#METABOLOMICS WORKBENCH sdasari_20150514_9491141_mwtab.txt DATATRACK_ID:244 STUDY_ID:ST000167 ANALYSIS_ID:AN000261 PROJECT_ID:PR000145 VERSION 1 CREATED_ON 2016-09-17 #PROJECT PR:PROJECT_TITLE Cold Storage of Rat Hepatocyte Spheroids PR:PROJECT_SUMMARY Cell-based therapies for liver disease rely on a high-quality supply of PR:PROJECT_SUMMARY and a means for storage during transportation from site of isolation to site of PR:PROJECT_SUMMARY Unfortunately, frozen cryopreservation is associated with unacceptable loss of PR:PROJECT_SUMMARY viability after thawing. The purpose of this study was to optimize conditions PR:PROJECT_SUMMARY cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were PR:PROJECT_SUMMARY by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of PR:PROJECT_SUMMARY culture in serum-free medium (SFM). Spheroids were then maintained in rocked PR:PROJECT_SUMMARY at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six PR:PROJECT_SUMMARY cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 ?M PR:PROJECT_SUMMARY A (CsA); SFM + 1 mM Def + 1 ?M CsA, University of Wisconsin (UW) solution PR:PROJECT_SUMMARY UW + 1 mM Def. Performance metrics after cold storage included viability, gene PR:PROJECT_SUMMARY albumin production, and functional activity of cytochrome P450 enzymes and urea PR:PROJECT_SUMMARY proteins. We observed that cold-induced injury was reduced significantly by the PR:PROJECT_SUMMARY of the iron chelator (Def) to both SFM and UW solution. Performance metrics PR:PROJECT_SUMMARY detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + PR:PROJECT_SUMMARY for 24 h were significantly increased from SFM alone and approached those in PR:PROJECT_SUMMARY conditions, while performance metrics after cold storage in SFM alone or cold PR:PROJECT_SUMMARY for 48 h were both significantly reduced. A serum-free medium supplemented with PR:PROJECT_SUMMARY allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than PR:PROJECT_SUMMARY loss in viability and functionality. Further research is warranted to optimize PR:PROJECT_SUMMARY solution for extended cold storage of hepatocyte spheroids. PR:INSTITUTE Mayo Clinic PR:DEPARTMENT Division of Experimental Surgery PR:LAST_NAME Nyberg PR:FIRST_NAME Scott PR:ADDRESS - PR:EMAIL Nyberg.scott@mayo.edu PR:PHONE - PR:FUNDING_SOURCE This work was funded by grants from NIH (RO1-DK56733), the Marriott Foundation, PR:FUNDING_SOURCE Wallace H. Coulter Foundation, the 12th Five-Year National Science and PR:FUNDING_SOURCE Major Project for Infectious Diseases, China (No. 2012 ZX 10002004-005, PR:FUNDING_SOURCE Liu), and Jiangsu province government, China (Yue Yu). The authors declare no PR:FUNDING_SOURCE of interest. #STUDY ST:STUDY_TITLE Cold Storage of Rat Hepatocyte Spheroids ST:STUDY_TYPE Storage condition testing ST:STUDY_SUMMARY The purpose of this study was to optimize conditions for cold storage of rat ST:STUDY_SUMMARY spheroids without freezing. Rat hepatocytes were isolated by a two-step ST:STUDY_SUMMARY method; hepatocyte spheroids were formed during 48 h of rocked culture in ST:STUDY_SUMMARY medium (SFM). Spheroids were then maintained in rocked culture at 37°C ST:STUDY_SUMMARY condition) or cold stored at 4°C for 24 or 48 h in six different cold storage ST:STUDY_SUMMARY SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 ?M cyclosporin A (CsA); SFM + ST:STUDY_SUMMARY mM Def + 