Mesenchymal Stem/Stromal Cells - Fujifilmisj.fujifilm.co.jp/products/regenerative-medicine/pdf/... · 2019-03-31 · Mesenchymal Stem/Stromal Cells Irvine Scientific, a member of

Mesenchymal Stem/Stromal Cells

®

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 commient to accelerate basic research and clinical applications in cell therapy and regenerative medicine.

ABOUT US

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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

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© 2014 Irvine Scientific www.irvinesci.com

PRIME-XV MatrIS FRecombinant Human Matrix ProteinCatalog # 31001

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 FibronectinHuman Plasma-Derived Fibronectin, Carrier FreeCatalog # 31002

FEATURES & BENEFITS• Validated for use in a variety of primary cell attachment and spreading applications• Carrier free

• Available in 1mg packaging

Figure 1PRIME-XV MatrIS F supports human bone marrow-derived mesenchymal stromal/stem cells (MSCs).Image was taken at 10X magnification.

Figure 2Human neural progenitor cells plated on PRIME-XV Human Fibronectin substrate retained NESTIN expres-sion (red). Nuclei were counterstained with DAPI (blue).

1 www.irvinesci.com © 2014 Irvine Scientific

PRIME-XV Attachment Substrates

Human Mesenchymal Stromal/Stem Cell (MSC)Expansion Serum-Free MediumCatalog # 91135

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 3Phenotypic 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 4Human 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.


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Passage Number

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PRIME-XV MSC Expansion SFM Serum-containing medium

PRIME-XV MSC Expansion SFM Serum-containing medium

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2 © 2014 Irvine Scientific www.irvinesci.com

Figure 5PRIME-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 lackedCD45 expression (B).

Figure 7 Immunosuppressive potential of human bone marrow-derived MSCs expanded in PRIME-XV MSC Expan-sion 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 wassuppressed when co-cultured with MSCs expanded in PRIME-XV MSC Expansion SFM (C) orcultured with MSCs expanded in serum-containing medium (D).

Figure 6Multi-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).

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Serum-containingmedium

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CD90 CD105 CD45

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PRIME-XV MSCExpansion SFM

Serum-containing Competitor 1 Competitor 2

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Isotype control Anti-CD staining

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CFSE Florescence

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PRIME-XV Hypothermic Preservation SolutionProtein-Free, Defined Hypothermic PreservationSolution for Cells and TissuesCatalog # 31003FEATURES & 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 10Human 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.

Figure 9Human bone marrow-derived MSCs before storage (A) and five days (B) under hypothermic storage. Cells imaged 24 hours post recovery (C).

Figure 8Flow cytometry analysis of human bone marrow-derived MSCs before and five days after hypothermic preservation using PRIME-XV Hypothermic Preservation Solution.


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Before hypothermic preservation

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Percent ViabilityIsotype control Anti-CD staining

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PRIME-XV Cryogenic Preservation SolutionProtein-Free, Defined Cryogenic PreservationSolution for Cells and Tissues Catalog # 31004FEATURES & 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 11Human 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.

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Before cryogenic preservation 24 hours after cryogenic preservation

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Control Solution

PRIME-XV Cryogenic Preservation Solution

Percent Viability

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The following protocols are optimized for the culture and processing of human MSCs derived from bone marrow using the following reagents:


PROTOCOLSMSC Expansion

Coating Thawing Subculturing

Biopreservation

Hypothermic Cryogenic

Analysis

Immune Modulation

MSC Isolation

Adipose Tissue Cord Blood Umbilical Cord

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PRIME-XV MSC Expansion SFM 91135 250mL

PRIME-XV MatrIS F 31001 200µg

PRIME-XV Human Fibronectin 31002 1mg

PRIME-XV Hypothermic Preservation Solution 31003 10mL

PRIME-XV Cryogenic Preservation Solution 31004 10mL

PBS with Ca2+ Mg2+ 9236 500mL

PBS without Ca2+ Mg2+ 9240 500mL

RPMI 9161 500mL

Product Catalog # Size

Coating

Attachment matrix substrates recommended for optimal consistency:

PRIME-XV MatrIS F Catalog # 31001Note: Reconstitute PRIME-XV MatrIS F to 100µg/mL stock solution in1X Dulbecco’s Phosphate Buffered Saline (PBS) (Irvine Scientific, Catalog # 9236)before use.

