On-Sensor Cryopreservation of Adherent Cells in Lab-on-a-Chip Systems
Authors: Ozsoylu Dua , Isik Tugba , Demir Mustafa Muammer , Kizildag Sefa , Schoening Michael J. , Wagner Torsten 
Published: July 2021

Introduction

Biochemical sensors are one of the essential tools for cell-based assays used in major research fields including drug discovery, cancer research, and immunology. Although significant efforts have been made to develop new technologies and tools, most existing systems in this field are still not suitable for “ready‐to‐use in-field” applications, as the cells inside the assay need vital parameters, such as oxygen, nutrients, and temperature and therefore will not survive during a transportation to an end user. Nevertheless, providing these parameters is not practicable during conventional transportation either. Those systems need to be assembled and prepared on-site, which often requires a cell culture laboratory setting and trained staff. Thus, there is an urgent need for preserving the whole optimized assay system to utilize them for concepts like “ready-to-use” or “on-demand-use”.
Cryopreservation, the process of freezing and preserving the cells at sub-zero temperatures is a well-known technique for cell culturing. It could be one possible solution to solve the transportation and storage challenges in the fields mentioned above. However, freezing of adherent cells is more prone to the cell membrane damage and cell detachment, due to ice crystal formation and a mismatch in the linear thermal expansion between the frozen cell membrane and the rigid substrate [1]. Therefore, innovative methods and tools must be implemented to overcome these challenges.
In this work, for the first time, the surface of a field-effect-based sensor was modified with flexible electrospun fibers to allow on-sensor cryopreservation. Furthermore, a protocol was developed to freeze and to thaw adherent cells in a microfluidic channel.

Method

The light-addressable potentiometric sensor (LAPS), a field-effect-based biochemical sensor, can detect the extracellular acidification of the cells due to its pH sensitive transducer layer (Ta2O5). The LAPS was fabricated as a structure of Al/p-Si/SiO2/Ta2O5 as described in ref. [2]. The sensor surface was modified with flexible polyethylene vinyl acetate (PEVA) fibers using an electrospinning method. The modified LAPS chip was then fixed to the underside of a bottomless microfluidic slide to form a miniaturized device (cryo-chip) (Figure 1(a) and (b)). A flexible polymer coverslip and a conventional LAPS were used as control samples. All samples were sterilized by 70% ethanol for one hour under aseptic conditions in a laminar flow cabinet to maintain the sterility. Chinese hamster ovary (CHO-K1) cells were adherently cultured inside the channels of the device with the mixture of Ham’s Nutrient Mixture F-12/Dulbecco’s Modified Eagle Medium (Ham’s F-12/DMEM, 1:2 mixture, pH 7) supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin. After the cell culture inside the channels were obtained for 48h under cell culture conditions in an incubator, the entire device was placed in a 3D-printed freezing container and frozen at -80oC using a cryoprotectant solution (90% fetal calf serum supplemented with 10% dimethyl sulfoxide). The samples were kept in the freezer overnight. The frozen device was then thawed rapidly by transferring them directly to the incubator (37oC). The cryoprotectant solution inside the channels was removed by washing with the fresh medium (37oC) four times, as soon as the ice crystals disappeared. Cell recovery and cell viability were analyzed before and after cryopreservation using a dye exclusion assay and a cell counting kit (CCK)-8 assay. In addition, as proof-of-concept measurements, the extracellular acidification of the cells was monitored by the LAPS chip, after the cryopreservation.

Results and Conclusions

The results showed that the sensor surface modified with the electrospun PEVA fibers exhibits enhanced biocompatibility, which promotes cell proliferation and spreading. In addition, the novel cryo-chip system using this hybrid structure (rigid/flexible) is effective compared to an un-modified LAPS surface for keeping cells viable during on-sensor cryopreservation. These findings showed, for the first time, that a cryopreservation on-chip is possible and that microfluidic systems including semiconductor based sensors can survive the cryopreservation treatment. Thus, this kind of cryo-chip systems enable on-sensor cryopreservation and measurement which can open up a new possibility for “ready-to-use, in field” applications. Being able to prepare the entire system at the manufacturing stage and to send them via a cold-chain transport further eliminate potential run-to-run and operator-to-operator variability, resulting in more reproducible results.

Acknowledgements

Dua Özsoylu (DÖ) would like to acknowledge the PhD research scholarship grant from Scientific and Technological Research Council of Turkey (TÜBITAK) under BIDEB 2214-A. The authors also gratefully thank the Federal Ministry of Education and Research of Germany (Opto-Switch FKZ: 13N12585). This study is a part of PhD thesis study of DÖ (thesis number DEU. HSI. PhD-2013970198) in Dokuz Eylül University, Institute of Health Sciences, Turkey.

References

[1] T. Rutt, N. Eskandari, M. Zhurova, J.A.W. Elliott, L.E. McGann, J.P. Acker, J.A. Nychka, Thermal expansion of substrate may affect adhesion of Chinese hamster fibroblasts to surfaces during freezing, Cryobiology. 86 (2019), 134–139. doi.org/10.1016/j.cryobiol.2018.10.006.
[2] D. Özsoylu, S. Kizildag, M.J. Schöning, T. Wagner, Effect of plasma treatment on the sensor properties of a light-addressable potentiometric sensor (LAPS), physica status solidi (a). 216 (2019), 1900259. doi.org/10.1002/pssa.201900259.

Figure 1: Photos of the cryo-chip system for on-sensor cryopreservation. a) The modified LAPS chip integrated in a microfluidic chip system and its complementary connections; b) culturing of the CHO-K1 cells inside the channels on the LAPS surface, which are modified by electrospun PEVA fibers.

Figure 1