1 ?M CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. ST:INSTITUTE Mayo Clinic ST:DEPARTMENT Division of Experimental Surgery ST:LAST_NAME Nyberg ST:FIRST_NAME Scott ST:ADDRESS - ST:EMAIL Nyberg.scott@mayo.edu ST:PHONE - ST:NUM_GROUPS 9 ST:TOTAL_SUBJECTS 45 #SUBJECT SU:SUBJECT_TYPE Animals SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10114 SU:GENOTYPE_STRAIN SpragueDawley rats SU:WEIGHT_OR_WEIGHT_RANGE 300400 g #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - 1 Treatment:20uM diazepam Replicate=2; factor description=20uM diazepam, 6-13-2012, Time 0 SUBJECT_SAMPLE_FACTORS - 17 Treatment:20uM diazepam Replicate=3; factor description=20uM diazepam, 6-20-2012, Time 0 SUBJECT_SAMPLE_FACTORS - 33 Treatment:20uM diazepam Replicate=1; factor description=20uM diazepam, 5-31-2012, Time 0 SUBJECT_SAMPLE_FACTORS - 20 Treatment:Control group 120h Replicate=2; factor description=Control group 120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 36 Treatment:Control group 120h Replicate=3; factor description=Control group 120h 6-3-2012 SUBJECT_SAMPLE_FACTORS - 4 Treatment:Control group 120h Replicate=1; factor description=Control group 120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 18 Treatment:Control group 72h Replicate=3; factor description=Control group 72h 6-21-2012 SUBJECT_SAMPLE_FACTORS - 2 Treatment:Control group 72h Replicate=2; factor description=Control group 72h 6-14-2012 SUBJECT_SAMPLE_FACTORS - 34 Treatment:Control group 72h Replicate=1; factor description=Control group 72h 6-1-2012 SUBJECT_SAMPLE_FACTORS - 19 Treatment:Control group 96h Replicate=3; factor description=Control group 96h 6-22-2012 SUBJECT_SAMPLE_FACTORS - 3 Treatment:Control group 96h Replicate=1; factor description=Control group 96h 6-15-2012 SUBJECT_SAMPLE_FACTORS - 35 Treatment:Control group 96h Replicate=2; factor description=Control group 96h 6-2-2012 SUBJECT_SAMPLE_FACTORS - 25 Treatment:SFM cold 24-120h Replicate=2; factor description=A SFM cold 24-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 9 Treatment:SFM cold 24-120h Replicate=1; factor description=A SFM cold 24-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 21 Treatment:SFM cold 24-96h Replicate=2; factor description=A SFM cold 24-96h 6-22-2012 SUBJECT_SAMPLE_FACTORS - 5 Treatment:SFM cold 24-96h Replicate=1; factor description=A SFM cold 24-96h 6-15-2012 SUBJECT_SAMPLE_FACTORS - 13 Treatment:SFM cold 48-120h Replicate=1; factor description=A SFM cold 48-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 29 Treatment:SFM cold 48-120h Replicate=2; factor description=A SFM cold 48-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 12 Treatment:SFM +CsA cold 24-120h Replicate=1; factor description=D SFM +CsA cold 24-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 28 Treatment:SFM +CsA cold 24-120h Replicate=2; factor description=D SFM +CsA cold 24-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 24 Treatment:SFM +CsA cold 24-96h Replicate=2; factor description=D SFM +CsA cold 24-96h 6-22-2012 SUBJECT_SAMPLE_FACTORS - 8 Treatment:SFM +CsA cold 24-96h Replicate=1; factor description=D SFM +CsA cold 24-96h 6-15-2012 SUBJECT_SAMPLE_FACTORS - 16 Treatment:SFM +CsA cold 48-120h Replicate=1; factor description=D SFM +CsA cold 48-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 32 Treatment:SFM +CsA cold 48-120h Replicate=2; factor description=D