OR

PRIME-XV Human FibronectinCatalog # 31002

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MSC Expansion

6-well plate 9.6 768µL per well

T-25 flask 25 2mL per flask

T-75 flask 75 6mL per flask

Culture Vessel Surface Area(cm2)

Volume of 5µg/mL PRIME-XV Attachment Substrate

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/cm2. Refer to the table for respective culture vessel:

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 daybefore use.

Aspirate out and discard diluted solution from culture vessels before the addition of cells.

Parafilm® is a registered trademark of American Can Company.

Thawing

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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 overnightat 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 mediummay 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% CO2 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.

Subculturing

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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 overnightat 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 theflask in all directions to disperse the TrypLE Express evenly over the cells.

Incubate the cells at 37°C, 5% CO2 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 arefully 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 4.5–5.0 x 105 cells into 20mL of pre-warmed PRIME-XV MSC Expansion SFM for each pre-coated T-75 flask. Note: It is recommended toseed cells at approximately 6,000 cells/cm2 in 0.3mL of media for 2-dimensionalpre-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% CO2 incubator.

Remove and discard spent media. Feed the cells with pre-warmed PRIME-XVMSC Expansion SFM every 2 days.


MSC Expansion

TrypLE is a registered trademark of Life Technologies.

Hypothermic Preservation

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Replace culture medium with chilled (2–10°C) PRIME-XV HypothermicPreservation 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.

Cryogenic Preservation

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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 0.5–10 × 106 cells/mL for standard cell culture protocols.A higher cell concentration is possible, if established by end users. Transfer cells to cryovials.

Incubate cell suspension at 2–8°C for approximately 10 minutes.

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°Cis only recommended for short-term storage up to weeks or months.

Transfer samples to liquid nitrogen for long term cryopreservation (below -130°C).

Biopreservation


MSC Immune Modulation on T cells

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Thaw and seed MSCs at 3,000 cells/cm2 in six-well plates. See Thawing Procedureon Page 8.

Allow MSCs to reach 80% confluence before adding the peripheral blood mononuclear cells (PBMCs) (approximately 2–3 days). MSCs should beapproximately 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 than30 minutes.

Thaw PBMCs according to vendor’s instructions. Incubate cells at 37°C, 5% CO2 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 finalconcentration 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.

MSC Analysis

11 www.irvinesci.com © 2014 Irvine ScientificCellTrace is a registered trademark of Life Technologies.


Adipose TissueRapid 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.

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Aspirate blood/saline into a 50mL conical tube.

Centrifuge at 400xg for 10 minutes at room temperature.

Resuspend pellet in 160mM NH4Cl for 5 minutes at room temperature.


Remove supernatant and resuspend pellet in 40% FBS/DMEM and plate.

Incubate at 37°C, 5% CO2 incubator overnight.

MSC Isolation

Adipose TissueStandard Isolation

The following protocol was simplified and adapted from Organogenesis 6:11–14, 2010and has not been validated by Irvine Scientific. For reference use only.

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Aspirate off saline and oil phases.

Wash ~250mL of fat 3–5 times for 5 minutes in PBS each wash, discarding lowerphase 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 NH4Cl and incubate for 10 minutes at roomtemperature.


Layer cells on Percoll or Histopaque gradient.


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% CO2 incubator overnight.


MSC Isolation


Cord Blood

The following protocol was simplified and adapted from Stem Cells 26:146–150, 2008 and has not been validated by Irvine Scientific. For reference use only.

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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 for30 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).


Remove supernatant and resuspend cells by adding culture medium and plate.

Incubate at 37°C, 5% CO2 overnight.

Ficoll-Paque is a registered trademark of GE Healthcare.

Umbilical Cord

The following protocol was simplified and adapted from Stem Cells 26:146–150, 2008 and has not been validated by Irvine Scientific. For reference use only.

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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.


Remove supernatant, add culture medium and plate.

Incubate at 37°C, 5% CO2 overnight.


MSC Isolation

© 2014 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.

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Documents

Mesenchymal Stem/Stromal Cells - Fujifilmisj.fujifilm.co.jp/products/regenerative-medicine/pdf/... · 2019-03-31 · Mesenchymal Stem/Stromal Cells Irvine Scientific, a member of