Summary of Study ST002469

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001595. The data can be accessed directly via it's Project DOI: 10.21228/M8J13B This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002469
Study TitleMesenchymal stromal cell (MSC) Metabolite MS study
Study TypeUntargeted Metabolite Study
Study SummaryMetabolomics and lipidomics workflows were used to analyze Mesenchymal stromal cell (MSC) metabolites. Metabolite abundances were used to model MSC potency results in IDO and T-cell proliferation assays.
Institute
Georgia Institute of Technology
DepartmentChemistry and Biochemistry
LaboratoryFernandez Lab
Last NameVan Grouw
First NameAlexandria
Address311 Ferst Dr. NW Atlanta, GA 30332
Emailagrouw3@gatech.edu
Phone7072391412
Submit Date2023-02-07
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2023-02-26
Release Version1
Alexandria Van Grouw Alexandria Van Grouw
https://dx.doi.org/10.21228/M8J13B
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001595
Project DOI:doi: 10.21228/M8J13B
Project Title:Development of a Robust Consensus Modeling Approach for Identifying Cellular and Media Metabolites Predictive of Mesenchymal Stromal Cell Potency
Project Type:MS Untargeted Analysis
Project Summary:Mesenchymal stromal cells (MSCs) have shown promise in regenerative medicine applications due in part to their ability to modulate immune cells, such as T cells. However, MSCs demonstrate significant functional heterogeneity in terms of their immunomodulatory function because of differences in MSC donor/tissue source, as well as non-standardized manufacturing approaches. As MSC metabolism plays a critical role in their ability to expand to therapeutic numbers ex vivo, we comprehensively profiled intracellular and extracellular metabolites throughout the expansion process to identify predictors of MSC immunomodulatory function (T cell modulation and indoleamine-2,3-dehydrogenase (IDO) activity). Here, we profiled media metabolites in a non-destructive manner through daily sampling and nuclear magnetic resonance (NMR), as well as MSC intracellular metabolites at the end of expansion using mass spectrometry (MS). Using a robust consensus machine learning approach, we were able to identify panels of metabolites predictive of MSC immunomodulatory function for 10 independent MSC lines.
Institute:Georgia Institute of Technology
Department:Chemistry and Biochemistry
Laboratory:Fernandez Lab
Last Name:Van Grouw
First Name:Alexandria
Address:311 Ferst Dr. NW Atlanta, GA 30332
Email:agrouw3@gatech.edu
Phone:7072391412
Funding Source:NSF Center for Cell Manufacturing Technologies

Subject:

Subject ID:SU002559
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Male and female
Cell Biosource Or Supplier:RoosterBio
Cell Strain Details:Mesenchymal Stromal Cells

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Media
SA247499alphaMEM_RB182Gibco MEM alpha
SA247500alphaMEM_iMSCGibco MEM alpha
SA247501alphaMEM_RB139Gibco MEM alpha
SA247502alphaMEM_RB174_02Gibco MEM alpha
SA247503alphaMEM_RB175Gibco MEM alpha
SA247504alphaMEM_RB71Gibco MEM alpha
SA247505alphaMEM_RB174_01Gibco MEM alpha
SA247506alphaMEM_RB179Gibco MEM alpha
SA247507alphaMEM_RB177Gibco MEM alpha
SA247508alphaMEM_RB183Gibco MEM alpha
SA247509RBio_RB175RoosterBasal-MSC media
SA247510RBio_RB182RoosterBasal-MSC media
SA247511RBio_iMSCRoosterBasal-MSC media
SA247512RBio_RB174_02RoosterBasal-MSC media
SA247513RBio_RB139RoosterBasal-MSC media
SA247514RBio_RB174_01RoosterBasal-MSC media
SA247515RBio_RB177RoosterBasal-MSC media
SA247516RBio_RB179RoosterBasal-MSC media
SA247517RBio_RB183RoosterBasal-MSC media
SA247518RBio_RB71RoosterBasal-MSC media
Showing results 1 to 20 of 20

Collection:

Collection ID:CO002552
Collection Summary:For intracellular lipidomic/metabolomic analysis, cell pellets were washed twice by resuspending in PBS and centrifuged at 10,000 rpm. All supernatant was removed and cell pellets were then stored at –80° C.
Sample Type:Bone marrow

Treatment:

Treatment ID:TR002571
Treatment Summary:Bone marrow-derived MSCs (BMMSCs) were purchased from RoosterBio, Inc. (Frederick, MD), and iMSCs were purchased from Fujifilm Cellular Dynamics Inc (Madison, WI). Prior to this study’s expansion, MSCs were previously expanded to an initial population doubling level (PDL0). Cryopreserved vials from each donor were thawed, and 106 MSCs were seeded into an initial T-150 tissue culture flask in complete media containing Gibco™ Minimum Essential Media α with nucleosides (Thermo Fisher Scientific, Waltham, MA), 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT), and 1% penicillin-streptomycin solution (10,000 U/mL; Sigma-Aldrich, St. Louis, MO) for a culture rescue period of 48 hr. The same lot of FBS was used throughout the study. MSCs were washed with endotoxin-free Dulbecco’s phosphate buffered saline (PBS) without calcium and magnesium (Millipore Sigma), harvested using 1X TrypLE™ Express Enzyme (Thermo Fisher Scientific), neutralized with complete media, and centrifuged 300g to create a cell pellet. MSCs were then resuspended in complete media and counted. Next, MSCs from each donor were seeded at 500 cells/cm2 into 10 T-75 flasks containing 10 mL complete media. Control flasks containing 10 mL complete media only were also prepared. All flasks were then transferred to a humidified incubator set to 37° C and 5% CO2. MSC conditioned medium (CM) sample collection of 300 µl was performed for each flask at approximately the same time each day (±1 hr) and total complete media exchange was performed every 3 days until MSCs achieved 70-80% confluence. All media samples were placed directly into –80° C storage until further analysis by NMR. MSCs were then harvested using the same procedure described above. Cell pellets were split for cryopreservation (and functional analysis, see below) or preparation for intracellular lipidomic/metabolic analysis. Cell pellets designated for cryopreservation were prepared into cryovials containing 106 MSCs in 1 mL CryoStor® CS 10 (Sigma-Aldrich) and stored at –80° C for 24 hr using controlled rate freezing containers. Vials were then transferred to the vapor phase in a liquid nitrogen cryogenic freezer until further analysis.