SFM +CsA cold 48-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 42 Treatment:SFM +CsA+Gly cold 24-120h Replicate=1; factor description=D SFM +CsA+Gly cold 24-120h 6-3-2012 SUBJECT_SAMPLE_FACTORS - 39 Treatment:SFM +CsA+Gly cold 24-96h Replicate=1; factor description=D SFM +CsA+Gly cold 24-96h 6-2-2012 SUBJECT_SAMPLE_FACTORS - 45 Treatment:SFM +CsA+Gly cold 48-120h Replicate=1; factor description=D SFM +CsA+Gly cold 48-120h 6-3-2012 SUBJECT_SAMPLE_FACTORS - 10 Treatment:SFM+Def cold 24-120h Replicate=2; factor description=B SFM+Def cold 24-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 26 Treatment:SFM+Def cold 24-120h Replicate=3; factor description=B SFM+Def cold 24-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 40 Treatment:SFM+Def cold 24-120h Replicate=1; factor description=A SFM+Def cold 24-120h 6-3-2012 SUBJECT_SAMPLE_FACTORS - 22 Treatment:SFM+Def cold 24-96h Replicate=3; factor description=B SFM+Def cold 24-96h 6-22-2012 SUBJECT_SAMPLE_FACTORS - 37 Treatment:SFM+Def cold 24-96h Replicate=1; factor description=A SFM+Def cold 24-96h 6-2-2012 SUBJECT_SAMPLE_FACTORS - 6 Treatment:SFM+Def cold 24-96h Replicate=2; factor description=B SFM+Def cold 24-96h 6-15-2012 SUBJECT_SAMPLE_FACTORS - 14 Treatment:SFM+Def cold 48-120h Replicate=2; factor description=B SFM+Def cold 48-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 30 Treatment:SFM+Def cold 48-120h Replicate=3; factor description=B SFM+Def cold 48-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 43 Treatment:SFM+Def cold 48-120h Replicate=1; factor description=A SFM+Def cold 48-120h 6-3-2012 SUBJECT_SAMPLE_FACTORS - 11 Treatment:SFM +Def +CsA cold 24-120h Replicate=2; factor description=C SFM +Def +CsA cold 24-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 27 Treatment:SFM +Def +CsA cold 24-120h Replicate=3; factor description=C SFM +Def +CsA cold 24-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 41 Treatment:SFM +Def +CsA cold 24-120h Replicate=1; factor description=B SFM +Def +CsA cold 24-120h 6-3-2012 SUBJECT_SAMPLE_FACTORS - 23 Treatment:SFM +Def +CsA cold 24-96h Replicate=3; factor description=C SFM +Def +CsA cold 24-96h 6-22-2012 SUBJECT_SAMPLE_FACTORS - 38 Treatment:SFM +Def +CsA cold 24-96h Replicate=1; factor description=B SFM +Def +CsA cold 24-96h 6-2-2012 SUBJECT_SAMPLE_FACTORS - 7 Treatment:SFM +Def +CsA cold 24-96h Replicate=2; factor description=C SFM +Def +CsA cold 24-96h 6-15-2012 SUBJECT_SAMPLE_FACTORS - 15 Treatment:SFM +Def +CsA cold 48-120h Replicate=2; factor description=C SFM +Def +CsA cold 48-120h 6-16-2012 SUBJECT_SAMPLE_FACTORS - 31 Treatment:SFM +Def +CsA cold 48-120h Replicate=3; factor description=C SFM +Def +CsA cold 48-120h 6-23-2012 SUBJECT_SAMPLE_FACTORS - 44 Treatment:SFM +Def +CsA cold 48-120h Replicate=1; factor description=B SFM +Def +CsA cold 48-120h 6-3-2012 #COLLECTION CO:COLLECTION_SUMMARY Hepatocytes were isolated from male SpragueDawley rats (300400 g; Harlan, CO:COLLECTION_SUMMARY IN, USA) by a two-step perfusion method as previously described (33). All CO:COLLECTION_SUMMARY yielded hepatocytes with viability exceeding 95% by trypan blue dye CO:COLLECTION_SUMMARY isolated hepatocytes were suspended in SFM, composed of Williams E CO:COLLECTION_SUMMARY with 0.2 U/ml insulin, 6 ?g/ml transferrin, 100 U/ml penicillin G, 100 mg/ml CO:COLLECTION_SUMMARY 3 g/L human albumin, 2.2 g/L sodium