{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000167","ANALYSIS_ID":"AN000261","VERSION":"1","CREATED_ON":"2016-09-17"},
"PROJECT":{"PROJECT_TITLE":"Cold Storage of Rat Hepatocyte Spheroids","PROJECT_SUMMARY":"Cell-based therapies for liver disease rely on a high-quality supply of and a means for storage during transportation from site of isolation to site of Unfortunately, frozen cryopreservation is associated with unacceptable loss of viability after thawing. The purpose of this study was to optimize conditions cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of culture in serum-free medium (SFM). Spheroids were then maintained in rocked at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 ?M A (CsA); SFM + 1 mM Def + 1 ?M CsA, University of Wisconsin (UW) solution UW + 1 mM Def. Performance metrics after cold storage included viability, gene albumin production, and functional activity of cytochrome P450 enzymes and urea proteins. We observed that cold-induced injury was reduced significantly by the of the iron chelator (Def) to both SFM and UW solution. Performance metrics detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + for 24 h were significantly increased from SFM alone and approached those in conditions, while performance metrics after cold storage in SFM alone or cold for 48 h were both significantly reduced. A serum-free medium supplemented with allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than loss in viability and functionality. Further research is warranted to optimize solution for extended cold storage of hepatocyte spheroids.","INSTITUTE":"Mayo Clinic","DEPARTMENT":"Division of Experimental Surgery","LAST_NAME":"Nyberg","FIRST_NAME":"Scott","ADDRESS":"-","EMAIL":"Nyberg.scott@mayo.edu","PHONE":"-","FUNDING_SOURCE":"This work was funded by grants from NIH (RO1-DK56733), the Marriott Foundation, Wallace H. Coulter Foundation, the 12th Five-Year National Science and Major Project for Infectious Diseases, China (No. 2012 ZX 10002004-005, Liu), and Jiangsu province government, China (Yue Yu). The authors declare no of interest."},
"STUDY":{"STUDY_TITLE":"Cold Storage of Rat Hepatocyte Spheroids","STUDY_TYPE":"Storage condition testing","STUDY_SUMMARY":"The purpose of this study was to optimize conditions for cold storage of rat spheroids without freezing. Rat hepatocytes were isolated by a two-step method; hepatocyte spheroids were formed during 48 h of rocked culture in medium (SFM). Spheroids were then maintained in rocked culture at 37°C condition) or cold stored at 4°C for 24 or 48 h in six different cold storage SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 ?M cyclosporin A (CsA); SFM + mM Def + 1 ?M CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def.","INSTITUTE":"Mayo Clinic","DEPARTMENT":"Division of Experimental Surgery","LAST_NAME":"Nyberg","FIRST_NAME":"Scott","ADDRESS":"-","EMAIL":"Nyberg.scott@mayo.edu","PHONE":"-","NUM_GROUPS":"9","TOTAL_SUBJECTS":"45"},
"SUBJECT":{"SUBJECT_TYPE":"Animals","SUBJECT_SPECIES":"Rattus norvegicus","TAXONOMY_ID":"10114","GENOTYPE_STRAIN":"SpragueDawley rats","WEIGHT_OR_WEIGHT_RANGE":"300400 g"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1",
"Factors":{"Treatment":"20uM diazepam"},
"Additional sample data":{"Replicate":"2","factor description":"20uM diazepam, 6-13-2012, Time 0"}
},
{
"Subject ID":"-",
"Sample ID":"17",
"Factors":{"Treatment":"20uM diazepam"},
"Additional sample data":{"Replicate":"3","factor description":"20uM diazepam, 6-20-2012, Time 0"}
},
{
"Subject ID":"-",
"Sample ID":"33",
"Factors":{"Treatment":"20uM diazepam"},
"Additional sample data":{"Replicate":"1","factor description":"20uM diazepam, 5-31-2012, Time 0"}