Sample Preparation:

Sampleprep ID:SP002565
Sampleprep Summary:Approximately one million MSCs were analyzed for each sample. Frozen cell pellets were thawed and washed prior to undergoing a modified Bligh-Dyer extraction to yield two phases. Extraction solvent (2:2:1 chloroform:methanol:water) and glass beads (400-600 µm) were added to cell pellets for extraction and homogenization in a TissueLyser II to 30 Hz for 6 minutes. Samples were then sonicated and centrifuged. Following extraction, 300 µL aliquots from each layer were transferred to new microcentrifuge tubes and solvent was dried using vacuum centrifugation. Dried organic phase samples were re-constituted in isopropyl alcohol, while dried aqueous phase samples were re-constituted in 80% methanol. Re-constitution was followed by sonication, centrifugation, and transfer to liquid chromatography (LC) vials for ultrahigh performance liquid chromatography mass spectrometry (UHPLC-MS) analysis. Media samples without cells were also analyzed as blanks to remove any features corresponding to remaining media components on the cells. Ten µL of media was subject to the same Bligh-Dyer extraction as above and extracts were run according to the instrumental methods listed above. A quality control (QC) sample for hydrophilic interaction chromatography (HILIC) and reverse phase datasets was created by pooling 20 µL from each experimental sample. The pooled QC injections were used for drift correction of peak areas. Sample queue was randomized with a mix of samples, QCs, and blanks.

Combined analysis:

Analysis ID AN004025 AN004026 AN004027 AN004028
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase HILIC HILIC
Chromatography system Q Exactive HF Hybrid Quadrupole-Orbitrap Q Exactive HF Hybrid Quadrupole-Orbitrap Orbitrap ID-X Tribrid mass spectrometer Orbitrap ID-X Tribrid mass spectrometer
Column AccucoreTM C30 column (2.1 × 150 mm, 2.6 µm particle size) AccucoreTM C30 column (2.1 × 150 mm, 2.6 µm particle size) Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um) Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap Thermo Orbitrap ID-X Tribrid Thermo Orbitrap ID-X Tribrid
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE
Units Peak Area Peak Area Peak Area Peak Area

Chromatography:

Chromatography ID:CH002975
Instrument Name:Q Exactive HF Hybrid Quadrupole-Orbitrap
Column Name:AccucoreTM C30 column (2.1 × 150 mm, 2.6 µm particle size)
Column Temperature:50
Flow Gradient:80% A 0-1min; 40% A 1-5min; 30% A 5-5.5min; 15% A 5.5-8min; 10% A 8-8.2min; 0% A 8.2-10.5 min; 80% A 10.7-12min
Flow Rate:0.4 mL/min
Solvent A:40% water/60% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Solvent B:10% acetonitrile/90% isopropyl alcohol; 10 mM ammonium formate; 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH002976
Instrument Name:Orbitrap ID-X Tribrid mass spectrometer
Column Name:Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:40
Flow Gradient:5% A 0-8 min; 60% A 8-10.4 min; 5% A 10.5-14min
Flow Rate:0.4 mL/min
Solvent A:80% water/20% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS003772
Analysis ID:AN004025
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mobile Phase A for Reverse Phase chromatography in positive mode was 40:60 water: acetonitrile with 10 mM ammonium formate and 0.1% formic acid and Mobile Phase B was 10:90 acetonitrile:isopropyl alcohol, with 10 mM ammonium formate and 0.1% formic acid.
Ion Mode:POSITIVE
  
MS ID:MS003773
Analysis ID:AN004026
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:For negative ionization mode, the mobile phases were 40:60 water:acetonitrile with 10 mM ammonium acetate (mobile phase A) and 10:90 acetonitrile:isopropyl alcohol, with 10 mM ammonium acetate (mobile phase B).
Ion Mode:NEGATIVE
  
MS ID:MS003774
Analysis ID:AN004027
Instrument Name:Thermo Orbitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:​Mobile Phase A used for HILIC chromatography was 80:20 water:MeCN with 10 mM ammonium formate and 0.1% formic acid. Mobile phase B for HILIC chromatography was acetonitrile with 0.1% formic acid. The flow rate was set at 0.4 mL/min.  The column temperature was set to 40 °C, the injection volume was 2 µL.
Ion Mode:POSITIVE
  
MS ID:MS003775
Analysis ID:AN004028
Instrument Name:Thermo Orbitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:​Mobile Phase A used for HILIC chromatography was 80:20 water:MeCN with 10 mM ammonium formate and 0.1% formic acid. Mobile phase B for HILIC chromatography was acetonitrile with 0.1% formic acid. The flow rate was set at 0.4 mL/min.  The column temperature was set to 40 °C, the injection volume was 2 µL.
Ion Mode:NEGATIVE
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