Mesenchymal Stem/Stromal Cells

Transcription

1 Mesenchymal Stem/Stromal Cells

2 ABOUT US Irvine Scientific, a member of JX Holdings group, is a worldwide leader in the design, manufacture and distribution of medical devices, including Cell Therapy, Industrial Cell Culture, Cytogenetic and Assisted Reproductive Technology products. We are a large scale producer of advanced quality cell culture media for the cell therapy, industrial bioprocess, medical and diagnostic markets. Our company s extensive experience in the design of culture media, compliance with ISO and FDA regulations for class II/III medical devices and industrial scale manufacturing capacity provides our customers with unique capabilities and support. Irvine Scientific delivers products worldwide to biopharmaceutical industry, research and medical laboratory communities. From our expertise accumulated for more than 40 years, we have steadily advanced as a global supplier of cell therapy media and ancillary reagents. We understand the importance of having an integrated workflow solution and pre-validated products when working with primary cells. Therefore, Irvine Scientific is proud to incorporate our cell therapy PRIME-XV product portfolio as part of our company s commitment to accelerate basic research and clinical applications in cell therapy and regenerative medicine Irvine Scientific

3 TABLE OF CONTENTS PRIME-XV Attachment Substrates PRIME-XV MSC EXPANSION SFM PRIME-XV Biopreservation Solutions PROTOCOLS MSC Expansion Coating Thawing Subculturing Biopreservation Hypothermic Cryogenic Analysis Immune Modulation MSC Isolation Adipose Tissue Cord Blood Umbilical Cord Irvine Scientific

4 PRIME-XV Attachment Substrates PRIME-XV MatrIS F Recombinant Human Matrix Protein Catalog # FEATURES & BENEFITS Proprietary attachment substrate for the culture of human stem/progenitor cells under serum-free conditions Recombinant human matrix protein to ensure lot-to-lot consistency Available in 200µg lyophilized packaging PRIME-XV Human Fibronectin Human Plasma-Derived Fibronectin, Carrier Free Catalog # FEATURES & BENEFITS Validated for use in a variety of primary cell attachment and spreading applications Carrier free Available in 1mg packaging Figure 1 PRIME-XV MatrIS F supports human bone marrow-derived mesenchymal stromal/stem cells (MSCs). Image was taken at 10X magnification. Figure 2 Human neural progenitor cells plated on PRIME-XV Human Fibronectin substrate retained NESTIN expression (red). Nuclei were counterstained with DAPI (blue) Irvine Scientific

5 PRIME-XV MSC EXPANSION SFM Human Mesenchymal Stromal/Stem Cell (MSC) Expansion Serum-Free Medium Catalog # FEATURES & BENEFITS Outperforms leading competitors and serum-containing media in expansion while maintaining MSC characteristics and multipotency Supports cell expansion of MSCs derived from different tissue sources Little to no adaptation required from serum-containing medium Available in one 250mL complete component and ready-to-use Manufactured under cgmp conditions Figure 3 Phenotypic morphology of human bone marrow-derived MSCs after prolonged passaging in PRIME-XV MSC EXPANSION SFM as compared to control, serum-containing medium. Images were taken at 10X magnification. Figure 4 Human bone marrow-derived MSCs (A) and adipose-derived MSCs (B) grown in PRIME-XV MSC EXPANSION SFM showed an efficient increase in cell expansion above the control, serum-containing medium over three passages. Fold increase was calculated as the ratio of final viable cell count by the initial seeded viable cell count at passage 1. Standard deviation was calculated as the square root of the variance among replicates Irvine Scientific 2

6 PRIME-XV MSC EXPANSION SFM Figure 6 Multi-lineage differentiation potential was retained in human bone marrow-derived MSCs cultured in PRIME-XV MSC EXPANSION SFM. Immunofluorescence analysis of FABP-4 (A), OSTEOCALCIN (B) and AGGRECAN (C) represent the adipogenic, osteogenic and chrondrogenic lineages, respectively. Nuclei were counterstained with DAPI (blue). Figure 5 PRIME-XV MSC EXPANSION SFM supported maximal human MSC expansion, compared to leading competitors media (A). Flow cytometry analysis of human bone marrow-derived MSCs propagated in PRIME-XV MSC EXPANSION SFM were positive for CD105 and CD90 cell surface markers but lacked CD45 expression (B) Irvine Scientific Figure 7 Immunosuppressive potential of human bone marrow-derived MSCs expanded in PRIME-XV MSC EXPANSION SFM. Cell proliferation assay was performed using CFSE-labeled human peripheral blood mononuclear cell (PBMC) derived CD3 + cells stimulated with phytohemagglutinin. Non-activated CD3 + cells without MSC co-culture were arrested at the parent generation (A). Activated CD3 + cells proliferated for 3 days without MSC co-culture (B). Proliferation of CD3 + cells stimulated with phytohemagglutinin was suppressed when co-cultured with MSCs expanded in PRIME-XV MSC EXPANSION SFM (C) or cultured with MSCs expanded in serum-containing medium (D).