},
{
"Subject ID":"-",
"Sample ID":"20",
"Factors":{"Treatment":"Control group 120h"},
"Additional sample data":{"Replicate":"2","factor description":"Control group 120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"36",
"Factors":{"Treatment":"Control group 120h"},
"Additional sample data":{"Replicate":"3","factor description":"Control group 120h 6-3-2012"}
},
{
"Subject ID":"-",
"Sample ID":"4",
"Factors":{"Treatment":"Control group 120h"},
"Additional sample data":{"Replicate":"1","factor description":"Control group 120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"18",
"Factors":{"Treatment":"Control group 72h"},
"Additional sample data":{"Replicate":"3","factor description":"Control group 72h 6-21-2012"}
},
{
"Subject ID":"-",
"Sample ID":"2",
"Factors":{"Treatment":"Control group 72h"},
"Additional sample data":{"Replicate":"2","factor description":"Control group 72h 6-14-2012"}
},
{
"Subject ID":"-",
"Sample ID":"34",
"Factors":{"Treatment":"Control group 72h"},
"Additional sample data":{"Replicate":"1","factor description":"Control group 72h 6-1-2012"}
},
{
"Subject ID":"-",
"Sample ID":"19",
"Factors":{"Treatment":"Control group 96h"},
"Additional sample data":{"Replicate":"3","factor description":"Control group 96h 6-22-2012"}
},
{
"Subject ID":"-",
"Sample ID":"3",
"Factors":{"Treatment":"Control group 96h"},
"Additional sample data":{"Replicate":"1","factor description":"Control group 96h 6-15-2012"}
},
{
"Subject ID":"-",
"Sample ID":"35",
"Factors":{"Treatment":"Control group 96h"},
"Additional sample data":{"Replicate":"2","factor description":"Control group 96h 6-2-2012"}
},
{
"Subject ID":"-",
"Sample ID":"25",
"Factors":{"Treatment":"SFM cold 24-120h"},
"Additional sample data":{"Replicate":"2","factor description":"A SFM cold 24-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"9",
"Factors":{"Treatment":"SFM cold 24-120h"},
"Additional sample data":{"Replicate":"1","factor description":"A SFM cold 24-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"21",
"Factors":{"Treatment":"SFM cold 24-96h"},
"Additional sample data":{"Replicate":"2","factor description":"A SFM cold 24-96h 6-22-2012"}
},
{
"Subject ID":"-",
"Sample ID":"5",
"Factors":{"Treatment":"SFM cold 24-96h"},
"Additional sample data":{"Replicate":"1","factor description":"A SFM cold 24-96h 6-15-2012"}
},
{
"Subject ID":"-",
"Sample ID":"13",
"Factors":{"Treatment":"SFM cold 48-120h"},
"Additional sample data":{"Replicate":"1","factor description":"A SFM cold 48-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"29",
"Factors":{"Treatment":"SFM cold 48-120h"},
"Additional sample data":{"Replicate":"2","factor description":"A SFM cold 48-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"12",
"Factors":{"Treatment":"SFM +CsA cold 24-120h"},
"Additional sample data":{"Replicate":"1","factor description":"D SFM +CsA cold 24-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"28",
"Factors":{"Treatment":"SFM +CsA cold 24-120h"},
"Additional sample data":{"Replicate":"2","factor description":"D SFM +CsA cold 24-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"24",
"Factors":{"Treatment":"SFM +CsA cold 24-96h"},
"Additional sample data":{"Replicate":"2","factor description":"D SFM +CsA cold 24-96h 6-22-2012"}
},
{
"Subject ID":"-",
"Sample ID":"8",
"Factors":{"Treatment":"SFM +CsA cold 24-96h"},
"Additional sample data":{"Replicate":"1","factor description":"D SFM +CsA cold 24-96h 6-15-2012"}
},
{
"Subject ID":"-",
"Sample ID":"16",
"Factors":{"Treatment":"SFM +CsA cold 48-120h"},
"Additional sample data":{"Replicate":"1","factor description":"D SFM +CsA cold 48-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"32",
