Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA009119

Local Sample ID:	42
Subject ID:	SU000186
Subject Type:	Animal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Genotype Strain:	SpragueDawley rats
Weight Or Weight Range:	300400 g
Species Group:	Mammal

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Subject:

Subject ID:	SU000186
Subject Type:	Animal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116
Genotype Strain:	SpragueDawley rats
Weight Or Weight Range:	300400 g
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
42	SA009119	FL002152	SFM +CsA+Gly cold 24-120h	Treatment

Collection:

Collection ID:	CO000172
Collection Summary:	Hepatocytes were isolated from male SpragueDawley rats (300400 g; Harlan, Indianapolis, IN, USA) by a two-step perfusion method as previously described (33). All harvests yielded hepatocytes with viability exceeding 95% by trypan blue dye exclusion. Freshly isolated hepatocytes were suspended in SFM, composed of Williams E supplemented with 0.2 U/ml insulin, 6 ?g/ml transferrin, 100 U/ml penicillin G, 100 mg/ml streptomycin, 3 g/L human albumin, 2.2 g/L sodium bicarbonate, 1 g/L L-carnitine, 2.0 mM L-glutamine, 100 nM dexamethasone, 40 ng/ml glucagon, 20 ng/ml Gly-His-Lys, 1,000 U/L heparin, 1 mg/L warfarin, and 5 ng/ml of mouse epidermal growth factor (EGF) (3). The cells, suspended in SFM, were placed in a spheroid box (33 × 28 × 6 cm) custom-made of polycarbonate by Mayo Division of Engineering and siliconized with Sigmacote for 30 min (20) and gently rocked continuously at a frequency of 10 cycles per minute (0.17 Hz) to induce spheroid formation and to maintain spheroids in suspension. Final hepatocyte concentration was 5 × 105 cells/ml per spheroid box. All culture conditions were maintained in a 5% CO2, 37°C incubator as previously described (20). Spheroids were centrifuged and resuspended in fresh culture media every 24 h.
Sample Type:	Liver

Treatment:

Treatment ID:	TR000192
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 treatment Cold 48 Indicates 48 hours cold storate prior to treatment -96h Indicates treated for 96 hours after cold storage -120h Indicates treated for 120 hours after cold storage
Treatment Compound:	University of Wisconsin (UW) solution\|UW + deferoxamine (Def)\| serum-free medium; Sigma-Aldrich (St. Louis, MO, USA)\|SFM + Def\|SFM + Def + cyclosporin A (CsA)\|SFM + CsA

Sample Preparation:

Sampleprep ID:	SP000186
Sampleprep Summary:	After 48 h of continuous rocking, the spheroids were washed with SFM at room temperature. The culture medium was changed to the cold storage medium (UW alone, UW + 1 mM Def; SFM alone; SFM + 1 mM Def; SFM + 1 mM Def + 1 ?M CsA; SFM + 1 ?M CsA) at room temperature. Spheroids were left in rocked culture without cold storage as control. Spheroids intended for cold storage were rocked in glass dishes (10 × 8 × 2 cm) custom-made by Mayo Division of Engineering and siliconized with Sigmacote for 30 min and then transferred into 50-ml conical tubes (BD Falcon; 1 × 106cells/ml, 20 ml in total) and put on ice for 1 h. Of note, spheroid box and glass dishes differ in size (i.e., volume capacity) but possess similar properties of spheroid formation and culture. Finally, tubes of spheroids were placed in a refrigerator to be cold stored at 4°C. After 24 or 48 h of cold storage treatment, the spheroids were centrifuged at 50 × g for 5 min, and the supernatant fluid was removed. Warm SFM (20 ml) at 37°C was added to each tube. The tubes were mixed gently prior to adding 20 ml of cell suspension to each glass culture dish and continued to rocking culture. Cultures were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere.

Sampleprep ID:

SP000186

Sampleprep Summary:

After 48 h of continuous rocking, the spheroids were washed with SFM at room temperature. The culture medium was changed to the cold storage medium (UW alone, UW + 1 mM Def; SFM alone; SFM + 1 mM Def; SFM + 1 mM Def + 1 ?M CsA; SFM + 1 ?M CsA) at room temperature. Spheroids were left in rocked culture without cold storage as control. Spheroids intended for cold storage were rocked in glass dishes (10 × 8 × 2 cm) custom-made by Mayo Division of Engineering and siliconized with Sigmacote for 30 min and then transferred into 50-ml conical tubes (BD Falcon; 1 × 106cells/ml, 20 ml in total) and put on ice for 1 h. Of note, spheroid box and glass dishes differ in size (i.e., volume capacity) but possess similar properties of spheroid formation and culture. Finally, tubes of spheroids were placed in a refrigerator to be cold stored at 4°C. After 24 or 48 h of cold storage treatment, the spheroids were centrifuged at 50 × g for 5 min, and the supernatant fluid was removed. Warm SFM (20 ml) at 37°C was added to each tube. The tubes were mixed gently prior to adding 20 ml of cell suspension to each glass culture dish and continued to rocking culture. Cultures were maintained in a humidified incubator at 37°C with a 5% CO2 atmosphere.

Combined analysis:

Analysis ID	AN000261
Analysis type	MS
Chromatography type
Chromatography system
Column
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name
Ion Mode	POSITIVE
Units	ng/ml

Chromatography:

Chromatography ID:	CH000184
Chromatography Summary:	Concentrations of diazepam and its two major metabolites (nordiazepam and temazepam) were determined by high-performance liquid chromatography (HPLC) with mass spectrometry detection in the CTSA Metabolomics Core lab at Mayo Clinic.

MS:

MS ID:	MS000210
Analysis ID:	AN000261
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE