few cells are plated, CFU-F may be undetectable or the number of colonies scored may be too low to give a reliable estimation of CFU-F. If too many cells are plated, the CFU-F may grow such that individual colonies cannot be determined. Human bone marrow-derived CFU-F colonies cultured in MesenCult™-XF medium are generally large enough to see with the naked eye following staining with Giemsa (see Fig. 3 ).


CFU-F cultured in MesenCult™-XF Medium have a slightly different morphology than CFU-F typically obtained when per-forming the CFU-F assay with the MesenCult™ Proliferation Kit (Human) (see Fig. 4a, b ). Be sure to monitor CFU-F colony size. MSCs cultured in MesenCult™-XF Medium proliferate faster than cells cultured in a traditional serum-based medium.

11. The proliferation potential of cells obtained from different bone marrow donors is highly variable. To ensure that cultures con-tain an optimal number of cells for expansion, it is recommended to seed two to three different cell densities. If too few cells are

Fig. 3. The circled colonies are easily visible macroscopically. It is important to look at the CFU-F cultures under a micro-scope for con fi rmation because some colonies may not take up enough stain and could be missed when scored macro-scopically. The CFU-F assay was performed in a 6-well plate (note the edges of the well in each image). A Lumenera In fi nity 2–3 C camera was used to capture the images using Image Pro 6.2 software.

Fig. 4. The morphology of CFU-F colonies generated when MSCs are cultured in MesenCult™-XF Medium ( a ) differs from the morphology of CFU-F generated when MSCs are cultured with MesenCult™ Proliferation Kit ( b ) containing FBS.


plated, cells grow too slowly and reach recommended splitting density too late (cells may start to detach from the surface). If too many cells are plated, the cells will reach con fl uence too fast and will become senescent and lose pluripotency.

12. The cells are ready to be passaged when they reach 80% con fl uence (see Fig. 5a, b ). Normally cells reach 70–80% con fl uence between 9 and 13 days after initial plating of pri-mary BM mononuclear cells, but this depends on the donor and initial plating density.

Monitor the color of the medium after day 7: if the medium appears acidic (yellowish in color) prior to reaching 80% con fl uence, a half-medium change can be performed by remov-ing 1/2 of the medium and replacing it with fresh Complete MesenCult™-XF Medium warmed to 37°C.

13. During development of this Xeno-free formulation, we tested different components of the culture system to promote opti-mal cell growth. The cells cultured in a serum- and Xeno-free formulation normally do not adhere easily due to the absence of FBS. FBS is a rich mixture of various proteins including extracellular matrix which can promote cell adherence. There are several critical factors to obtain optimal cell adherence and growth when culturing MSCs in a xeno-Free medium. They are as follows: (a) Culture dish. It is essential to use tissue-culture-treated

dishes with strong adherence properties. There are slight differences in the strength of cell attachment between tis-sue-culture-treated dishes from different suppliers. The ones that promote best cell adherence are from Corning (Catalog #3516) or BD (Falcon™ Catalog #353109 and Falcon™ Catalog #353136).

Fig. 5. It is important that cells are passaged in MesenCult™-XF medium when they reach 80% con fl uence. Figures a and b depict cells at an optimal density for passaging.


(b) Attachment substrate (coating solution). This is one of the most important steps to ensure strong cell adherence. The attachment substrate which is an extracellular matrix should be mixed gently (inverting the tube). Repeated mixing with a pipette is not recommended (as this may interfere with the stability of the substrate). Following the proper coating procedure is critical to achieve strong cell adherence.

(c) Gentle dissociation solution. During subculture it is critical to use a gentle dissociation solution to ensure cell surface molecules are intact. A strong detachment enzyme can lead to lack of cell adherence following subculture.

(d) Cell plating concentrations . The cell plating densities sug-gested in this protocol have been optimized for this par-ticular culture system (attachment substrate, medium formulation, and dissociation enzyme). For the CFU-F assay, plating different cell densities will ensure that the resulting numbers of colonies can be scored. The prolif-erative potential of CFU-F from various BM samples is widely variable. If too few cells are plated, CFU-F may be undetectable or the number of colonies scored may be too low to give a reliable estimation of CFU-F. If too many cells are plated, too many CFU-F may grow such that individual colonies cannot be determined. For the expan-sion assay, plating too few or too many cells can result in poor proliferation. If using other attachment substrates and dissociation enzymes (not as suggested in this proto-col) with MesenCult™-XF, further optimization is required to obtain optimal cell adherence and cell growth.

14. For a T-25 cm 2 fl ask pipette the 6 mL cell suspension into a 14 mL polystyrene tube and wash each fl ask by adding 5 mL MEM Alpha using a pipette to recover remaining cells. Add MEM Alpha with a 2 mL pipette to bring the total volume to 12 mL. For a T-75 cm 2 fl ask, collect the 12 mL cell suspension with a pipette, transfer to a 50 mL conical tube and wash each fl ask with 8 mL MEM Alpha to recover remaining cells. Add MEM Alpha with a 10 mL pipette to the tube to bring the total volume to 30 mL. It is important to add additional medium when washing the cells so the MesenCult™-ACF Enzymatic Dissociation Solution is suf fi ciently washed from the cells. Continue to step 11 under Subheading 3.2.4 .

15. BM-derived mesenchymal cells cultured in MesenCult™-XF medium have less hematopoietic contaminating-cells com-pared to cells cultured in MesenCult™- proliferation medium containing FBS (see Fig. 6a, b ).