"Factors":{"Treatment":"SFM +CsA cold 48-120h"},
"Additional sample data":{"Replicate":"2","factor description":"D SFM +CsA cold 48-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"42",
"Factors":{"Treatment":"SFM +CsA+Gly cold 24-120h"},
"Additional sample data":{"Replicate":"1","factor description":"D SFM +CsA+Gly cold 24-120h 6-3-2012"}
},
{
"Subject ID":"-",
"Sample ID":"39",
"Factors":{"Treatment":"SFM +CsA+Gly cold 24-96h"},
"Additional sample data":{"Replicate":"1","factor description":"D SFM +CsA+Gly cold 24-96h 6-2-2012"}
},
{
"Subject ID":"-",
"Sample ID":"45",
"Factors":{"Treatment":"SFM +CsA+Gly cold 48-120h"},
"Additional sample data":{"Replicate":"1","factor description":"D SFM +CsA+Gly cold 48-120h 6-3-2012"}
},
{
"Subject ID":"-",
"Sample ID":"10",
"Factors":{"Treatment":"SFM+Def cold 24-120h"},
"Additional sample data":{"Replicate":"2","factor description":"B SFM+Def cold 24-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"26",
"Factors":{"Treatment":"SFM+Def cold 24-120h"},
"Additional sample data":{"Replicate":"3","factor description":"B SFM+Def cold 24-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"40",
"Factors":{"Treatment":"SFM+Def cold 24-120h"},
"Additional sample data":{"Replicate":"1","factor description":"A SFM+Def cold 24-120h 6-3-2012"}
},
{
"Subject ID":"-",
"Sample ID":"22",
"Factors":{"Treatment":"SFM+Def cold 24-96h"},
"Additional sample data":{"Replicate":"3","factor description":"B SFM+Def cold 24-96h 6-22-2012"}
},
{
"Subject ID":"-",
"Sample ID":"37",
"Factors":{"Treatment":"SFM+Def cold 24-96h"},
"Additional sample data":{"Replicate":"1","factor description":"A SFM+Def cold 24-96h 6-2-2012"}
},
{
"Subject ID":"-",
"Sample ID":"6",
"Factors":{"Treatment":"SFM+Def cold 24-96h"},
"Additional sample data":{"Replicate":"2","factor description":"B SFM+Def cold 24-96h 6-15-2012"}
},
{
"Subject ID":"-",
"Sample ID":"14",
"Factors":{"Treatment":"SFM+Def cold 48-120h"},
"Additional sample data":{"Replicate":"2","factor description":"B SFM+Def cold 48-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"30",
"Factors":{"Treatment":"SFM+Def cold 48-120h"},
"Additional sample data":{"Replicate":"3","factor description":"B SFM+Def cold 48-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"43",
"Factors":{"Treatment":"SFM+Def cold 48-120h"},
"Additional sample data":{"Replicate":"1","factor description":"A SFM+Def cold 48-120h 6-3-2012"}
},
{
"Subject ID":"-",
"Sample ID":"11",
"Factors":{"Treatment":"SFM +Def +CsA cold 24-120h"},
"Additional sample data":{"Replicate":"2","factor description":"C SFM +Def +CsA cold 24-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"27",
"Factors":{"Treatment":"SFM +Def +CsA cold 24-120h"},
"Additional sample data":{"Replicate":"3","factor description":"C SFM +Def +CsA cold 24-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"41",
"Factors":{"Treatment":"SFM +Def +CsA cold 24-120h"},
"Additional sample data":{"Replicate":"1","factor description":"B SFM +Def +CsA cold 24-120h 6-3-2012"}
},
{
"Subject ID":"-",
"Sample ID":"23",
"Factors":{"Treatment":"SFM +Def +CsA cold 24-96h"},
"Additional sample data":{"Replicate":"3","factor description":"C SFM +Def +CsA cold 24-96h 6-22-2012"}
},
{
"Subject ID":"-",
"Sample ID":"38",
"Factors":{"Treatment":"SFM +Def +CsA cold 24-96h"},
"Additional sample data":{"Replicate":"1","factor description":"B SFM +Def +CsA cold 24-96h 6-2-2012"}
},
{
"Subject ID":"-",
"Sample ID":"7",
"Factors":{"Treatment":"SFM +Def +CsA cold 24-96h"},
"Additional sample data":{"Replicate":"2","factor description":"C SFM +Def +CsA cold 24-96h 6-15-2012"}
},
{
"Subject ID":"-",
"Sample ID":"15",
