{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002469","ANALYSIS_ID":"AN004027","VERSION":"1","CREATED_ON":"February 9, 2023, 7:40 am"},

"PROJECT":{"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"},

"STUDY":{"STUDY_TITLE":"Mesenchymal stromal cell (MSC) Metabolite MS study","STUDY_SUMMARY":"Metabolomics 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","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","STUDY_TYPE":"Untargeted Metabolite Study","PHONE":"7072391412"},

"SUBJECT":{"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"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"RB139",
"Sample ID":"alphaMEM_RB139",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"0.96","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"8.10E+06","Initial_Population_Doubling_Level":"12.2","Final_Population_Doubling_Level":"16.6","Batch":"1","Tcell_proliferation_01_CD4":"81.58","Tcell_proliferation_01_CD8":"88.55","Tcell_proliferation_02_CD4":"50.59","Tcell_proliferation_02_CD8":"35.42","IDO_Activity":"49.31"}
},
{
"Subject ID":"RB174",
"Sample ID":"alphaMEM_RB174_01",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"0.57","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"9.80E+06","Initial_Population_Doubling_Level":"13.06","Final_Population_Doubling_Level":"17.8","Batch":"1","Tcell_proliferation_01_CD4":"75.15","Tcell_proliferation_01_CD8":"78.96","Tcell_proliferation_02_CD4":"50.49","Tcell_proliferation_02_CD8":"38.16","IDO_Activity":"38.90"}
},
{
"Subject ID":"RB177",
"Sample ID":"alphaMEM_RB177",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"-1.62","Sex":"Male","Age":"22","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"4.23E+06","Initial_Population_Doubling_Level":"15.1","Final_Population_Doubling_Level":"18.6","Batch":"1","Tcell_proliferation_01_CD4":"73.11","Tcell_proliferation_01_CD8":"70.64","Tcell_proliferation_02_CD4":"35.57","Tcell_proliferation_02_CD8":"29.35","IDO_Activity":"70.43"}
},
{
"Subject ID":"RB179",
"Sample ID":"alphaMEM_RB179",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"1.27","Sex":"Male","Age":"21","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"8.20E+06","Initial_Population_Doubling_Level":"12.6","Final_Population_Doubling_Level":"17.1","Batch":"1","Tcell_proliferation_01_CD4":"85.35","Tcell_proliferation_01_CD8":"84.80","Tcell_proliferation_02_CD4":"45.71","Tcell_proliferation_02_CD8":"34.36","IDO_Activity":"22.49"}
},
{
"Subject ID":"RB183",
"Sample ID":"alphaMEM_RB183",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"0.50","Sex":"Female","Age":"26","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"5.30E+06","Initial_Population_Doubling_Level":"12","Final_Population_Doubling_Level":"15.8","Batch":"1","Tcell_proliferation_01_CD4":"83.28","Tcell_proliferation_01_CD8":"80.65","Tcell_proliferation_02_CD4":"48.26","Tcell_proliferation_02_CD8":"35.15","IDO_Activity":"53.76"}
},
{
"Subject ID":"RB71",
"Sample ID":"alphaMEM_RB71",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"2.13","Sex":"Female","Age":"18-30","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"7.50E+06","Initial_Population_Doubling_Level":"12.72","Final_Population_Doubling_Level":"17","Batch":"2","Tcell_proliferation_01_CD4":"86.95","Tcell_proliferation_01_CD8":"89.51","Tcell_proliferation_02_CD4":"55.34","Tcell_proliferation_02_CD8":"45.80","IDO_Activity":"46.26"}
},
{
"Subject ID":"RB174",
