研究目的
To design and optimize a simple, static preservation system for shipping tissue-engineered cell sheets, specifically retinal pigment epithelium (RPE) cell sheets, to minimize costs, human error, and mechanical damage during transit.
研究成果
The designed shipping system effectively minimizes physical and chemical stress on RPE cell sheets during transit, maintaining cell integrity and function. It reduces costs and human error, facilitating the sharing of engineered tissues between laboratories and hospitals to enhance patient care in regenerative medicine.
研究不足
The system may not be applicable to more fragile cell types or tissues beyond RPE sheets. Additional tests with other tissue types are required. The study did not test air shipping, and the weight of the system could be further optimized for shorter distances.
1：Experimental Design and Method Selection:
The study involved designing a shipping system with insulated containers and latent heat storage materials to maintain stable temperatures. Methods included turbulence tests, transepithelial electrical resistance (TER) measurements, temperature and mechanical stress monitoring, cell death staining, ELISA for secretion detection, and gene expression analysis.
2：Sample Selection and Data Sources:
Human iPS cell-derived and fetal RPE cell sheets were used, cultured under specific conditions. Samples were prepared and tested before and after shipping.
3：List of Experimental Equipment and Materials:
Equipment included heat insulating containers (iP-TEC? Premier BOX-V
4：5), latent heat storage materials (iP-TEC? Temperature Stabilizer-24), vortex mixer (Vortex-Genie 2), Millicell? ERS-2 Voltohmmeter, temperature loggers (Thermochron Type-SL), gravity-shock recorder (G-MEN DR 20), fluorescence microscope, and various culture media and reagents. Experimental Procedures and Operational Workflow:
Procedures involved culturing RPE cells, preparing shipping vessels with optimized medium volume, performing transit tests by car and train, monitoring temperature and acceleration, and analyzing cell sheets post-shipping for morphology, TER, viability, gene expression, and secretion.
5：Data Analysis Methods:
Data were analyzed using statistical tests like Steel Dwass test for TER comparisons, and qualitative assessments for other parameters.
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iP-TEC Premier BOX-V8.5
BOX-V8.5
Sanplatec
Heat insulating container for shipping cell sheets, maintaining stable temperature.
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iP-TEC Temperature Stabilizer-24
Temperature Stabilizer-24
Sanplatec
Latent heat storage material to maintain temperature in the shipping container.
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Vortex-Genie 2
Vortex-Genie 2
Scientific Industries
Vortex mixer used for turbulence tests to induce mechanical stress.
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Millicell ERS-2 Voltohmmeter
ERS-2
Merck
Measure transepithelial electrical resistance (TER) to evaluate tight junction function.
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Thermochron Type-SL
Type-SL
KN Laboratories
Temperature logger to monitor temperature during shipping.
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G-MEN DR 20
DR 20
Sric Corporation
Gravity-shock recorder to monitor mechanical stress and acceleration during shipping.
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Transwell insert
Corning
Cell culture insert used to hold RPE cell sheets in the shipping vessel.
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StemFit AK03
AK03
Ajinomoto
Culture medium for human iPS cells.
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iMatrix-511
Nippi
Laminin-511 E8 coating for cell culture plates.
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KnockOut Serum Replacement
Invitrogen
Supplement for differentiation medium.
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SYTOX Green Nucleic Acid Stain
Thermo Fisher Scientific
Stain for cell death detection.
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Human VEGF-A Platinum ELISA Kit
eBioscience
Kit for ELISA detection of VEGF secretion.
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Human PEDF ELISA kit
BioVender
Kit for ELISA detection of PEDF secretion.
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RNeasy Micro Kit
Qiagen
Kit for RNA isolation.
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SuperScript III Reverse Transcriptase Kit
Invitrogen
Kit for cDNA preparation.
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