Bioreactor for in vitro optical fluorescence and magnetic resonance spectroscopy
Benjamin L Cox1,2,3, Joseph M Szulczewski3,4, Kai D Ludwig1, Erin B Adamson1, Robert A Swader2, Sarah A Erickson-Bhatt2,3,4, Patricia J Keely4, Kevin W Eliceiri1,2,3,5, and Sean B Fain1,6

1Medical Physics, University of Wisconsin - Madison, Madison, WI, United States, 2Medical Engineering, Morgridge Institute for Research, Madison, WI, United States, 3Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin - Madison, Madison, WI, United States, 4Cell and Regenerative Biology, University of Wisconsin - Madison, Madison, WI, United States, 5Biomedical Engineering, University of Wisconsin - Madison, Madison, WI, United States, 6Radiology, University of Wisconsin - Madison, Madison, WI, United States


The design and implementation of a novel bioreactor capable of facilitating both magnetic resonance spectroscopy (MRS) and optical fluorescence microscopy for complementary metabolic information is described. Fluorescence lifetime imaging (FLIM) of nicotinamide adenine dinucleotide (NADH) and hyperpolarized [1-13C] pyruvic acid (PA) MRS of human breast cancer cells in a 3D collagen matrix is demonstrated. The system provides a novel test-bed for simulating cell-matrix and cell-cell interactions in a 3D microenvironment for investigating multi-scale cellular metabolism in vitro.


Tumor metabolism is increasingly recognized as an important feature and potential therapy target in cancer1. However, many aspects of the metabolism of tumor cells and their interactions with the surrounding microenvironment are not fully understood. Fluorescence lifetime imaging (FLIM)2 based optical microscopy and hyperpolarized 13C magnetic resonance spectroscopy (MRS)3 are imaging methods that enable in vitro and in vivo imaging of cellular metabolism. FLIM can measure the rate of decay of a molecule’s fluorescence, including the metabolic cofactor nicotinamide adenine dinucleotide (NADH) whose fluorescence lifetime depends on its protein binding state allowing visualization of metabolic processes that are dominant within a cell4. Hyperpolarized MRS most commonly uses hyperpolarized [1-13C] pyruvic acid to measure metabolic products of glycolysis, especially lactate production globally for in vitro cell culture and at the tumor or organ level for in vivo studies3. As these techniques occur on very different scales they can yield complementary information at the cellular and macroscopic scales to investigate cell-cell and cell-matrix interactions and possible effects on global metabolism (Figure 1). Here, we present a multi-modal and multi-scale bioreactor capable of combined MRS and optical microscopy in 3D in vitro cell culture. The ability to image in a 3D cell culture configuration enables investigation of fundamental components mimicking the in vivo microenvironment.


The bioreactor (Figure 2) was designed using SolidWorks software (Dassault Systemes, Velizy-Villacoublay, France). All parts will be made open-source, including the designs for the four custom components. Pictures of the experimental setup for both optical microscopy (Figure 3) and MRS experiments (Figure 4) are shown. Key supporting systems are labeled, including the heating system used for temperature control during MRS experiments (a temperature controlled box around the microscope itself is used for optical microscopy).

Collagen and green fluorescent protein (GFP) expressing MDA-MB-231 human breast cancer cells were used for all experiments as previously described5. In brief, stock collagen was neutralized for 1 hour with 2x HEPES buffer, then further diluted to 3mg/mL with cells and media. 4-8 million cells/mL were used. After polymerization of collagen, cells were allowed to grow overnight in an incubator prior to experiments. Optical imaging was performed on a custom multiphoton laser scanning system built around an inverted Nikon TE300 microscope6. MRS experiments were performed in a 4.7T pre-clinical MRI scanner (Agilent, Palo Alto, CA) using a temporally resolved pulse acquire sequence.

