tb496

BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide: Synthetic and Animal-free Surface for Culture of Human Bone Marrow-derived Mesenchymal Stem Cells in StemPro® MSC SFM Xeno-free Medium

Jeff Partridge, Katie Slater, Paula Flaherty, Susan Qian, and Deepa SaxenaBD Biosciences, Two Oak Park, Bedford, MA 01730 US

Application Note Contents

1 Introduction

2 Materials and Methods

4 Results and Discussion

7 Conclusions

7 References

IntroductionMesenchymal stem cells (MSCs) are an important tool in regenerative medicine and tissue engineering. These multipotent stem cells have the ability to differentiate into bone cells (osteocytes), cartilage cells (chondrocytes) and fat cells (adipocytes).1 Optimal ex vivo expansion of MSCs requires either a bovine serum-containing media or a coating of the culture vessel with a human or animal-derived extracellular matrix (ECM) protein. Biological proteins can introduce a source of variability and contaminants into the culture, masking cell signaling events, and requiring additional screening to ensure overall cell quality. Growing concerns about introducing human- and animal-derived pathogens into the culture necessitate the need for an animal-free (xeno-free and human origin components-free) culture environment to enable high quality cells with therapeutic potential.

BD PureCoat™ ECM Mimetic Cultureware provides a pre-coated, synthetic and animal component-free alternative to the use of complex ECM or biological proteins coatings. The BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide has been proven to work in the expansion of hMSCs in defined and xeno-free culture environments.2 The Fibronectin mimetic surface is a pre-coated, synthetic, xeno-free, animal-free, and room temperature stable surface. Fibronectin peptide is covalently immobilized on the cultureware surface to present in a functionally active orientation to the cells. The peptide consists of the RGD amino acid sequence from the Fibronectin cell binding domain that facilitates cell attachment.3 MSC growth and morphology on the Fibronectin mimetic surface has been proven comparable to cells grown on human-origin matrix-coated surfaces.2 Cells maintain their differentiation capability and can be successfully differentiated into osteogenic and adipogenic lineages following multiple passages on the Fibronectin mimetic surface. In addition, cells exhibit the characteristic MSC marker profile, as determined by flow cytometry.4

The BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide has been evaluated for use with commercially available serum-free and xeno-free media. In this Application Note, we show performance in the StemPro MSC SFM Xeno-free media. The data show that BD PureCoat ECM Mimetic Cultureware Fibronectin peptide provides a ready to use alternative to self-coating with comparable cell attachment and functionality.

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October 2012

Application Note 496


Materials and MethodsReagents

Human bone marrow-derived MSCs were purchased from Lonza (Cat. No. PT-2501). Cell culture media, dissociation reagents and differentiation kits were purchased from Gibco, Life Technologies. Cells were cultured in StemPro® MSC SFM Xeno-free (Life Technologies Cat. No. A10675-01) on BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide 6-well plate (BD Cat. No. 356240). BD Falcon™ 6-well cell culture plates (BD Cat. No. 353224) coated with CELLstart™ CTS™ substrate (Life Technologies Cat. No. A10142) served as a control and is also referred to as coating matrix. Cells were passaged using TrypLE™ Select CTS reagent, a dissociation solution (Life Technologies Cat. No. 12859), as per supplier’s instructions. MSC immunophenotypic analysis was performed using the BD Stemflow™ hMSC Analysis Kit (BD Cat. No. 562245). StemPro Adipogenesis Differentiation Kit (Life Technologies Cat. No. PT-2501 A10070-01) was used for differentiation into Adipogenic lineage. Differentiation into Osteogenic lineage was performed using StemPro Osteogenesis Differentiation Kit (Life Technologies Cat. No. PT-2501 A10072-01). Mineralization was quantified with OsteoImage™ Mineralization Assay Kit (Lonza Cat. No. PA-1503). Adipocytes were stained with Oil Red O (Sigma O-0625). Paraformaldehyde 16% solution (EMS Cat. No. 15710) was diluted to 4% with 1 X Dulbecco Phosphate Buffer Saline (DPBS).