7 PRIME-XV Biopreservation Solutions PRIME-XV Hypothermic Preservation Solution Protein-Free, Defined Hypothermic Preservation Solution for Cells and Tissues Catalog # FEATURES & BENEFITS Animal component-free, protein-free solution Supports storage and shipping stability for biologics under hypothermic (2 8 C) conditions Reduces cellular stress response from chilling and re-warming of cells and tissues Provides ph buffering, osmotic/oncotic support and energy substrates to balance intracellular states at low temperatures No additional components are needed Manufactured under cgmp conditions Available in 10mL packaging Figure 9 Human bone marrow-derived MSCs before storage (A) and five days (B) under hypothermic storage. Cells imaged 24 hours post recovery (C). Figure 8 Flow cytometry analysis of human bone marrow-derived MSCs before and five days after hypothermic preservation using PRIME-XV Hypothermic Preservation Solution. Figure 10 Human bone marrow-derived MSCs remained highly viable after five days preserved using PRIME-XV Hypothermic Preservation Solution. Cell viability was calculated 24 hours post recovery Irvine Scientific 4

8 PRIME-XV Biopreservation Solutions PRIME-XV Cryogenic Preservation Solution Protein-Free, Defined Cryogenic Preservation Solution for Cells and Tissues Catalog # FEATURES & BENEFITS Animal component-free, protein-free solution Enables a variety of cell types to preserve at -80 C to -196 C temperature environments Reduces post-preservation necrosis and apoptosis compared to commercial and home-brew isotonic formulations No additional components are needed Manufactured under cgmp conditions Available in 10mL packaging Figure 11 Human bone marrow-derived MSCs had high plating efficiency (A) and viability (B) after cryogenic preservation in PRIME-XV Cryogenic Preservation Solution. Control solution represents growth medium + 10% DMSO Irvine Scientific

9 PROTOCOLS MSC Expansion Coating Thawing Subculturing Biopreservation Analysis MSC Isolation Hypothermic Cryogenic Immune Modulation Adipose Tissue Cord Blood Umbilical Cord The following protocols are optimized for the culture and processing of human MSCs derived from bone marrow using the following reagents: 2013 Irvine Scientific 6

10 MSC Expansion Coating Attachment matrix substrates recommended for optimal consistency: PRIME-XV MatrIS F Catalog # Note: Reconstitute PRIME-XV MatrIS F to 100µg/mL stock solution in 1X Dulbecco s Phosphate Buffered Saline (PBS) (Irvine Scientific, Catalog # 9236) before use. OR PRIME-XV Human Fibronectin Catalog # Prepare a 5µg/mL working solution in PBS (Irvine Scientific, Catalog # 9236) of PRIME-XV MatrIS F or PRIME-XV Human Fibronectin. Add the diluted product solution to culture vessel at a final volume per surface area of 0.08mL/cm 2. Refer to the table for respective culture vessel: 3. Incubate culture vessels at room temperature for 3 hours or overnight at 2 8 C. The culture vessel must be sealed with Parafilm to avoid drying if stored at 2 8 C overnight. It is recommended to coat culture vessels the day of use or the day before use. 4. Aspirate out and discard diluted solution from culture vessels before the addition of cells Irvine Scientific Parafilm is a registered trademark of American Can Company.

11 Thawing Pre-coat the tissue culture vessel with 5µg/mL of PRIME-XV MatrIS F or PRIME-XV Human Fibronectin for 3 hours at room temperature or overnight at 2 8 C. See Coating Procedure on Page 7. Pre-warm PRIME-XV MSC EXPANSION SFM (Irvine Scientific, Catalog # 91135) to 37 C for no more than 30 minutes. Repeated warming of medium may reduce product performance. Rapidly thaw frozen vial of cells in a 37 C water bath. Sample should be thawed with gentle swirling until all visible ice has melted. Thaw time for a 1mL sample in a cryovial is approximately 3 minutes. Note: DO NOT allow sample to warm above chilled temperatures (0 10 C). Cryovials should be cool to the touch when removed from the water bath. Pipet the entire content of the cryovial into a 15mL conical tube. Carefully add 5 10mL of pre-warmed PRIME-XV MSC EXPANSION SFM at an approximate rate of 3 5 drops per 10 seconds and gently swirl after each addition. Transfer the entire content of the conical tube into a pre-coated tissue culture vessel. Incubate the cells at 37 C, 5% CO 2 incubator. Aspirate off media and feed the cells with pre-warmed PRIME-XV MSC EXPANSION SFM 24 hours post-thaw. Remove and discard spent media every two days, and feed cells with pre-warmed PRIME-XV MSC EXPANSION SFM. Subculture when cells reach 80 90% confluence. Do not allow the cultures to become completely or over confluent. Subculturing under suboptimal conditions may affect product performance Irvine Scientific 8

