Nano-Electrochemical Characterization of a 3D Bioprinted Cervical Tumor Model

Abstract

Current cancer research is limited by the availability of reliable in vivo and in vitro models that are able to reproduce the fundamental hallmarks of cancer. Animal experimentation is of paramount importance in the progress of research, but it is becoming more evident that it has several limitations due to the numerous differences between animal tissues and real, in vivo human tissues. 3D bioprinting techniques have become an attractive tool for many basic and applied research fields. Concerning cancer, this technology has enabled the development of three-dimensional in vitro tumor models that recreate the characteristics of real tissues and look extremely promising for studying cancer cell biology. As 3D bioprinting is a relatively recently developed technique, there is still a lack of characterization of the chemical cellular microenvironment of 3D bioprinted constructs. In this work, we fabricated a cervical tumor model obtained by 3D bioprinting of HeLa cells in an alginate-based matrix. Characterization of the spheroid population obtained as a function of culturing time was performed by phase-contrast and confocal fluorescence microscopies. Scanning electrochemical microscopy and platinum nanoelectrodes were employed to characterize oxygen concentrations-a fundamental characteristic of the cellular microenvironment-with a high spatial resolution within the 3D bioprinted cervical tumor model; we also demonstrated that the diffusion of a molecular model of drugs in the 3D bioprinted construct, in which the spheroids were embedded, could be measured quantitatively over time using scanning electrochemical microscopy.

Keywords: 3D bioprinting; cellular microenvironment; cervical tumor model; hypoxia; scanning electrochemical microscopy.

Figure 1

Alginate bioink optimization. (A) Printed grid of alginate bioink with a ratio between alginate solution of 3.5% w/v and 0.67% w/v CaCl₂ equal to 25:9, Pr > 1. (B) Printed half-grid of alginate bioink with ratio 25:10, Pr < 1. (C) Cube of (1 × 1 × 0.15) cm³ made of alginate bioink before the crosslinking process. (D) Cube of (1 × 1 × 0.15) cm³ made of alginate bioink without D-mannitol and following the crosslinking process with 0.67% w/v CaCl₂. (E) Cube of (1 × 1 × 0.15) cm³ made of alginate bioink with D-mannitol after the crosslinking process with 0.67% w/v CaCl₂. (F) 3D bioprinted sample of cervical tumor model: a cube of (1 × 1 × 0.15) cm³ made of alginate bioink, inside of which are three printed lines of the bioink containing HeLa cells.

Figure 2

Live/dead assay with calcein-AM/propidium iodide. (A,B) Living cells (green, calcein-AM) and dead cells (red, propidium iodide) in a 3D bioprinted cervical tumor model at 3 h (A) and 24 h (B) after printing. (C) Viability of 3D bioprinted HeLa cells at 3 h, 24 h, 48 h, and 72 h after printing. Population size, n, i.e., the number of cells evaluated for each time point, is reported for each bar of the graph. Levels of statistical significance are calculated as detailed in Section 2.9 and are indicated as follow: ****, p < 0.0001.

Figure 3

HeLa spheroid formation in a 3D alginate matrix, printed according to the optimized procedure, during four weeks of culture. (A) HeLa cell distribution and organization 1 day after printing; the green arrow indicates a single cell. (B) Representative example of cellular organization at 7 days after printing; the green arrow indicates an area where spheroid organization is starting. (C) 3D bioprinted matrix 10 days after printing: the green arrow indicates a HeLa spheroid. (D,E) 18 days (D) and 23 days (E) after printing; the green arrows indicate spheroids. HeLa spheroids are increasing, both in number and average dimensions. (F) HeLa spheroids and surrounding matrix 32 days after printing: a green arrow indicates a spheroid with a diameter of 470 μm; the degree of cellularization of the sample ulteriorly increases.

Figure 4

Analyses of HeLa spheroid diameter over several weeks (at day 5, 10, 14, 19, 24, 34). (A,B) Frequency distribution of spheroid diameter (A) and mean diameter in the spheroid population ((B), mean ± standard deviation) with increasing culturing time. The number of spheroids at each time point are reported as: day 5, 22 spheroids; day 10, 107 spheroids; day 14, 113 spheroids; day 19, 72 spheroids; day 24, 86 spheroids; day 34, 91 spheroids. Levels of statistical significance are calculated as detailed in Section 2.9 and are indicated as follow: ***, p < 0.001; ****, p < 0.0001.

Figure 5

Histological images of HeLa spheroids in the 3D bioprinted alginate matrix. HE staining of HeLa spheroids in the alginate matrix after 11 (A,C) and 18 (B,D) days of culturing. Magnification factor: (A,B), 40×; (C,D), 20×.

Figure 6

Confocal fluorescence images of Hela spheroids. E-cadherin immunolocalization (red, antibody conjugated with AlexaFluor 568) and nuclei staining (blue, DAPI) at 11 (A–C) and 18 (D–F) days of culturing. Colocalization by merging of the two channels at 11 (C) and 18 (F) days of culture.

Figure 7

Phase-contrast images during oxygen content SECM measurements inside single spheroids. (A,B) Lateral penetration in the side of a spheroid with diameter of ~250 µm; before (A) and after (B) penetration. The nanoelectrode tip is indicated in (B) with a blue arrow; the optical focus is adjusted to resolve the nanoelectrode tip in the different locations. In (A,B) the nanoelectrode profile is contoured with a red dashed line. (C,D) Bright field images of vertical penetration inside spheroids with a diameter of ~450 µm; spheroid before (C) and after (D) penetration. The black dot indicated by the green arrow in (D) is the active part of the nanoelectrode; the grey hole indicated by the blue arrow is the upper part seen in perspective. The upper part of the nanoelectrode did not penetrate the spheroid; the optical focus was adjusted to make the nanoelectrode tip visible.

Figure 8

SECM measurements of oxygen concentration inside spheroids with a diameter of ~250 µm. (A) The three different measurements were made inside the same spheroids in adjacent sections. The blue curve is oxygen concentration measured in the longer spheroid diameter (blue arrow in panel (B)); black and red curves are measurements performed on more peripheral parts of the spheroid (black and red arrow, respectively, in the panel (B). Measurements were performed using a 200–500 nm Pt UME at −0.7 V vs. Ag/AgCl (KCl 3 M) with a scan rate of 10 μm/s in PBS solution. These measurements are representative of three measurements performed on three different spheroids. The current detected at the nanoelectrode has been normalized (i_norm) with respect to that measured at a fixed distance from the bottom of the Petri dish and outside the spheroid, in this case this distance was 28 μm.

Figure 9

(A) Cyclic Voltammetries of FeMeOH diffusion in 3D bioprinted tissue. Measurement of FeMeOH diffusion in the 3D alginate sample followed by cyclic voltammetry over time. Oxidation current increased as FeMeOH concentration increased over time (the direction of the blue arrow indicates increasing time). (B) Diffusion coefficient curve of FeMeOH (black curve) in the alginate 3D sample obtained from repeated cyclic voltammetries; the best fitting (red curve) using the Equation (1) was calculated using OriginPro 9.1 software. Values of free parameters for the best fit are C₀ = (1.705 ± 0.003) mM and D = (2.57 ± 0.02) cm² s⁻¹. The distance of the microelectrode from the nearest construct/solution boundary, x, was kept as x = 0.8 mm as a non-adjustable parameter during best fit. This value was measured from the SECM approach curve to the bottom of the Petri dish.