研究目的
To design and apply a novel bioreactor that connects fluorescence lifetime imaging microscopy (FLIM) and magnetic resonance spectroscopy (MRS) for studying cellular metabolism in 3D cell cultures, enabling complementary measurements at cellular and population scales.
研究成果
The bioreactor successfully integrates FLIM and MRS for metabolic studies in 3D cell cultures, demonstrating feasibility with glucose deprivation in 4 T1 cells. It enables multiscale imaging, bridging cellular and population-level metabolic changes, and provides a platform for future research on metabolic heterogeneity and interventions.
研究不足
Limitations include potential heterogeneity in drug perfusion into the collagen gel, reduced signal due to limited diffusion depth, use of a horizontal bore magnet limiting field strength and coil sensitivity, and need for further evaluation of long-term cell viability and gas environment control.
1：Experimental Design and Method Selection:
The study involved designing a bioreactor to maintain 3D cell cultures in a collagen matrix, compatible with both FLIM and MRS. Methods included FLIM for NADH fluorescence lifetime measurement and MRS for hyperpolarized 13C-pyruvate metabolism monitoring. A diffusion model for pyruvate into collagen gel was developed.
2：Sample Selection and Data Sources:
4 T1 murine breast cancer cells were cultured in 3D collagen gels. Samples were prepared with normal or glucose-free medium.
3：List of Experimental Equipment and Materials:
Custom bioreactor parts (e.g., polypropylene body, 3D-printed components), commercial parts (e.g., cover glass, rubber bands), and supporting equipment (e.g., multiphoton microscope, MRI scanner) were used. Materials included collagen, RPMI 1640 medium, and hyperpolarized pyruvate.
4：Experimental Procedures and Operational Workflow:
Cells were embedded in collagen gels in the bioreactor. FLIM was performed using a multiphoton microscope with 740 nm excitation, followed by MRS on a 4.7 T MRI scanner after injecting hyperpolarized pyruvate. Temperature was controlled at 36±1°C.
5：7 T MRI scanner after injecting hyperpolarized pyruvate. Temperature was controlled at 36±1°C. Data Analysis Methods:
5. Data Analysis Methods: FLIM data were analyzed using SPCImage software for mean fluorescence lifetimes and component fitting. MRS data were processed in MATLAB for Lac:Pyr ratios. Statistical analysis used Wilcoxon rank-sum test in R.
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ULS 6.75 Laser Cutter
ULS 6.75
Universal Laser Systems, Inc.
Laser cutter used to fabricate the optical window silicone gasket from silicone sheet.
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MRI Scanner
4.7 T preclinical horizontal bore
Agilent
Used for magnetic resonance spectroscopy (MRS) to measure hyperpolarized 13C-pyruvate metabolism in 3D cell cultures.
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HyperSense Dynamic Nuclear Polarizer
HyperSense
Oxford Instruments
Used to polarize [1-13C] pyruvic acid for hyperpolarized MRS experiments.
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Cover Glass
#1.5
Corning Life Sciences
Serves as the optical window for FLIM imaging, allowing light transmission for microscopy.
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Bandpass Filter
450/70
Semrock
Used in the FLIM setup to collect emitted light from NADH fluorescence.
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Haas Super Mini Mill 2
Super Mini Mill 2
Haas Automation, Inc.
Computer numerical control machining center used to fabricate the bioreactor body from polypropylene stock material.
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Perfactory 3 3D Printer
Perfactory 3
EnvisionTEC Inc.
3D printer used to fabricate custom components of the bioreactor, specifically the optical window compression piece and bioreactor top piece.
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Multiphoton Laser Scanning Microscope
Custom built around Nikon TE300
Nikon
Used for fluorescence lifetime imaging microscopy (FLIM) to measure NADH fluorescence lifetimes in 3D cell cultures.
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Volume Coil
Dual-tuned 1H/13C volume coil
Doty Scientific
Used for MR signal acquisition in the MRI scanner.
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Temperature Probe
MR temperature probe
SA Instruments Inc.
Used for temperature feedback during MRS experiments to maintain sample at 36±1°C.
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Diaphragm Pump
6-V DC diaphragm pump
UXCell
Used for pumping hot water through the bioreactor to maintain temperature during experiments.
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