12 MSC Expansion Subculturing Pre-coat the tissue culture vessel with 5µg/mL of PRIME-XV MatrIS F or PRIME-XV Human Fibronectin for 3 hours at room temperature or overnight at 2 8 C. See Coating Procedure on Page 7. Pre-warm PRIME-XV MSC EXPANSION SFM (Irvine Scientific, Catalog # 91135) to 37 C for no more than 30 minutes. Repeated warming of medium may reduce product performance. Remove spent media from T-75 flask culture and gently rinse cells once with 10mL of PBS (Irvine Scientific, Catalog # 9240) for each T-75 flask. Add 3mL of room temperature TrypLE Express to each T-75 flask, and tilt the flask in all directions to disperse the TrypLE Express evenly over the cells. Incubate the cells at 37 C, 5% CO 2 incubator. Monitoring periodically for cell detachment by observing the cells under the microscope. Cells will start to round and detach. Tap the side of the flask to aid the detachment of the cells and return culture to the incubator. Repeat the above process until at least 90% of cells are fully detached. This process takes approximately 5 10 minutes. Add 5mL of PRIME-XV MSC EXPANSION SFM to the flask. Disperse the cells by pipetting the media over the entire growing surface of the flask, and transfer the contents to a 15mL conical tube. Centrifuge cells down at 400xg for 5 minutes. Aspirate off supernatant. Resuspend the cell pellet in a small amount of pre-warmed PRIME-XV MSC EXPANSION SFM and count the cells with a cell counter. Resuspend x 10 5 cells into 20mL of pre-warmed PRIME-XV MSC EXPANSION SFM for each pre-coated T-75 flask. Note: It is recommended to seed cells at approximately 6,000 cells/cm 2 in 0.3mL of media for 2-dimensional pre-coated culture vessels. Gently aspirate off PRIME-XV attachment substrate solution from the flask and slowly add the cell suspension to a T-75 flask. Avoid scraping the coated surface when aspirating. Incubate the cells at 37 C, 5% CO 2 incubator. Remove and discard spent media. Feed the cells with pre-warmed PRIME-XV MSC EXPANSION SFM every 2 days Irvine Scientific TrypLE is a registered trademark of Life Technologies.

13 Biopreservation Hypothermic Preservation Cryogenic Preservation Replace culture medium with chilled (2 10 C) PRIME-XV Hypothermic Preservation Solution and maintain at 2 8 C. At the end of cold storage period, simply remove samples from cold environment, discard the cold PRIME-XV Hypothermic Preservation Solution. Replace with pre-warmed (20 37 C) culture medium of choice to recover Prepare cell suspension using cell specific protocol (mechanical or enzymatic dissociation) and centrifuge cells appropriately to obtain a cell pellet. Methods for generating a cell suspension should be determined by individual users. Remove supernatant as much as possible to reduce diluting PRIME-XV Cryogenic Preservation Solution. Add cold (2 8 C) PRIME-XV Cryogenic Preservation Solution to a cell concentration range of cells/ml for standard cell culture protocols. A higher cell concentration is possible, if established by end users. Transfer cells to cryovials. 4. Incubate cell suspension at 2 8 C for approximately 10 minutes. 5. Lower sample temperature to -80 C using a controlled rate freezer (-1 C/minute) or similar procedure for most mammalian cell systems. Note: Freezing device or isopropanol container should be pre-cooled to 2 8 C. Freeze time (-80 C) using isopropanol containers is recommended to be 3 4 hours. Sample storage at -80 C is only recommended for short-term storage up to weeks or months. 6. Transfer samples to liquid nitrogen for long term cryopreservation (below -130 C) Irvine Scientific 10