For MRS experiments, [1-13C] pyruvic acid (PA) was hyperpolarized as previously described7,8, but using a modified dissolution protocol. Briefly, 30µL aliquots of [1- 13C] PA (Cambridge Isotope Laboratories Inc., Tewksbury, MA) and 15mM trityl radical (Ox063, GE Healthcare) were polarized in a Hypersense polarizer (Oxford Instruments) for ~1 hour. Samples were dissoluted with 4 mL of solvent containing 1.2mL 426 mM NaOH, 1.4mL 400 mM Tris buffer and 1.4mL 250mg/L EDTA. [1-13C] PA was drawn off and 400µL were rapidly injected into the sample volume of the bioreactor. Dynamic global spectra (FA=10º, TR=3000ms) were acquired with a 13C surface coil (Varian) with transmit and receive frequencies centered on the expected pyruvate resonance.


Optical microscopy data (Figure 3) shows the fluorescence signal from cells labeled by expression of GFP signal overlaid on second harmonic generation (SHG) signal of the collagen gel matrix which comprises the 3D microenvironment. Cellular metabolism is indicated by NADH fluorescence intensity and NADH fluorescence lifetime images. Note that the NADH fluorescence lifetimes vary across individual cells, indicating regions of both free (short lifetimes) and protein-bound (long lifetimes) NADH typical of normal cellular metabolism.

Dynamic spectra and a corresponding time-averaged spectrum (Figure 4) from a MRS experiment are also shown. Lactate, alanine and urea peaks are labeled in the time-averaged spectrum. Lactate production, in particular, is visible, but low. Circulating warm water worked well for temperature control, maintaining a sample temperature of 37±0.5 oC during all experiments.


Alanine is relatively high compared to the lactate peak, which most likely reflects the cells in a glucose-rich and fully oxygenated state consistent with the longer observed fluorescence lifetimes observed at the cellular level. Future work will aim to study the effect of the collagen matrix, fasting, and oxygenation in different cancer cell lines.


A bioreactor system enabling combined MRS and optical studies of 3D cell cultures in vitro is demonstrated. Hyperpolarized [1-13C] PA MRS (global metabolism) and SHG (collagen), GFP (protein expression), and NADH fluorescence lifetime (cellular metabolism) optical data are shown. This system represents a novel approach to achieving multimodal and multiscale measurement of metabolism in 3D cell cultures that mimic the cancer microenvironment in vitro.


The authors would like to thank the Laboratory for Optical and Computational Instrumentation (LOCI) and the Morgridge Institute for Research for their continued support of this project. The authors would also like to acknowledge support from the Morgridge Postoctoral Program, the Departments of Medical Physics and Radiology at the authors' institution, the AAPM Graduate Fellowship, and GE Healthcare.


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Schematic showing the bioreactor concept: Combining techniques on different scales like MRS and FLIM allows the collection of complementary metabolic and morphological information on both the atomic and cellular imaging scales. Scale bar is 50 microns.

Block diagram of the bioreactor layout with key systems labeled (Top). Warm water (Blue) is pumped around the sample volume (Pink) using a circulating pump. Media is slowly streamed through the sample volume using a peristaltic pump. An optical window assembly on the bottom of the bioreactor allows for optical imaging on an inverted microscope. Injections can be performed through a tube into the top of the sample volume for MRS experiments. Computer aided design (CAD) rendering of the bioreactor assembly (Bottom right) and an exploded view of that assembly (Bottom left).

Picture of the underside of the bioreactor with the collagen gel visible through the cover glass of the optical window (A). Sample optical data using a 20x air immersion objective (B-D) and using a 40x air immersion objective (E-G). Fluorescent signal from green fluorescent protein (GFP) in the cell line overlaid on second harmonic generation (SHG) signal of the collagen matrix (B,E). Intrinsic fluorescence from the reduced form of nicotinamide adenine dinucleotide (NADH) (C,F). Fluorescence lifetime of the NADH fluorescence (D,G).

Picture of the bioreactor setup during an MRS experiment (A). The temperature probe (red arrow), media flow lines (pink arrows), hot water flow lines (blue arrows) and injection tube (green arrow) are displayed. Dynamic spectra, taken over ~3 minutes, of MDA-231 human breast cancer cells embedded in a collagen matrix (B). Time-averaged spectra (C) with peaks labeled. The urea peak is from a thermal reference phantom placed adjacent to the sample during scanning.

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)