"Factors":{"Treatment":"SFM +Def +CsA cold 48-120h"},
"Additional sample data":{"Replicate":"2","factor description":"C SFM +Def +CsA cold 48-120h 6-16-2012"}
},
{
"Subject ID":"-",
"Sample ID":"31",
"Factors":{"Treatment":"SFM +Def +CsA cold 48-120h"},
"Additional sample data":{"Replicate":"3","factor description":"C SFM +Def +CsA cold 48-120h 6-23-2012"}
},
{
"Subject ID":"-",
"Sample ID":"44",
"Factors":{"Treatment":"SFM +Def +CsA cold 48-120h"},
"Additional sample data":{"Replicate":"1","factor description":"B SFM +Def +CsA cold 48-120h 6-3-2012"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Hepatocytes were isolated from male SpragueDawley rats (300400 g; Harlan, IN, USA) by a two-step perfusion method as previously described (33). All yielded hepatocytes with viability exceeding 95% by trypan blue dye isolated hepatocytes were suspended in SFM, composed of Williams E with 0.2 U/ml insulin, 6 ?g/ml transferrin, 100 U/ml penicillin G, 100 mg/ml 3 g/L human albumin, 2.2 g/L sodium bicarbonate, 1 g/L L-carnitine, 2.0 mM 100 nM dexamethasone, 40 ng/ml glucagon, 20 ng/ml Gly-His-Lys, 1,000 U/L 1 mg/L warfarin, and 5 ng/ml of mouse epidermal growth factor (EGF) (3). The suspended in SFM, were placed in a spheroid box (33 × 28 × 6 cm) custom-made polycarbonate by Mayo Division of Engineering and siliconized with Sigmacote 30 min (20) and gently rocked continuously at a frequency of 10 cycles per (0.17 Hz) to induce spheroid formation and to maintain spheroids in suspension. hepatocyte concentration was 5 × 105 cells/ml per spheroid box. All culture were maintained in a 5% CO2, 37°C incubator as previously described (20). were centrifuged and resuspended in fresh culture media every 24 h."},
"TREATMENT":{"TREATMENT_SUMMARY":"UW alone|UW + 1 mM Def|SFM alone|SFM + 1 mM Def|SFM + 1 mM Def + 1 µM CsA|SFM 1 µM CsA","TREATMENT_PROTOCOL_FILENAME":"PMID-23507348-Liu-Nydberg-CellTrans-2014.pdf","TREATMENT_PROTOCOL_COMMENTS":"Treatment annotation Cold 24 Indicates 24 hours cold storate prior to 48 Indicates 48 hours cold storate prior to treatment -96h Indicates treated 96 hours after cold storage -120h Indicates treated for 120 hours after cold","TREATMENT_COMPOUND":"University of Wisconsin (UW) solution|UW + deferoxamine (Def)| serum-free Sigma-Aldrich (St. Louis, MO, USA)|SFM + Def|SFM + Def + cyclosporin A + CsA"},
"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"After 48 h of continuous rocking, the spheroids were washed with SFM at room The culture medium was changed to the cold storage medium (UW alone, UW + 1 mM SFM alone; SFM + 1 mM Def; SFM + 1 mM Def + 1 ?M CsA; SFM + 1 ?M CsA) at room Spheroids were left in rocked culture without cold storage as control. intended for cold storage were rocked in glass dishes (10 × 8 × 2 cm) by Mayo Division of Engineering and siliconized with Sigmacote for 30 min and transferred into 50-ml conical tubes (BD Falcon; 1 × 106cells/ml, 20 ml in and put on ice for 1 h. Of note, spheroid box and glass dishes differ in size volume capacity) but possess similar properties of spheroid formation and Finally, tubes of spheroids were placed in a refrigerator to be cold stored at After 24 or 48 h of cold storage treatment, the spheroids were centrifuged at × g for 5 min, and the supernatant fluid was removed. Warm SFM (20 ml) at was added to each tube. The tubes were mixed gently prior to adding 20 ml of suspension to each glass culture dish and continued to rocking culture. were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere."},
"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Concentrations of diazepam and its two major metabolites (nordiazepam and were determined by high-performance liquid chromatography (HPLC) with mass detection in the CTSA Metabolomics Core lab at Mayo Clinic.","CHROMATOGRAPHY_TYPE":"-","INSTRUMENT_NAME":"-","COLUMN_NAME":"-"},
