Multilayer Microfluidic Chip with Electrospun Biomaterial Mesh for Tumor Cell Retention and Culture

Introduction

Cancer remains a leading cause of mortality worldwide, with nearly 10 million deaths in 2020. Projections estimate up to 7,000 colorectal cancer (CRC) deaths by 2040 due to screening delays, highlighting the need for early diagnostic tools [1].

Circulating tumor cells (CTCs) are key biomarkers of tumor progression and metastasis, providing real-time insights [2]. However, their rarity and the complexity of biological fluids present significant challenges for isolation and analysis. Conventional methods lack sensitivity and often involve laborious processing. Microfluidic technologies, especially when integrated with functional biomaterials, offer a promising platform for rare biomarker detection [3].

Here, we present a MULTILAYER MICROFLUIDIC CHIP (ONCO-CTC) integrating a removable electrospun fiber net (EFN) to retain tumor cell models with high efficiency while maintaining viability. The chip supports cell culture on EFN for downstream analysis, representing a promising approach for liquid biopsy applications.

Materials and Methods

The ONCO-CTC was fabricated via standard photolithography, comprising four bonded polydimethylsiloxane (PDMS) layers with a central chamber housing an EFN [4]. EFNs were produced by electrospinning 15% w/v polycaprolactone in chloroform:dimethylformamide (8:2) at 0.7 mL/h and 16 kV, then punched into 6 mm discs. SEM and microCT were used to assess fiber morphology, pore size, porosity, and interconnectivity. HCT-116 CRC cells (200,000 cells/mL) were injected manually into the ONCO-CTC. Cell counts at inlet and outlet determined retention efficiency. EpCAM-FITC and DAPI staining confirmed captured cells. EFNs were removed and cultured in plates. Viability after 72 h was assessed using PrestoBlue® and Calcein AM/PI. A commercial ScreenCell^® kit served as reference.

 Results

SEM revealed randomly oriented fibers with layered internal structure. MicroCT showed mean pore size of 13.9 ± 0.8 µm, porosity of 13.1 ± 0.9%, and interconnectivity of 88.5 ± 1.1%, confirming the scaffold's suitability for selective cell retention and nutrient diffusion.

Fig. 1 presents the structural layout of the ONCO-CTC, composed of four bonded PDMS layers with precisely engineered inlet, outlet, and central chamber geometries. The central chamber (8 mm in diameter) houses the EFN disc, and the top and lateral views depict spatial flow distribution (250–3000 µm height; 35,000 µm length) optimized to minimize shear stress and facilitate efficient cell trapping. The modular design enables both insertion and retrieval of the EFN, supporting downstream biological applications.

The ONCO-CTC demonstrated a cell retention efficiency of 94% ± 2.6, significantly higher than that of the commercial ScreenCell^® system (82% ± 10.9). As shown in Fig. 2, quantitative analysis confirmed the device's reproducible performance across replicates, and it shows fluorescence microscopy of EpCAM-positive cells retained on EFNs.

After 72 h of in vitro culture, the viability of cells retained in the EFNs was markedly higher (613.4 ± 126) compared to those on ScreenCell^® membranes (15.4 ± 0.5, p < 0.01), demonstrating the EFN’s ability to preserve cellular metabolic activity and support prolonged cell survival.

 Discussion

The integration of EFN within the ONCO-CTC provides a multifunctional platform for tumor cell retention and biological analysis. The EFNs' controlled porosity, high interconnectivity, and fibrous architecture contributed not only to efficient cell trapping but also to the preservation of cellular viability over extended culture periods.

Compared to conventional filtration systems [2-3], the ONCO-CTC's modular architecture enabled a stable and low-shear flow environment suitable for gentle cell trapping, while facilitating scaffold insertion and retrieval. This flexibility enhances its adaptability for various diagnostic or research applications. Furthermore, the significantly higher retention efficiency and viability observed, relative to a commercial benchmark, highlight the advantages of coupling microfluidics with tailored biomaterials.

These findings show ONCO-CTC supports post-capture biology, enabling downstream molecular analysis, drug testing, or single-cell profiling for liquid biopsy.

 Conclusions

 The ONCO-CTC demonstrates the utility of smart biomaterials in microfluidic platforms for CTC retention and culture. Its efficiency, modularity, and compatibility with biological analysis support its potential in diagnostic and translational applications. Future validation with patient samples is planned.

References

(1)  van den Puttelaar R. et al, 2023, 10.1158/1055-9965.EPI-22-0544

(2)  Gu X. et al, 2024, 10.1038/s41392-024-01938-6

(3)  Mishra A. et al, 2025, 10.1038/s41467-024-55140-x

(4)  Casanova M.R. et al, 2024, Patent application No. 119865

Acknowledgments: This research was supported by the UID/50026:3B's-Biomaterials, Biodegradables and Biomimetics Research Group, University of Minho (3B's Res. Group/UM) and the EU-EC through the ONCOSCREEN (ID: 101097036), EngVIPO (ID: 101183041) and CAN TARGET (NORTE2030-FEDER-02705300) projects.