Summary of Study ST000167

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 PR000145. The data can be accessed directly via it's Project DOI: 10.21228/M87596 This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST000167
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 hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 ?M cyclosporin A (CsA); SFM + 1 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
Email	Nyberg.scott@mayo.edu
Submit Date	2015-05-14
Num Groups	9
Total Subjects	45
Raw Data Available	No
Analysis Type Detail	LC-MS
Release Date	2015-06-28
Release Version	1

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Project:

Project ID:	PR000145
Project DOI:	doi: 10.21228/M87596
Project Title:	Cold Storage of Rat Hepatocyte Spheroids
Project Summary:	Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 ?M cyclosporin A (CsA); SFM + 1 mM Def + 1 ?M CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.
Institute:	Mayo Clinic
Department:	Division of Experimental Surgery
Last Name:	Nyberg
First Name:	Scott
Email:	Nyberg.scott@mayo.edu
Funding Source:	This work was funded by grants from NIH (RO1-DK56733), the Marriott Foundation, the Wallace H. Coulter Foundation, the 12th Five-Year National Science and Technology Major Project for Infectious Diseases, China (No. 2012 ZX 10002004-005, Hongling Liu), and Jiangsu province government, China (Yue Yu). The authors declare no conflicts of interest.

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:

Subject type: Animal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA009095	33	20uM diazepam
SA009096	17	20uM diazepam
SA009097	1	20uM diazepam
SA009098	36	Control group 120h
SA009099	20	Control group 120h
SA009100	4	Control group 120h
SA009101	18	Control group 72h
SA009102	2	Control group 72h
SA009103	34	Control group 72h
SA009104	35	Control group 96h
SA009105	19	Control group 96h
SA009106	3	Control group 96h
SA009107	25	SFM cold 24-120h
SA009108	9	SFM cold 24-120h
SA009109	21	SFM cold 24-96h
SA009110	5	SFM cold 24-96h
SA009111	29	SFM cold 48-120h
SA009112	13	SFM cold 48-120h
SA009113	12	SFM +CsA cold 24-120h
SA009114	28	SFM +CsA cold 24-120h
SA009115	24	SFM +CsA cold 24-96h
SA009116	8	SFM +CsA cold 24-96h
SA009117	32	SFM +CsA cold 48-120h
SA009118	16	SFM +CsA cold 48-120h
SA009119	42	SFM +CsA+Gly cold 24-120h
SA009120	39	SFM +CsA+Gly cold 24-96h
SA009121	45	SFM +CsA+Gly cold 48-120h
SA009131	26	SFM+Def cold 24-120h
SA009132	10	SFM+Def cold 24-120h
SA009133	40	SFM+Def cold 24-120h
SA009134	22	SFM+Def cold 24-96h
SA009135	37	SFM+Def cold 24-96h
SA009136	6	SFM+Def cold 24-96h
SA009137	30	SFM+Def cold 48-120h
SA009138	43	SFM+Def cold 48-120h
SA009139	14	SFM+Def cold 48-120h
SA009122	27	SFM +Def +CsA cold 24-120h
SA009123	11	SFM +Def +CsA cold 24-120h
SA009124	41	SFM +Def +CsA cold 24-120h
SA009125	23	SFM +Def +CsA cold 24-96h
SA009126	7	SFM +Def +CsA cold 24-96h
SA009127	38	SFM +Def +CsA cold 24-96h
SA009128	31	SFM +Def +CsA cold 48-120h
SA009129	15	SFM +Def +CsA cold 48-120h
SA009130	44	SFM +Def +CsA cold 48-120h

Showing results 1 to 45 of 45

Showing results 1 to 45 of 45

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 Name:	-
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	POSITIVE