A Reliable Flow-Based Method for the Accurate Measure of Mass Density, Size and Weight of Live 3D Tumor Spheroids

Abstract

Gathering precise information on mass density, size and weight of cells or cell aggregates, is crucial for applications in many biomedical fields with a specific focus on cancer research. Although few technical solutions have been presented for single-cell analysis, literature does not cover this aspect for 3D models such as spheroids. Since the research interest on such samples is notably rising, here we describe a flow-apparatus, and the associated physical method and operative protocol for the accurate measurements of mass density, size and weight. The technique is based on the detection of the terminal velocity of a free-falling sample into a specifically conceived analysis flow-channel. Moreover, in order to demonstrate the accuracy and precision of the presented flow-device, analyses were initially carried out on standardized polystyrene beads. Finally, to display the application of the proposed system for biological samples, mass density, size and weight of live SW620 tumor spheroids were analyzed. The combined measurements of such parameters can represent a step toward a deeper understanding of 3D culture models.

Physical Characterization of Colorectal Cancer Spheroids and Evaluation of NK Cell Infiltration Through a Flow-Based Analysis

“…We could also measure the results of effector lymphocyte activity on CRC spheroids. Although controversial for many years, the role of NK cells in anti-tumor immune response is documented ( 31 , 32 , 40 , 41 ). In CRC, the contemporary infiltration of T and NK cells is apparently linked to a better prognosis and an anti-cancer response mediated by NK cell activation has been reported in a metastatic CRC patient ( 42 , 43 ).…”

“…The adopted method is based on tracking the sample’s motion, when free-falling into a vertical flow-channel while the flow is at rest, to calculate its terminal velocity. This is achieved by using customized software that relies on a specific physical method, combined with detailed statistical analyses ( 31 ). Briefly, as shown in Supplementary Figure S3 , the device is composed of a fluidic-core chip, equipped with a bright-field imaging setup, a peristaltic pump, a temperature sensor and a flow-circuit for the introduction and elimination of samples.…”

“…All these check points insure the conformity of the analyzed samples in term of shape and size. CRC spheroids were fixed with PFA 4% overnight at 4°C, resuspended in 3.5mL of Dulbecco's phosphate-buffered saline (DPBS), 1X w/o Ca 2+ & Mg 2+ (Corning ® Life Sciences) at low concentration (<200 spheroids/mL), transferred in a centrifuge conical tube and then analyzed according to the previous protocol performed by Cristaldi et al for biological samples (31). A minimum of 10 single spheroids was analyzed for every test condition and values were extrapolated from at least 10 repetitions.…”

Advanced biomedical applications based on emerging 3D cell culturing platforms

“…Another recent attractive work on microfluidic devices was introduced by Cristaldi et al, which could gather crucial information about cell spheroids, such as density, size, and weight. 101 This technique is based on the detection of the terminal velocity of a free-falling sample in a specifically conceived analysis flow-channel. The device was proven to be capable of measuring cell spheroids with a size ranging from 20 to 200 mm in diameter.…”

DataSheet_1.zip

“…Moreover, data are often collected in one step, limiting sample replication and analysis repetition essential for accurate biometric studies. We have recently developed a precise and rapid technique, that enables the simultaneous determination of weight, size and mass density of sphere-like samples (31).…”