bicarbonate, 1 g/L L-carnitine, 2.0 mM CO:COLLECTION_SUMMARY 100 nM dexamethasone, 40 ng/ml glucagon, 20 ng/ml Gly-His-Lys, 1,000 U/L CO:COLLECTION_SUMMARY 1 mg/L warfarin, and 5 ng/ml of mouse epidermal growth factor (EGF) (3). The CO:COLLECTION_SUMMARY suspended in SFM, were placed in a spheroid box (33 × 28 × 6 cm) custom-made CO:COLLECTION_SUMMARY polycarbonate by Mayo Division of Engineering and siliconized with Sigmacote CO:COLLECTION_SUMMARY 30 min (20) and gently rocked continuously at a frequency of 10 cycles per CO:COLLECTION_SUMMARY (0.17 Hz) to induce spheroid formation and to maintain spheroids in suspension. CO:COLLECTION_SUMMARY hepatocyte concentration was 5 × 105 cells/ml per spheroid box. All culture CO:COLLECTION_SUMMARY were maintained in a 5% CO2, 37°C incubator as previously described (20). CO:COLLECTION_SUMMARY were centrifuged and resuspended in fresh culture media every 24 h. #TREATMENT TR:TREATMENT_SUMMARY UW alone|UW + 1 mM Def|SFM alone|SFM + 1 mM Def|SFM + 1 mM Def + 1 µM CsA|SFM TR:TREATMENT_SUMMARY 1 µM CsA TR:TREATMENT_PROTOCOL_FILENAME PMID-23507348-Liu-Nydberg-CellTrans-2014.pdf TR:TREATMENT_PROTOCOL_COMMENTS Treatment annotation Cold 24 Indicates 24 hours cold storate prior to TR:TREATMENT_PROTOCOL_COMMENTS 48 Indicates 48 hours cold storate prior to treatment -96h Indicates treated TR:TREATMENT_PROTOCOL_COMMENTS 96 hours after cold storage -120h Indicates treated for 120 hours after cold TR:TREATMENT_COMPOUND University of Wisconsin (UW) solution|UW + deferoxamine (Def)| serum-free TR:TREATMENT_COMPOUND Sigma-Aldrich (St. Louis, MO, USA)|SFM + Def|SFM + Def + cyclosporin A TR:TREATMENT_COMPOUND + CsA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After 48 h of continuous rocking, the spheroids were washed with SFM at room SP:SAMPLEPREP_SUMMARY The culture medium was changed to the cold storage medium (UW alone, UW + 1 mM SP:SAMPLEPREP_SUMMARY SFM alone; SFM + 1 mM Def; SFM + 1 mM Def + 1 ?M CsA; SFM + 1 ?M CsA) at room SP:SAMPLEPREP_SUMMARY Spheroids were left in rocked culture without cold storage as control. SP:SAMPLEPREP_SUMMARY intended for cold storage were rocked in glass dishes (10 × 8 × 2 cm) SP:SAMPLEPREP_SUMMARY by Mayo Division of Engineering and siliconized with Sigmacote for 30 min and SP:SAMPLEPREP_SUMMARY transferred into 50-ml conical tubes (BD Falcon; 1 × 106cells/ml, 20 ml in SP:SAMPLEPREP_SUMMARY and put on ice for 1 h. Of note, spheroid box and glass dishes differ in size SP:SAMPLEPREP_SUMMARY volume capacity) but possess similar properties of spheroid formation and SP:SAMPLEPREP_SUMMARY Finally, tubes of spheroids were placed in a refrigerator to be cold stored at SP:SAMPLEPREP_SUMMARY After 24 or 48 h of cold storage treatment, the spheroids were centrifuged at SP:SAMPLEPREP_SUMMARY × g for 5 min, and the supernatant fluid was removed. Warm SFM (20 ml) at SP:SAMPLEPREP_SUMMARY was added to each tube. The tubes were mixed gently prior to adding 20 ml of SP:SAMPLEPREP_SUMMARY suspension to each glass culture dish and continued to rocking culture. SP:SAMPLEPREP_SUMMARY were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Concentrations of diazepam and its two major metabolites (nordiazepam and CH:CHROMATOGRAPHY_SUMMARY were determined by high-performance liquid chromatography (HPLC) with mass CH:CHROMATOGRAPHY_SUMMARY detection in the CTSA Metabolomics Core lab at Mayo