"Sample ID":"alphaMEM_RB174_02",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"0.92","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"8.43E+06","Initial_Population_Doubling_Level":"13.06","Final_Population_Doubling_Level":"17.6","Batch":"2","Tcell_proliferation_01_CD4":"73.37","Tcell_proliferation_01_CD8":"79.08","Tcell_proliferation_02_CD4":"49.34","Tcell_proliferation_02_CD8":"40.72","IDO_Activity":"21.98"}
},
{
"Subject ID":"RB175",
"Sample ID":"alphaMEM_RB175",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"0.96","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"6.24E+06","Initial_Population_Doubling_Level":"12","Final_Population_Doubling_Level":"16.1","Batch":"2","Tcell_proliferation_01_CD4":"74.21","Tcell_proliferation_01_CD8":"75.19","Tcell_proliferation_02_CD4":"50.93","Tcell_proliferation_02_CD8":"42.01","IDO_Activity":"20.45"}
},
{
"Subject ID":"RB182",
"Sample ID":"alphaMEM_RB182",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"-1.21","Sex":"Female","Age":"26","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"3.65E+06","Initial_Population_Doubling_Level":"11.48","Final_Population_Doubling_Level":"14.8","Batch":"2","Tcell_proliferation_01_CD4":"65.76","Tcell_proliferation_01_CD8":"61.28","Tcell_proliferation_02_CD4":"48.56","Tcell_proliferation_02_CD8":"36.16","IDO_Activity":"54.69"}
},
{
"Subject ID":"iMSC",
"Sample ID":"alphaMEM_iMSC",
"Factors":{"Media":"Gibco MEM alpha"},
"Additional sample data":{"Functional_Composite_Score":"-4.47","Sex":"not available","Age":"not available","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"1.86E+06","Initial_Population_Doubling_Level":"not available","Final_Population_Doubling_Level":"not available","Batch":"2","Tcell_proliferation_01_CD4":"60.06","Tcell_proliferation_01_CD8":"62.65","Tcell_proliferation_02_CD4":"11.82","Tcell_proliferation_02_CD8":"17.11","IDO_Activity":"80.56"}
},
{
"Subject ID":"RB139",
"Sample ID":"RBio_RB139",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"1.05","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"1.51E+07","Initial_Population_Doubling_Level":"12.2","Final_Population_Doubling_Level":"17.5","Batch":"1","Tcell_proliferation_01_CD4":"55.84","Tcell_proliferation_01_CD8":"64.27","Tcell_proliferation_02_CD4":"68.63","Tcell_proliferation_02_CD8":"66.47","IDO_Activity":"8.87"}
},
{
"Subject ID":"RB174",
"Sample ID":"RBio_RB174_01",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"0.53","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"2.55E+07","Initial_Population_Doubling_Level":"13.06","Final_Population_Doubling_Level":"19.1","Batch":"1","Tcell_proliferation_01_CD4":"56.11","Tcell_proliferation_01_CD8":"60.94","Tcell_proliferation_02_CD4":"58.77","Tcell_proliferation_02_CD8":"53.87","IDO_Activity":"2.48"}
},
{
"Subject ID":"RB177",
"Sample ID":"RBio_RB177",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"-1.35","Sex":"Male","Age":"22","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"2.12E+07","Initial_Population_Doubling_Level":"15.1","Final_Population_Doubling_Level":"20.9","Batch":"1","Tcell_proliferation_01_CD4":"51.37","Tcell_proliferation_01_CD8":"45.73","Tcell_proliferation_02_CD4":"54.93","Tcell_proliferation_02_CD8":"46.49","IDO_Activity":"14.93"}
},
{
"Subject ID":"RB179",
"Sample ID":"RBio_RB179",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"-0.10","Sex":"Male","Age":"21","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"2.66E+07","Initial_Population_Doubling_Level":"12.6","Final_Population_Doubling_Level":"18.7","Batch":"1","Tcell_proliferation_01_CD4":"55.07","Tcell_proliferation_01_CD8":"61.90","Tcell_proliferation_02_CD4":"49.48","Tcell_proliferation_02_CD8":"42.74","IDO_Activity":"0.16"}
},
{
"Subject ID":"RB183",