MSC Culture

BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide is pre-coated and ready to use. BD Falcon 6-well cell culture plates were coated with human-origin matrix as per supplier’s instructions. MSCs were thawed from the frozen stock and spun at 200 x g for 5 minutes, resuspended in culture medium, counted using the Vi-CELL™ cell counter (Beckman Coulter) and seeded at a density of 5,000 cells/cm2 in 2 mL culture medium/well in 6-well plate. Cells were incubated in a humidified incubator at 37ºC with 5% CO2. Cells were passaged once they reached ~60-80% confluence. Passaging was performed following instruction from the media kit. Briefly, culture media was aspirated and cells were washed once with DPBS and 0.5 mL dissociation solution was added to each well (6-well format), cells were examined under the microscope; when cells were detached from the surface, 1 mL culture medium was added to each well. Cells were then transferred to a polystyrene BD Falcon tube (BD Cat. No. 352097). The remaining cells were recovered by rinsing each well with 1 mL culture medium. Cell suspension was centrifuged at 200 x g for 5 min, supernatant was removed, and the cells were resuspended in culture medium. Cells were counted and seeded again for expansion.

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October 2012



Materials and Methods (continued)

MSC Differentiation and Analysis

After 5 passages on mimetic or coating matrix surfaces, cells were differentiated into osteogenic and adipogenic lineages following the protocol from differentiation kits. MSCs were induced to osteogenic lineage for 9 days and mineral deposition was quantified by a fluorescence based detection method following supplier’s instructions. Adipogenic differentiation was performed for 1 week. Cells differentiated to adipogenic lineage were fixed and stained with Oil Red O following supplier’s instructions. Uninduced controls were maintained in MSC culture medium.

Characterization of Markers by Flow Cytometry

Cells were grown for 5 passages and expression of markers was analyzed by flow cytometry using the Human MSC Analysis Kit. Cells were dissociated using TrypLE Select CTS, washed with PBS with 10% fetal bovine serum and resuspended in PBS/bovine serum at a concentration of 5x106 cells/mL. Cells were stained with antibodies CD90, CD105, CD73, CD34, CD11b, CD19, CD45 and HLA-DR. For staining, 0.01 mL cell suspension was transferred to each tube and antibodies were added. Tubes were incubated in the dark for 30 minutes on ice. Cells were washed twice with PBS containing 10% fetal bovine serum, and then resuspended in 0.3-0.5 mL of the same buffer. Cells were analyzed using a BD FACSCalibur™ flow cytometer, and data were analyzed using BD CellQuest™ 3.0 software.

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October 2012



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October 2012



Figure 1. Phase contrast images of human bone marrow-derived MSCs at different passages in a defined and xeno-free medium. Bottom row represents images from the BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide. Top row shows MSCs cultured on human-origin, matrix-coated plates. Note comparable morphology on both surfaces.

Table 1. Cumulative population doubling of hMSCs on Fibronectin mimetic surface and human-origin coating matrix. Cell passaging was based on confluence. Expansion of MSCs on both surfaces is comparable.

Passage 1

HFN

Mim

etic

Co

atin

g M

atri

x

Passage 4

Results and DiscussionMSC Culture and Population Doubling

MSCs from the frozen stock were cultured in serum-free and xeno-free culture medium. Cells did not require any pre-adaptation on the Fibronectin mimetic surface and exhibited similar morphology on human-origin coating matrix and mimetic surface (Figure 1). After MSCs reached 60-80% confluence, cells were passaged. At each passage cells were counted and population doubling was determined. Cumulative population doubling over the period of culture has been shown in Table 1. Population doublings on the Fibronectin mimetic and human-origin coating matrix were comparable over a period of 20-21 days, demonstrating that Fibronectin mimetic cultureware supported good attachment and expansion of MSCs for multiple passages and suitability for MSC culture.

Culture Condition Days in Culture Cumulative Population Doubling

Coating matrix 20 15.63

Fibronectin matrix 21 16.64

Analysis of MSC Markers by Flow Cytometry

After MSCs were cultured on Fibronectin mimetic surface for 5 passages, cells were collected by enzymatic dissociation, stained for established MSC markers using the respective antibodies, and then analyzed by flow cytometry. Immunophenotyping analysis captured in Figure 2 demonstrated that the MSC population was positive for CD90, CD105, and CD73 markers and negative for CD34, CD11b, CD19, CD45 and HLA-DR. A similar marker profile was exhibited by cells cultured on the human-origin matrix-coated surface. MSCs cultured on Fibronectin mimetic surface exhibited the ISCT-established marker profile.

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October 2012



Figure 2. Immunophenotyping of MSCs after 3 passages on human-origin coating matrix or Fibronectin mimetic surfaces. Cells exhibited the ISCT established marker profile with >95% MSC population expressing CD73, CD90, and CD105, and lacking expression of CD34, CD45, CD11b, CD19, and HLA DR (negative cocktail PE).