14 MSC Analysis MSC Immune Modulation on T cells Thaw and seed MSCs at 3,000 cells/cm 2 in six-well plates. See Thawing Procedure on Page 8. Allow MSCs to reach 80% confluence before adding the peripheral blood mononuclear cells (PBMCs) (approximately 2 3 days). MSCs should be approximately 500,000 cells per well before proceeding. Pre-warm complete RPMI Medium 1640 (Irvine Scientific, Catalog # 9161) containing 10% fetal bovine serum (MSC qualified) to 37 C for no more than 30 minutes. Thaw PBMCs according to vendor s instructions. Incubate cells at 37 C, 5% CO 2 for 3 hours to recover. After recovery, stain PBMCs with CellTrace CFSE according to manufacturer s instructions. After staining, wash the PBMCs three times using fresh, room temperature complete RMPI medium. On the last wash, resuspend the PBMC cell pellet in minimal volume of complete RMPI medium. Based on MSC cell counts, co-culture labeled PBMCs with MSCs in a six-well plate in a 1:10 ratio (MSC:PBMCs). Add phytohemagglutinin to a final concentration of 20µg/mL in a total volume of 4mL complete RPMI per well. Prepare a negative control sample by plating 250,000 PBMCs per well in a six-well plate with 4mL complete RPMI medium. Prepare a positive control sample by plating 250,000 PBMCs per well in a six-well plate with 4mL complete RPMI medium supplemented with a final concentration of 20µg/mL phytohemagglutinin. On day three and day five of co-culture, PBMC derived CD3 + cell proliferation can be measured by using a flow cytometer with 488nm excitation and emission filters appropriate for fluorescein Irvine Scientific CellTrace is a registered trademark of Life Technologies.

15 MSC Isolation Adipose Tissue Rapid Isolation The following protocol was simplified and adapted from Organogenesis 6:11 14, 2010 and has not been validated by Irvine Scientific. For reference use only Aspirate blood/saline into a 50mL conical tube. Centrifuge at 400xg for 10 minutes at room temperature. Resuspend pellet in 160mM NH 4 Cl for 5 minutes at room temperature. Centrifuge at 400xg for 10 minutes at room temperature. Remove supernatant and resuspend pellet in 40% FBS/DMEM and plate. Incubate at 37 C, 5% CO 2 incubator overnight Irvine Scientific 12

16 MSC Isolation Adipose Tissue Standard Isolation The following protocol was simplified and adapted from Organogenesis 6:11 14, 2010 and has not been validated by Irvine Scientific. For reference use only Aspirate off saline and oil phases. Wash ~250mL of fat 3 5 times for 5 minutes in PBS each wash, discarding lower phase until clear. Add collagenase and incubate 1 4 hours at 37 C on a shaker. Add 10% FBS to neutralize collagenase. Centrifuge digested fat at 800xg for 10 minutes. Aspirate floating adipocytes, lipids and liquid, leaving stromal vascular fraction (SVF) pellet. Resuspend SVF pellet in 160mM NH 4 Cl and incubate for 10 minutes at room temperature. Centrifuge at 400xg for 10 minutes at room temperature. Layer cells on Percoll or Histopaque gradient. Centrifuge at 1000xg for 30 minutes at room temperature. Wash cells twice with PBS and centrifuge at 400xg for 10 minutes between each wash. Resuspend cell pellet in PBS and filter cells through 100µM nylon mesh. Pass cells through 40µM mesh. Centrifuge at 400xg for 10 minutes. Resuspend cell pellet in 40% FBS/DMEM and plate. Incubate at 37 C, 5% CO 2 incubator overnight Irvine Scientific

17 Cord Blood The following protocol was simplified and adapted from Stem Cells 26: , 2008 and has not been validated by Irvine Scientific. For reference use only Dilute cord blood with RPMI Medium 1640 with a 3:1 ratio (3 parts cord blood to one part RPMI). Isolate mononuclear cells (MNCs) by density gradient centrifugation at 400g for 30 minutes at room temperature using Ficoll-Paque Premium according to the manufacturer s instructions. Transfer MNCs to new centrifuge tube and add PBS with a 1:3 ratio (1 part MNCs to 3 parts PBS). Centrifuge at 400xg for 10 minutes at room temperature. Remove supernatant and resuspend cells by adding culture medium and plate. Incubate at 37 C, 5% CO 2 overnight. Ficoll-Paque is a registered trademark of GE Healthcare Irvine Scientific 14

18 MSC Isolation Umbilical Cord The following protocol was simplified and adapted from Stem Cells 26: , 2008 and has not been validated by Irvine Scientific. For reference use only Wash umbilical cords (UCs) in a hypochlorite solution (1:3). Rinse UCs with PBS. Store UCs in 10% FBS/DMEM-low glucose for up to 12 hours. Wash UCs three times with PBS. Inject vein and arteries with 3mL 0.1% collagenase in PBS. Incubate for 20 minutes at 37 C. Inject 5mL DMEM-low glucose with 10% FBS. Harvest cells by massaging the cord tissue. Centrifuge at 300xg for 10 minutes at room temperature. Remove supernatant, add culture medium and plate. Incubate at 37 C, 5% CO 2 overnight Irvine Scientific

19 2013 Irvine Scientific. All rights reserved. For research or further manufacturing use only. Not intended for any diagnostic or therapeutic use in humans or animals. All trademarks mentioned herein are the property of Irvine Scientific or their respective owners.

20 2013 Irvine Scientific P/N 10417CT Rev. 1