Chemical and Biological Sensor Capsules for Real-Time Measurement of Cell Properties in Bioreactors
Authors: Jin Decarle S. , Zhu Xingyuan , Brightbill Eleanor L. , Brown Billyde , Vogel Eric M. 
Published: July 2021

Introduction

Real-time bioprocess monitoring and control is needed for the scalable production and deployment of cancer cell therapies at reasonable cost. For example, CAR-T cell therapy shows promise as an effective treatment for cancer with high (83%) remission rates.1 However, the cost of these treatments is too expensive for their mass adoption.2 One reason for the steep price is the difficulty scaling production while ensuring delivery of an efficacious therapy to the patient. One factor preventing scalability is the lack of effective process analytical technology. As a potential solution to this issue, we have developed a fully integrated, wireless, 3D-printed sensor capsule to be used for multiplexed sensing of critical quality attributes (CQAs), namely pH, glucose, and lactate concentrations within cell bioreactors. CQAs are used not only as metrics for cell viability, but also as determinants of a cell’s ability to deliver efficacious treatment. Unlike current process monitoring technology, capsule sensors are buoyant and can be propelled using impellers within the bioreactor. This allows for measurements uniformly inside the bioreactor. To our knowledge, this is the first time that 3D-printed, wireless sensor capsules have been developed for use in cell bioreactors for cancer cell therapy.

Methods

The capsule consists of electrochemical sensors, and read-out and wireless transmission electronics integrated into a capsule made of a biocompatible polymer (Figure 1). The different sensors for glucose, lactate, and pH along with an Ag on-chip reference electrodes were fabricated using standard lithographic techniques on silicon substrates. The reference electrode consisted of a thin film of Ag deposited by e-beam evaporation that was later coated with polyvinyl butyral (PVB) to improve stability.3 Glucose and lactate sensing was achieved amperometrically using enzymes, glucose oxidase or lactase oxidase respectively, immobilized on a Pt working electrode biased to 0.7 V. pH sensing was achieved by depositing a pH-sensitive oxide on a gold electrode. A pH-sensitive Al2O3 or HfO2 layer was deposited through atomic layer deposition.
Figure 1 shows the capsule and sensor design. The capsule packaging was 3D printed using a Stratasys Connex 350 and was designed to be fully insulated from solution except for an opening on the top revealing the various sensors. The sensor chip is installed within the cap of the capsule which can be screwed off, but with wired connection to the electronics housed in the body of the capsule. This allows for simple replacement of the multiplexed sensor chip while retaining the capsule electronics, rechargeable battery, and packaging for reuse.

Results and Conclusions

Before testing within the capsule, the sensors were tested using microfluidics. Figure 2 shows the performance of the Ag/PVB on-chip pseudo reference electrode (pseudo-RE). The pseudo-RE shows a stable potential in different pH buffer solutions and shows similar sensitivity results for pH sensing when compared to a commercial reference electrode. By having a stable microfabricated pseudoRE, the sensing components will not be a limiting factor for miniaturization of the capsule. Figure 3 shows the response of the pH, lactate, and glucose sensors at different concentrations. The pH sensor shows high sensitivity (~53 mV/pH) over the range of pH 3 to 7. Glucose and lactate sensing was tested in 1X PBS. The glucose sensor shows a linear response to glucose concentrations from 0 to 25 mM validating the use of the sensor within cell culture media.4 The glucose sensor shows the same linear range when tested in human mesenchymal stem cell culture. In this case, the glucose concentration was first tested using a commercial sensor and then diluted to targeted glucose concentrations by dilution with PBS. The lactate sensor shows sensitivity in the concentration range of 1 to 5 mM with saturation occurring around ~6 mM. The capsule’s wireless sensing capability was validated using pH sensing in a beaker. The pH sensing results in Figure 4 were obtained wirelessly from the capsule and were nearly identical to the results measured using our microfluidic system and conventional parameter analyzer.
This work has successfully demonstrated wireless sensing using a 3D-printed sensor capsule and functionality of electrochemical sensors for multiple CQAs for cell growth in bioreactors. These sensor capsules can enable scalability of the cell manufacturing process while ensuring delivery of an efficacious treatment. Future work will focus on capsule sensor validation in cell growth media and eventually monitoring of cell growth in bioreactors.

References

[1] NOVARITIS. "Novartis Receives First Ever FDA Approval for a CAR-T Cell Therapy, Kymriah (TM)(CTL019), for Children and Young Adults with B-cell ALL That Is Eefractory or Has Relapsed At least Twice." (2019).
[2] Dolgin, Elie. "Bringing down the cost of cancer treatment." Nature 555.7695 (2018).
[3] Guinovart, Tomàs, et al. "A reference electrode based on polyvinyl butyral (PVB) polymer for decentralized chemical measurements." Analytica chimica acta 821 (2014): 72-80.
[4] “Glucose in Cell Culture.” Sigma Aldrich <https://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-expert/glucose.html>

Figure 1