Co

atin

g M

atri

xFi

bro

nec

tin

Mim

etic

040

8012

016

020

00

4080

120

160

200

040

8012

016

020

0

040

8012

016

020

0

040

8012

016

020

0

040

8012

016

020

0

040

8012

016

020

0

040

8012

016

020

0

Coun

tsCo

unts

Coun

ts

Coun

ts

Coun

ts

Coun

ts

Coun

ts

Coun

ts

100 101 102 103 104

CD90 FITC

CD90 FITC

99.8%

99.8%

97.9%

97.9%

99.9%

99.9%

CD105 PerCP-Cy5.5

CD105 PerCP-Cy5.5

CD73 APC

CD73 APC

negative cocktail PE

negative cocktail PE100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

100 101 102 103 104100 101 102 103 104100 101 102 103 104100 101 102 103 104

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October 2012



Differentiation to Osteogenic Lineage

After 5 passages, MSCs were differentiated into osteogenic lineage. Post-differentiation, mineralization by osteocytes was also quantified by a fluorescence-based staining method that detects the hydroxyapatite portion of the mineralized bone matrix. As shown in Figure 3, cells differentiated after culture on the Fibronectin mimetic surface exhibited mineral deposits comparable to cultures on human-origin coating matrix. No bone forming activity was detected in uninduced controls. This experiment has demonstrated that MSCs remained multipotent and successfully differentiated into osteogenic lineage when cultured on the Fibronectin mimetic surface.

Differentiation to Adipogenic Lineage

Adipogenesis was induced after 5 passages as described in Materials and Methods. Post-differentiation, cells were fixed and stained with Oil Red O. MSCs differentiated into adipocytes and deposited lipid vacuoles, which exhibit red staining with Oil Red O (Figure 4). Uninduced cells did not develop lipid vacuoles. Thus, multipotent MSCs were successfully differentiated into adipogenic lineage after multiple passages on the Fibronectin mimetic surface.

Figure 3. Quantitative analysis of mineral deposition to assess osteogenic differentiation. Cells from the Fibronectin mimetic surface successfully differentiated into osteogenic lineage as demonstrated by their mineralization ability. Uninduced controls did not show mineral deposition.

RFU

(49

2/52

0)

Quantification of Mineralization

Coating matrix Fibronectin matrix

4000

3000

2000

1000

0

Differentiated Undifferentiated

Figure 4. Qualitative analysis of adipogenesis by Oil Red O staining to assess adipogenic differentiation of MSCs. Cells were cultured on Fibronectin mimetic and human-origin coating matrix and differentiated to adipogenic lineage. Adipocytes exhibited staining of lipid vacuoles. Uninduced controls did not show staining.

Coating MatrixUninduced ControlFibronectin Mimetic

Conclusions• BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide supported

MSC attachment and growth for multiple passages in the StemPro® xeno-free and serum-free medium.

• The MSCs retained their characteristic marker profile after culture on the Fibronectin mimetic surface.

• The MSCs remained multipotent, and successfully differentiated into adipogenic and osteogenic lineages when cultured on the Fibronectin mimetic surface.

• BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide can be used for the culture of MSCs where a defined environment is desirable. This surface has been demonstrated to have ‘drop-in’ compatibility with commercially available media.2

• BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide is a versatile surface that is suitable for culture of other fibronectin-dependent cell types (including endothelial colony forming cells, as well as CHO and Vero cells)5,6 without the need for pre-adaptation.

References1. Pittenger MF, et al, Science 284:43-147 (1999).

2. Application note 494 - BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide: Novel Synthetic, Animal-free Surface for Culture of Human Bone Marrow-derived Mesenchymal Stem Cells.

3. Johansson S, et al, Frontiers in Bioscience 2:d126-146 (1997).

4. Dominici M, et al, Cytotherapy 8:315-317 (2006).

5. Application note 492 - BD PureCoat™ ECM Mimetic Cultureware: Novel Synthetic, Animal-free Surfaces for Human Endothelial Colony Forming Cell Expansion.

6. Application note 495 - BD PureCoatTM ECM Mimetic Cultureware: Animal-free, Synthetic Surfaces for Serum-free Culture of Adherent Cells.

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October 2012



BD Biosciences296 Concord RoadBillerica, MA 01821 USATel: [email protected]/bdbiosciences

CELLstart, CTS, StemPro, TrypLE™ are trademarks of Life Technologies, Inc.

OsteoImage is a trademark of Lonza Group Ltd.

Vi-CELL is a trademark of Beckman Coulter, Inc.

BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2012 BD

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