Clinic. CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Mayo Clinic Metabolomics Core #MS MS:INSTRUMENT_NAME - MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ng/ml MS_METABOLITE_DATA_START Samples 1 17 33 20 36 4 18 2 34 19 3 35 25 9 21 5 13 29 12 28 24 8 16 32 42 39 45 10 26 40 22 37 6 14 30 43 11 27 41 23 38 7 15 31 44 Factors Treatment:20uM diazepam Treatment:20uM diazepam Treatment:20uM diazepam Treatment:Control group 120h Treatment:Control group 120h Treatment:Control group 120h Treatment:Control group 72h Treatment:Control group 72h Treatment:Control group 72h Treatment:Control group 96h Treatment:Control group 96h Treatment:Control group 96h Treatment:SFM cold 24-120h Treatment:SFM cold 24-120h Treatment:SFM cold 24-96h Treatment:SFM cold 24-96h Treatment:SFM cold 48-120h Treatment:SFM cold 48-120h Treatment:SFM +CsA cold 24-120h Treatment:SFM +CsA cold 24-120h Treatment:SFM +CsA cold 24-96h Treatment:SFM +CsA cold 24-96h Treatment:SFM +CsA cold 48-120h Treatment:SFM +CsA cold 48-120h Treatment:SFM +CsA+Gly cold 24-120h Treatment:SFM +CsA+Gly cold 24-96h Treatment:SFM +CsA+Gly cold 48-120h Treatment:SFM+Def cold 24-120h Treatment:SFM+Def cold 24-120h Treatment:SFM+Def cold 24-120h Treatment:SFM+Def cold 24-96h Treatment:SFM+Def cold 24-96h Treatment:SFM+Def cold 24-96h Treatment:SFM+Def cold 48-120h Treatment:SFM+Def cold 48-120h Treatment:SFM+Def cold 48-120h Treatment:SFM +Def +CsA cold 24-120h Treatment:SFM +Def +CsA cold 24-120h Treatment:SFM +Def +CsA cold 24-120h Treatment:SFM +Def +CsA cold 24-96h Treatment:SFM +Def +CsA cold 24-96h Treatment:SFM +Def +CsA cold 24-96h Treatment:SFM +Def +CsA cold 48-120h Treatment:SFM +Def +CsA cold 48-120h Treatment:SFM +Def +CsA cold 48-120h diazepam 4481.5725 5736.6630 5623.3315 1624.1825 956.1976 726.7814 1633.6693 1621.7624 1264.7138 1897.2045 1045.1085 1328.6425 2411.7352 2148.4238 1810.5472 1908.3052 2474.8026 2351.5498 2776.9633 2073.6053 2116.1659 2116.6115 2727.2512 2099.1453 1318.7897 1900.1499 2066.6119 662.5650 2781.7872 2529.0427 2011.8566 1396.4370 1444.2694 2114.3240 2328.9302 1788.3991 1685.9612 2494.7350 1947.2209 2389.4712 1697.0849 1699.9506 1649.3030 1956.9368 2769.8399 nordiazepam 120.4401 320.1140 331.5166 144.0672 426.4867 188.2435 111.2503 565.4192 469.4874 3.5452 23.2656 23.8233 19.4455 5.4840 7.5175 6.5456 2.4504 27.2201 8.9072 4.3846 3.6653 183.1366 152.2262 45.5407 256.2231 62.0669 419.4401 102.7936 127.1815 239.4604 82.5938 47.6665 40.2152 669.9298 47.8024 227.3917 105.4923 119.4808 302.1144 69.3379 50.0816 57.3156 oxazepam 33.3808 329.6473 529.4960 13.1518 263.3279 93.3722 18.0641 696.8769 365.7868 2.2263 0.4201 0.0000 76.7970 23.1458 0.9956 289.0782 6.3126 165.7055 1.6718 36.1355 136.4053 6.6902 0.1394 0.8202 354.0988 4.5315 88.7497 1.6816 21.7052 180.4597 5.1440 0.3173 1.4070 temezepam 313.1072 309.0023 247.3904 84.5079 268.8166 157.2228 209.4556 314.0752 388.5075 2.4173 85.1736 6.1321 17.2721 2.0203 2.1027 21.6403 0.8599 7.1088 6.6813 1.7196 1.1660 283.6135 119.1452 20.6709 231.7318 145.8304 517.8487 33.7706 94.2949 223.2863 71.1168 15.7583 16.5374 463.0140 105.2445 431.0494 36.8710 108.4368 277.1309 50.7277 16.1774 26.7043 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name moverz_quant ri ri_type pubchem_id inchi_key kegg_id other_id other_id_type diazepam MAYO_ID nordiazepam MAYO_ID oxazepam MAYO_ID temezepam MAYO_ID METABOLITES_END #END