Acknowledgments

The authors would like to thank Betty Hoac and Jacky Yau for technical assistance, Bert Wognum and Emer Clarke for technical advice, Terry Thomas and Allen Eaves for continuous support.

References

Fig. 6. Passage 0 human bone marrow-derived mesenchymal stem cells show less hematopoietic cell contamination when cultured in MesenCult™-XF Medium ( a ) compared to serum-based medium ( b ).

1. Bruder SP, Jaiswal N, Haynesworth SE (1997) Growth kinetics, self-renewal, and osteogenic potential of puri fi ed human mesenchymal stem cells during extensive subcultivation and fol-lowing cryopreservation. J Cell Biochem 64:278–294

2. Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF (1998) Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 4:415–428

3. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesen-chymal stem cells. Science 284:143147

4. Erices A, Conget P, Minguell JJ (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109:235–242

5. Debari C, Dell’Accio F, Tylazanowski P et al (2001) Multipotent mesenchymal stem cells form adult human synovial membrane. Arthritis Rheum 44:1928–1942

6. Kuznetsov SA, Mankani MH, Gronthos S et al (2001) Circulating skeletal stem cells. J Cell Biol 153:113–114

7. Tondreau T, Meuleman N, Delforge A et al (2005) Mesenchymal stem cells derived from CD133 positive cells in mobilized peripheral blood and cord blood: proliferation, Oct4 expression, and plasticity. Stem Cells 23:1105–1112

8. Roberts IA, Campagnoli IA, Kumar S et al (2001) Identi fi cation of mesenchymal stem/progenitor cells in human fi rst trimester fetal blood, liver and bone marrow. Blood 98:2396–2402

9. Int’l Anker PS, Scherjon SA, Kleiburg-van der Keur C et al (2003) Amniotic fl uid as a novel source of mesenchymal stem cells for thera-peutic transplantation. Blood 102:1548–1549

10. Zuk PA, Zhu M, Ashjian P et al (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13:4279–4295

11. Crisan M, Chen CW, Corselli M, Andriolo G, Lazzari L, Péault B (2009) Perivascular multi-potent progenitor cells in human organs. Ann N Y Acad Sci 1176:118–123

12. Friedenstein AJ, Chailakhjan RK, Lalykina KS (1970) The development of fi broblast colonies in monolayer cultures of guinea-pig bone mar-row and spleen cells. Cell Tissue Kinet 3:393–403


13. Friedenstein AJ (1980) Stromal mechanisms of bone marrow: cloning in vitro and trans-plantation in vivo. Haematol Blood Transfus 25:19–29

14. Clarke E, McCann SR (1989) Age dependent in vitro stromal growth. Bone Marrow Transplant 4:596–597

15. Gronthos S, Graves SE, Ohta S, Simmons PJ (1994) The STRO-1+ fraction of adult human bone marrow contains the osteogenic precur-sors. Blood 84:4164–4173

16. Sekiya I, Vuoristo JT, Larson BL, Prockop DJ (2002) In vitro cartilage formation by human adult stem cells from bone marrow stroma de fi nes the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A 99:4397–4402

17. Russell KC, Phinney DG, Lacey MR, Barrilleaux BL, Meyertholen KE, O’Connor KC (2010) In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells 28:788–798

18. Phinney DG, Prockop DJ (2007) Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells 25:2896–2902

19. Deng J, Petersen BE, Steindler DA, Jorgensen ML, Laywell ED (2006) Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplanta-tion. Stem Cells 24:1054–1064

20. Tsuji H, Miyoshi S, Ikegami Y, Hida N, Asada H, Togashi I, Suzuki J, Satake M, Nakamizo H, Tanaka M, Mori T, Segawa K, Nishiyama N, Inoue J, Makino H, Miyado K, Ogawa S, Yoshimura Y, Umezawa A (2010) Xenografted human amniotic membrane-derived mesen-chymal stem cells are immunologically toler-ated and transdifferentiated into cardiomyocytes. Circ Res 106:1613–1623

21. Horwitz EM, Prockop DJ, Fitzpatrick LA, Koo WWK, Gordon PL, Neel M, Sussman M, Orchard P, Marx JC, Pyeritz RE, Brenner MK (1999) Transplantability and therapeutic

effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 5:309–313

22. Lazarus HM, Koc ON, Devine SM, Curtin P, Maziarz RT, Holland HK, Shpall EJ, McCarthy P, Atkinson K, Cooper BW, Gerson SL, Laughlin MJ, Loberiza FR Jr, Moseley AB, Bacigalupo A (2005) Cotransplantation of HLA-identical sibling culture-expanded mes-enchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 11:389–398

23. Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringdén O, Group for Blood and Marrow Transplantation (2008) Mesenchymal stem cells for treatment of ste-roid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 371:1579–86

24. Meuleman N, Tondreau T, Delforge A et al (2006) Human marrow mesenchymal stem cell culture: serum-free medium allows better expansion than classical alpha-MEM medium. Eur J Haematol 76:309–316

25. Kocaoemer A, Kern S, Kluter H, Bieback K (2007) Human AB-serum and thrombin-acti-vated platelet-rich plasma are suitable alterna-tives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells 25:1270–1278

26. Schallmoser K, Bartmann C, Rohde E et al (2007) Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47:1436–1446

27. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM (2001) Puri fi cation and ex vivo expansion of postnatal human marrow meso-dermal progenitor cells. Blood 98:2615–2625

28. D’Ippolito G, Diabira S, Howard GA, Menei P, Roos BA, Schiller PC (2004) Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 117:2971–2981

[methods in molecular biology] basic cell culture protocols volume 946 || isolation, enumeration,...

Documents