"Sample ID":"RBio_RB183",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"0.69","Sex":"Female","Age":"26","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"7.80E+06","Initial_Population_Doubling_Level":"12","Final_Population_Doubling_Level":"16.4","Batch":"1","Tcell_proliferation_01_CD4":"63.36","Tcell_proliferation_01_CD8":"64.09","Tcell_proliferation_02_CD4":"61.39","Tcell_proliferation_02_CD8":"54.84","IDO_Activity":"10.14"}
},
{
"Subject ID":"RB71",
"Sample ID":"RBio_RB71",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"1.50","Sex":"Female","Age":"18-30","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"1.50E+07","Initial_Population_Doubling_Level":"12.72","Final_Population_Doubling_Level":"12.72","Batch":"2","Tcell_proliferation_01_CD4":"63.09","Tcell_proliferation_01_CD8":"65.10","Tcell_proliferation_02_CD4":"67.70","Tcell_proliferation_02_CD8":"64.11","IDO_Activity":"6.33"}
},
{
"Subject ID":"RB174",
"Sample ID":"RBio_RB174_02",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"1.72","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"1.29E+07","Initial_Population_Doubling_Level":"13.06","Final_Population_Doubling_Level":"13.06","Batch":"2","Tcell_proliferation_01_CD4":"66.93","Tcell_proliferation_01_CD8":"77.71","Tcell_proliferation_02_CD4":"60.63","Tcell_proliferation_02_CD8":"56.73","IDO_Activity":"3.97"}
},
{
"Subject ID":"RB175",
"Sample ID":"RBio_RB175",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"1.44","Sex":"Male","Age":"25","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"1.92E+07","Initial_Population_Doubling_Level":"12","Final_Population_Doubling_Level":"12","Batch":"2","Tcell_proliferation_01_CD4":"62.30","Tcell_proliferation_01_CD8":"70.30","Tcell_proliferation_02_CD4":"61.80","Tcell_proliferation_02_CD8":"58.35","IDO_Activity":"2.21"}
},
{
"Subject ID":"RB182",
"Sample ID":"RBio_RB182",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"-0.32","Sex":"Female","Age":"26","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"1.47E+07","Initial_Population_Doubling_Level":"11.48","Final_Population_Doubling_Level":"11.48","Batch":"2","Tcell_proliferation_01_CD4":"60.47","Tcell_proliferation_01_CD8":"60.44","Tcell_proliferation_02_CD4":"53.46","Tcell_proliferation_02_CD8":"45.15","IDO_Activity":"11.97"}
},
{
"Subject ID":"iMSC",
"Sample ID":"RBio_iMSC",
"Factors":{"Media":"RoosterBasal-MSC media"},
"Additional sample data":{"Functional_Composite_Score":"-5.16","Sex":"not available","Age":"not available","Total_Cells_Seeded":"3.75E+05","Final_Cell_Yield":"1.90E+07","Initial_Population_Doubling_Level":"not available","Final_Population_Doubling_Level":"not available","Batch":"2","Tcell_proliferation_01_CD4":"37.12","Tcell_proliferation_01_CD8":"23.57","Tcell_proliferation_02_CD4":"25.68","Tcell_proliferation_02_CD8":"21.86","IDO_Activity":"21.54"}
}
],
"COLLECTION":{"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_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."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Orbitrap ID-X Tribrid mass spectrometer","COLUMN_NAME":"Waters ACQUITY UPLC BEH Amide (150 x 2.1mm,1.7um)","SOLVENT_A":"80% water/20% acetonitrile; 10 mM ammonium formate; 0.1% formic acid","SOLVENT_B":"100% acetonitrile; 0.1% formic acid","FLOW_GRADIENT":"5% A 0-8 min; 60% A 8-10.4 min; 5% A 10.5-14min","FLOW_RATE":"0.4 mL/min","COLUMN_TEMPERATURE":"40"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Orbitrap ID-X tribrid","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","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.","MS_RESULTS_FILE":"ST002469_AN004027_Results.txt UNITS:Peak Area Has m/z:Neutral masses Has RT:No RT units:No RT data"}

}