Chemical and Biological Sensor Capsules for Real-Time Measurement of Cell Properties in Bioreactors
Authors: Jin Decarle Sibi , Zhu Xingyuan , Brightbill Eleanor L , Brown Billyde , Vogel Eric M. 
Published: May 2020

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 (70-90%) 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 technologies. 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, lactate, and IL-2 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 as well as data acquisition and wireless transmission electronics integrated into a capsule made of a biocompatible polymer (Figure 1). The sensors for glucose, lactate, pH, and IL-2 along with Ag on-chip pseudo-reference electrode (pseudo-RE) were fabricated using standard lithographic techniques on silicon substrates. The reference electrode consisted of a thin-film of Ag that was later coated with polyvinyl butyral (PVB) to improve stability.3 IL-2 and pH sensing was achieved using gold electrodes. A pH-sensitive Al2O3 or HfO2 layer is deposited through atomic layer deposition on the pH sensor to achieve pH-sensitivity. Glucose and lactate sensing was achieved amperometrically using enzymes, glucose or lactase oxidase respectively, immobilized on a Pt electrode biased to 0.7 V. Detection could also be achieved potentiometrically by using the pH sensor to detect changes in pH caused by conversion of the enzyme product, H2O2, to H+.

Figure 1 shows the capsule and sensor design. The capsule 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 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 sensor chip while retaining the capsule components 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 pseudo-RE. The pseudo-RE shows a stable potential in different pH buffer solutions and similar sensitivity results when compared to a commercial reference electrode for pH sensing. By having a stable microfabricated pseudoRE, the sensing components will not limit 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 1-6 mM with saturation at ~8 mM validating the use of the sensor within cell culture media.4 The lactate sensor shows sensitivity in the concentration range of 1-5 mM with saturation occurring around ~6 mM. The division of T cells requires aerobic glycolysis where glucose is converted to lactate. However, not all glucose is expected to be converted. The lactate concentration expected to be produced is ~1 mM.5 Selectivity towards IL-2 is achieved using anti-IL2 antibodies. Figure 4a shows the functionalization scheme for detection of IL-2. A thiol self-assembled monolayer (SAM) is attached to the gold electrode and then modified for amine conjugation using NHS/EDC. Anti-IL2 can then bind to the SAM and unbound sites are blocked with ethanolamine. Figure 4b reveals antibody attachment occurs optimally at pH 5. Surface plasmon resonance shows successful attachment of 10 ug/mL of IL-2 to the functionalized gold sensor. The next step is to demonstrate IL-2 sensing potentiometrically. The capsule’s wireless sensing capability was validated using pH sensing in a beaker. The pH sensing results in Figure 5b were obtained wirelessly from the capsule and were nearly identical to the results measured using our microfluidic system and Keithley-4200 lab instrument (Fig. 5a).

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 monitoring of cell growth in bioreactors.

References

[1] A. Ronson et al. "Treatment of relapsed/refractory acute lymphoblastic leukemia in adults." Current oncology reports (2016). doi:10.1007/s11912-016-0519-8

[2] E. Dolgin, "Bringing down the cost of cancer treatment." Nature (2018). doi:10.1038/d41586-018-02483-3

[3] G. Tomàs et al. "A reference electrode based on PVB polymer for decentralized chemical measurements." Analytica chimica acta (2014) doi:10.1016/j.aca.2014.02.028

[4] J.R. Sarah et al. "Glucose uptake is limiting in T-cell activation and requires CD28-mediated Akt-dependent and independent pathways." The Journal of Immunology (2008): doi: 10.4049/jimmunol.180.7.4476

[5] G.T. James et al. "Extracellular lactate: a novel measure of T cell proliferation." The Journal of Immunology (2018) doi:10.4049/jimmunol.1700886

Figure 1