Colorectal Adenocarcinoma Cell Culture in a Microfluidically Controlled Environment with a Static Molecular Gradient of Polyphenol.

Roman G Szafran,Kazimierz Gąsiorowski,Benita Wiatrak

doi:10.3390/molecules26113215

Abstract

To study the simultaneous effect of the molecular gradient of polyphenols (curcumin, trans-resveratrol, and wogonin) and biological factors released from tumor cells on apoptosis of adjacent cells, a novel microfluidic system was designed and manufactured. The small height/volume of microfluidic culture chambers and static conditions allowed for establishing the local microenvironment and maintaining undisturbed concentration profiles of naturally secreted from cells biochemical factors. In all trials, we observe that these conditions significantly affect cell viability by stimulating cell apoptosis at lower concentrations of polyphenols than in traditional multiwell cultures. The observed difference varied between 20.4–87.8% for curcumin, 11.0–37.5% for resveratrol, and 21.7–62.2% for wogonin. At low concentrations of polyphenols, the proapoptotic substances released from adjacent cells, like protein degradation products, significantly influence cell viability. The mean increase in cell mortality was 38.3% for microfluidic cultures. Our research has also confirmed that the gradient microsystem is useful in routine laboratory tests in the same way as a multiwell plate and may be treated as its replacement in the future. We elaborated the new repetitive procedures for cell culture and tests in static gradient conditions, which may become a gold standard of new drug investigations in the future.

Highlights

All living cells can sense their environment [1]
We describe the new repetitive procedures of cell seeding, gradient generation, cell culture, and apoptosis detection, which may become in the future a gold standard for drug tests in molecular gradients
All the chip construction materials were cut or engraved using a commercial CO2 laser marker (Versa Laser VLS 2.30, Universal Laser System Inc., Scottsdale, AZ, USA) equipped with a 30 W CO2 pulsed laser source equipped with a high power density focusing optic (HPDFO)

Summary

Introduction

All living cells can sense their environment [1]. Concentration gradients of molecules play a crucial role in living organisms controlling multiple processes, including morphogenesis, wound healing, immune response, vessel pathfinding, and axonal guidance [2].The concentration gradients of biomolecules are responsible for cell differentiation and migration. All living cells can sense their environment [1]. Concentration gradients of molecules play a crucial role in living organisms controlling multiple processes, including morphogenesis, wound healing, immune response, vessel pathfinding, and axonal guidance [2]. The concentration gradients of biomolecules are responsible for cell differentiation and migration. A human body is built of about 1013 cells making up tissues and organs. Cells in tissue are exposed to a concentration gradient of biologically active substances that penetrate from the blood into the cell’s environment. Depending on the distance of a particular cell from the delivery point, some cells are exposed to high and others to low concentrations of the substance, so its metabolic and/or phenotypic effects may differ. Cells exposed to a bioactive substance can secrete different amounts of another proapoptotic substance into their environment

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