BD Biosciences October 2012

Application Note 496

Page 1

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

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 Saxena
BD Biosciences, Two Oak Park, Bedford, MA 01730 US

Application Note
Contents 1 Introduction 2 4 Materials and Methods Results and Discussion

Introduction
Mesenchymal 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.

7 Conclusions

7 References

PA-1503). StemPro Adipogenesis Differentiation Kit (Life Technologies Cat. MSC immunophenotypic analysis was performed using the BD Stemflow™ hMSC Analysis Kit (BD Cat. No. No. Cells were cultured in StemPro® MSC SFM Xeno-free (Life Technologies Cat. counted using the Vi-CELL™ cell counter (Beckman Coulter) and seeded at a density of 5. BD Falcon™ 6-well cell culture plates (BD Cat. PT-2501 A10070-01) was used for differentiation into Adipogenic lineage. supernatant was removed. No. 356240). a dissociation solution (Life Technologies Cat. Cell culture media. No.BD Biosciences October 2012 Application Note 496 Page 2 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 Materials and Methods Reagents Human bone marrow-derived MSCs were purchased from Lonza (Cat. Cell suspension was centrifuged at 200 x g for 5 min. Life Technologies. No. Paraformaldehyde 16% solution (EMS Cat. Passaging was performed following instruction from the media kit. Adipocytes were stained with Oil Red O (Sigma O-0625). resuspended in culture medium.000 cells/cm 2 in 2 mL culture medium/well in 6-well plate. No. No. MSC Culture BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide is pre-coated and ready to use. PT-2501). The remaining cells were recovered by rinsing each well with 1 mL culture medium. No. 353224) coated with CELLstart™ CTS™ substrate (Life Technologies Cat. 562245). and the cells were resuspended in culture medium. Differentiation into Osteogenic lineage was performed using StemPro Osteogenesis Differentiation Kit (Life Technologies Cat. PT-2501 A10072-01). 352097). 1 mL culture medium was added to each well. culture media was aspirated and cells were washed once with DPBS and 0. dissociation reagents and differentiation kits were purchased from Gibco. cells were examined under the microscope. No. BD Falcon 6-well cell culture plates were coated with human-origin matrix as per supplier’s instructions. No. 12859). Cells were passaged using TrypLE™ Select CTS reagent. when cells were detached from the surface. A10675-01) on BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide 6-well plate (BD Cat. A10142) served as a control and is also referred to as coating matrix. 15710) was diluted to 4% with 1 X Dulbecco Phosphate Buffer Saline (DPBS). Cells were then transferred to a polystyrene BD Falcon tube (BD Cat. MSCs were thawed from the frozen stock and spun at 200 x g for 5 minutes. Mineralization was quantified with OsteoImage™ Mineralization Assay Kit (Lonza Cat. Cells were incubated in a humidified incubator at 37ºC with 5% CO2. Cells were counted and seeded again for expansion. No. Briefly. No. Cells were passaged once they reached ~60-80% confluence.5 mL dissociation solution was added to each well (6-well format). . as per supplier’s instructions.

3-0.BD Biosciences October 2012 Application Note 496 Page 3 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 Materials and Methods (continued) MSC Differentiation and Analysis After 5 passages on mimetic or coating matrix surfaces.01 mL cell suspension was transferred to each tube and antibodies were added. CD34.5 mL of the same buffer. CD19. CD105. Cells were analyzed using a BD FACSCalibur™ flow cytometer. CD73. cells were differentiated into osteogenic and adipogenic lineages following the protocol from differentiation kits. and then resuspended in 0. washed with PBS with 10% fetal bovine serum and resuspended in PBS/bovine serum at a concentration of 5x106 cells/mL. 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. MSCs were induced to osteogenic lineage for 9 days and mineral deposition was quantified by a fluorescence based detection method following supplier’s instructions. Uninduced controls were maintained in MSC culture medium. Cells were washed twice with PBS containing 10% fetal bovine serum.0 software. For staining. and data were analyzed using BD CellQuest™ 3. . Adipogenic differentiation was performed for 1 week. Cells differentiated to adipogenic lineage were fixed and stained with Oil Red O following supplier’s instructions. CD45 and HLA-DR. Cells were dissociated using TrypLE Select CTS. Tubes were incubated in the dark for 30 minutes on ice. 0. CD11b. Cells were stained with antibodies CD90.

demonstrating that Fibronectin mimetic cultureware supported good attachment and expansion of MSCs for multiple passages and suitability for MSC culture. Cell passaging was based on confluence. Note comparable morphology on both surfaces. 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. Expansion of MSCs on both surfaces is comparable. matrix-coated plates. Figure 1. Cumulative population doubling of hMSCs on Fibronectin mimetic surface and human-origin coating matrix. Top row shows MSCs cultured on human-origin. After MSCs reached 60-80% confluence. Population doublings on the Fibronectin mimetic and human-origin coating matrix were comparable over a period of 20-21 days. HFN Mimetic Coating Matrix Culture Condition Coating matrix Fibronectin matrix Days in Culture 20 21 Cumulative Population Doubling 15.63 16. Phase contrast images of human bone marrow-derived MSCs at different passages in a defined and xeno-free medium. Passage 1 Passage 4 Table 1.64 . 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). cells were passaged.BD Biosciences October 2012 Application Note 496 Page 4 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 Results and Discussion MSC Culture and Population Doubling MSCs from the frozen stock were cultured in serum-free and xeno-free culture medium. Bottom row represents images from the BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide.