"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Mayo Clinic Metabolomics Core"},
"MS":{"INSTRUMENT_NAME":"-","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"POSITIVE"},
"MS_METABOLITE_DATA":{
"Units":"ng/ml",
"Data":[{"Metabolite":"diazepam","1":"4481.5725","17":"5736.6630","33":"5623.3315","20":"1624.1825","36":"956.1976","4":"726.7814","18":"1633.6693","2":"1621.7624","34":"1264.7138","19":"1897.2045","3":"1045.1085","35":"1328.6425","25":"2411.7352","9":"2148.4238","21":"1810.5472","5":"1908.3052","13":"2474.8026","29":"2351.5498","12":"2776.9633","28":"2073.6053","24":"2116.1659","8":"2116.6115","16":"2727.2512","32":"2099.1453","42":"1318.7897","39":"1900.1499","45":"2066.6119","10":"662.5650","26":"2781.7872","40":"2529.0427","22":"2011.8566","37":"1396.4370","6":"1444.2694","14":"2114.3240","30":"2328.9302","43":"1788.3991","11":"1685.9612","27":"2494.7350","41":"1947.2209","23":"2389.4712","38":"1697.0849","7":"1699.9506","15":"1649.3030","31":"1956.9368","44":"2769.8399"},{"Metabolite":"nordiazepam","1":"","17":"","33":"","20":"120.4401","36":"320.1140","4":"331.5166","18":"144.0672","2":"426.4867","34":"188.2435","19":"111.2503","3":"565.4192","35":"469.4874","25":"3.5452","9":"23.2656","21":"23.8233","5":"19.4455","13":"5.4840","29":"7.5175","12":"6.5456","28":"2.4504","24":"27.2201","8":"8.9072","16":"4.3846","32":"3.6653","42":"183.1366","39":"152.2262","45":"45.5407","10":"256.2231","26":"62.0669","40":"419.4401","22":"102.7936","37":"127.1815","6":"239.4604","14":"82.5938","30":"47.6665","43":"40.2152","11":"669.9298","27":"47.8024","41":"227.3917","23":"105.4923","38":"119.4808","7":"302.1144","15":"69.3379","31":"50.0816","44":"57.3156"},{"Metabolite":"oxazepam","1":"","17":"","33":"","20":"33.3808","36":"329.6473","4":"529.4960","18":"13.1518","2":"263.3279","34":"93.3722","19":"18.0641","3":"696.8769","35":"365.7868","25":"","9":"2.2263","21":"","5":"0.4201","13":"","29":"","12":"","28":"","24":"","8":"","16":"0.0000","32":"","42":"76.7970","39":"23.1458","45":"0.9956","10":"289.0782","26":"6.3126","40":"165.7055","22":"1.6718","37":"36.1355","6":"136.4053","14":"6.6902","30":"0.1394","43":"0.8202","11":"354.0988","27":"4.5315","41":"88.7497","23":"1.6816","38":"21.7052","7":"180.4597","15":"5.1440","31":"0.3173","44":"1.4070"},{"Metabolite":"temezepam","1":"","17":"","33":"","20":"313.1072","36":"309.0023","4":"247.3904","18":"84.5079","2":"268.8166","34":"157.2228","19":"209.4556","3":"314.0752","35":"388.5075","25":"2.4173","9":"85.1736","21":"6.1321","5":"17.2721","13":"2.0203","29":"2.1027","12":"21.6403","28":"0.8599","24":"7.1088","8":"6.6813","16":"1.7196","32":"1.1660","42":"283.6135","39":"119.1452","45":"20.6709","10":"231.7318","26":"145.8304","40":"517.8487","22":"33.7706","37":"94.2949","6":"223.2863","14":"71.1168","30":"15.7583","43":"16.5374","11":"463.0140","27":"105.2445","41":"431.0494","23":"36.8710","38":"108.4368","7":"277.1309","15":"50.7277","31":"16.1774","44":"26.7043"}],
"Metabolites":[{"Metabolite":"diazepam","moverz_quant":"","ri":"","ri_type":"","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"MAYO_ID"},{"Metabolite":"nordiazepam","moverz_quant":"","ri":"","ri_type":"","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"MAYO_ID"},{"Metabolite":"oxazepam","moverz_quant":"","ri":"","ri_type":"","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"MAYO_ID"},{"Metabolite":"temezepam","moverz_quant":"","ri":"","ri_type":"","pubchem_id":"","inchi_key":"","kegg_id":"","other_id":"","other_id_type":"MAYO_ID"}]
}
}