A DIY Bioreactor for in Situ Metabolic Tracking in 3D Cell Models via Hyperpolarized 13C NMR Spectroscopy

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a valuable diagnostic tool limited by low sensitivity due to low nuclear spin polarization. Hyperpolarization techniques, such as dissolution dynamic nuclear polarization, significantly enhance sensitivity, enabling real-time tracking of cellular metabolism. However, traditional high-field NMR systems and bioreactor platforms pose challenges, including the need for specialized equipment and fixed sample volumes. This study introduces a scalable, 3D-printed bioreactor platform compatible with low-field NMR spectrometers, designed to accommodate bioengineered 3D cell models. The bioreactor is fabricated using biocompatible materials and features a microfluidic system for media recirculation, ensuring optimal culture conditions during NMR acquisition and cell maintenance. We characterized the NMR compatibility of the bioreactor components and confirmed minimal signal distortion. The bioreactor's efficacy was validated using HeLa and HepG2 cells, demonstrating prolonged cell viability and enhanced metabolic activity in 3D cultures compared to 2D cultures. Hyperpolarized [1-¹³C] pyruvate experiments revealed distinct metabolic profiles for the two cell types, highlighting the bioreactor's ability to discern metabolic profiles among samples. Our results indicate that the bioreactor platform supports the maintenance and analysis of 3D cell models in NMR studies, offering a versatile and accessible tool for metabolic and biochemical research in tissue engineering. This platform bridges the gap between advanced cellular models and NMR spectroscopy, providing a robust framework for future applications in nonspecialized laboratories. The design files for the 3D printed components are shared within the text for easy download and customization, promoting their use and adaptation for further applications.

Figure 1

Bioreactor design: 3D renderings of the custom cap piece (A, B), stopper (D, E) and schematic of their assembly in an NMR tube (C). (F) Photos showing the individual components and their assembly.

Figure 2

Detection area characterization in the 1.4 T NMR benchtop Pulsar instrument. Schematic representation of the maximum detection region in an NMR tube determined by ¹H NMR water analysis.

Figure 3

Validation of the bioreactor NMR compatibility. The ¹H NMR spectra of the 10% H₂O in D₂O samples are shown in either blue (shimming after the addition of each BR component) or red (only shimming at the beginning of the experiment before adding any BR components). The four variations tested were: (A) NMR tube (no bioreactor components). (B) PDMS spacer. (C) PDMS spacer and bioreactor stopper. (D) PDMS spacer, bioreactor stopper, and two CMC-scaffolds (i.e., all components of the bioreactor in the NMR-active region).

Figure 4

Validation of the 3D cell model and bioreactor recirculation system. (A) Structure of the cryogel through brightfield optical microscopy. (B) Structure of the cryogel through microscope confocal imaging of the fluoresceinamine stained cryogels. (C) HeLa cell clusters self-assembled in the cryogel after seeding as seen through confocal imaging. Nuclei in blue (Hoechst 33342), cytoskeleton microtubules in green (ViaFluor 488) and apoptotic cells in red (Propidium Iodide). (D) Metabolic activity of HeLa cells cultured in 2D (in a plate) or 3D (in a CMC scaffold) for up to 11 days, measured using alamarBlue. (E) Metabolic viability of the 3D constructs assessed using alamarBlue after 20 h with and without media recirculation in the BR or NMR tube, respectively. (F) Metabolic activity of the cell-laden scaffolds 1 h after injection of non-HP pyruvate solution, with and without flow in the BR. Data are normalized to cell number at the time of seeding and are presented as mean ± SD of at least three independent experiments.

Figure 5

Picture of the setup used for 3D cell construct experiments. The BR containing the 3D cell model was placed inside the 1.4 T benchtop and connected to a reservoir of fresh cell media at culture conditions inside and incubator. The peristaltic pump at 0.2 mL/min was used for media recirculation between the reservoir and the BR.

Figure 6

Metabolic study in the bioreactor of HeLa and HepG2 cell models. (A) Representative dynamically acquired ¹³C NMR spectra of HepG2-laden 3D scaffolds every 5 s after injection of hyperpolarized pyruvate solution. (B) Plot of the integral values of the pyruvate and lactate signals from the ¹³C NMR spectra of a single experiment over time. (C) Sum-up of the dynamic spectra containing metabolic data presented in panel A. The SNR on the ¹³C pyruvate resonance was 750:1. (D) Calculated lactate-to-pyruvate ratios obtained from the sum-up spectra for either HeLa 2D and 3D models (N = 3 and 5 respectively) or HepG2 2D and 3D models (N = 10 and 5 respectively). Data are presented as mean ± SD.