and HLA DR (negative cocktail PE). CD11b. CD11b. CD45 and HLA-DR. cells were collected by enzymatic dissociation.9% 99. CD105. Immunophenotyping analysis captured in Figure 2 demonstrated that the MSC population was positive for CD90.9% 40 40 40 0 0 0 100 101 102 CD90 FITC 103 104 100 101 102 103 CD105 PerCP-Cy5. and CD73 markers and negative for CD34. MSCs cultured on Fibronectin mimetic surface exhibited the ISCTestablished marker profile. Figure 2.BD Biosciences October 2012 Application Note 496 Page 5 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 Analysis of MSC Markers by Flow Cytometry After MSCs were cultured on Fibronectin mimetic surface for 5 passages.5 104 100 101 102 CD73 APC 103 104 0 100 40 Counts 80 120 160 200 101 102 103 negative cocktail PE 104 200 200 200 Coating Matrix Counts 80 120 160 Counts 80 120 160 Counts 80 120 160 99. CD19.9% 40 40 40 0 0 0 100 101 102 CD90 FITC 103 104 100 101 102 103 CD105 PerCP-Cy5.8% 97. CD45.8% 97. 200 200 200 Fibronectin Mimetic Counts 80 120 160 Counts 80 120 160 Counts 80 120 160 99.5 104 100 101 102 CD73 APC 103 104 0 100 40 Counts 80 120 160 200 101 102 103 negative cocktail PE 104 . Immunophenotyping of MSCs after 3 passages on human-origin coating matrix or Fibronectin mimetic surfaces. and CD105. Cells exhibited the ISCT established marker profile with >95% MSC population expressing CD73.9% 99. A similar marker profile was exhibited by cells cultured on the human-origin matrix-coated surface. and lacking expression of CD34. stained for established MSC markers using the respective antibodies. CD19. and then analyzed by flow cytometry. CD90.

No bone forming activity was detected in uninduced controls. 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. cells differentiated after culture on the Fibronectin mimetic surface exhibited mineral deposits comparable to cultures on human-origin coating matrix. which exhibit red staining with Oil Red O (Figure 4). Uninduced controls did not show mineral deposition. Qualitative analysis of adipogenesis by Oil Red O staining to assess adipogenic differentiation of MSCs. Uninduced cells did not develop lipid vacuoles. As shown in Figure 3. This experiment has demonstrated that MSCs remained multipotent and successfully differentiated into osteogenic lineage when cultured on the Fibronectin mimetic surface. Figure 4. Post-differentiation.BD Biosciences October 2012 Application Note 496 Page 6 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 Differentiation to Osteogenic Lineage After 5 passages. Postdifferentiation. Quantification of Mineralization 4000 RFU (492/520) 3000 2000 1000 0 Coating matrix Fibronectin matrix Differentiated Undifferentiated Differentiation to Adipogenic Lineage Adipogenesis was induced after 5 passages as described in Materials and Methods. Uninduced controls did not show staining. Adipocytes exhibited staining of lipid vacuoles. multipotent MSCs were successfully differentiated into adipogenic lineage after multiple passages on the Fibronectin mimetic surface. Fibronectin Mimetic Uninduced Control Coating Matrix . MSCs differentiated into adipocytes and deposited lipid vacuoles. Cells were cultured on Fibronectin mimetic and human-origin coating matrix and differentiated to adipogenic lineage. cells were fixed and stained with Oil Red O. MSCs were differentiated into osteogenic lineage. Figure 3. mineralization by osteocytes was also quantified by a fluorescence-based staining method that detects the hydroxyapatite portion of the mineralized bone matrix. Thus.

5.2772 labware@bd. et al. Cytotherapy 8:315-317 (2006). Pittenger MF. Application note 492 . • • • • References 1.6 without the need for pre-adaptation. Johansson S. Synthetic Surfaces for Serum-free Culture of Adherent Cells.com/bdbiosciences . TrypLE™ are trademarks of Life Technologies.BD PureCoat™ ECM Mimetic Cultureware: Novel Synthetic. Application note 494 . and successfully differentiated into adipogenic and osteogenic lineages when cultured on the Fibronectin mimetic surface. The MSCs remained multipotent. Vi-CELL is a trademark of Beckman Coulter. Dominici M. 4.2 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. The MSCs retained their characteristic marker profile after culture on the Fibronectin mimetic surface. © 2012 BD A12P038 BD Biosciences 296 Concord Road Billerica. Inc. 6.BD PureCoatTM ECM Mimetic Cultureware: Animal-free. Science 284:43-147 (1999). StemPro. Animal-free Surfaces for Human Endothelial Colony Forming Cell Expansion.com bd. Animal-free Surface for Culture of Human Bone Marrow-derived Mesenchymal Stem Cells. MA 01821 USA Tel: 855. 3. et al. as well as CHO and Vero cells)5. Application note 495 . Frontiers in Bioscience 2:d126-146 (1997). Dickinson and Company. BD. This surface has been demonstrated to have ‘drop-in’ compatibility with commercially available media. BD PureCoat ECM Mimetic Cultureware Fibronectin Peptide can be used for the culture of MSCs where a defined environment is desirable. BD Logo and all other trademarks are property of Becton. CELLstart.BD PureCoat™ ECM Mimetic Cultureware Fibronectin Peptide: Novel Synthetic. et al.236. OsteoImage is a trademark of Lonza Group Ltd. 2. Inc.BD Biosciences October 2012 Application Note 496 Page 7 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 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. CTS.

Sign up to vote on this title
UsefulNot useful

Master Your Semester with Scribd & The New York Times

Special offer: Get 4 months of Scribd and The New York Times for just $1.87 per week!

Master Your Semester with a Special Offer from Scribd & The New York Times