2.1.

Materials

Normal human dermal fibroblasts (HFIB) were bought from PromoCell (Labclinics, S.A., Barcelona, Spain). Cell imaging plates were acquired from Ibidi GmbH (Ibidi, Munich, Germany). Agarose was purchased from Grisp (Porto, Portugal). Cell culture plates, T-flasks, and cell culture consumables were obtained from ThermoFisher Scientific (Porto, Portugal). Dulbecco’s modified Eagle’s medium F12 (DMEM-F12), formamide ($\ge 99.5\%$), paraformaldehyde (PFA), phosphate-buffered saline (PBS), trypsin, ethylenediaminetetraacetate (EDTA), PEG 4000, 8000, and 10,000 Da were purchased from Sigma-Aldrich (Sintra, Portugal). Fetal bovine serum (FBS) was obtained from Biochrom AG (Berlin, Germany). PI was purchased from Invitrogen (Carlsbad, California).

2.2.

Methods

2.2.3.

Whole spheroids clearing using the ${\text{Clear}}^{\mathrm{T}2}$ method

The ${\text{Clear}}^{\mathrm{T}2}$ clearing method variations (${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, and ${\text{Clear}}^{\mathrm{T}2}/10$ that use PEG with an MW of 4000, 8000, and 10,000 Da, respectively) were performed accordingly to previous works (see Fig. 1 for the pipeline overview of the method).^22,26

Fig. 1

Pipeline overview of the spheroids formation, labeling with the PI, and clearing with ${\text{Clear}}^{\mathrm{T}2}$. The ${\text{Clear}}^{\mathrm{T}2}$ clearing procedure was performed using PEGs with different MWs: ${\text{Clear}}^{\mathrm{T}2}/4$ (PEG 4000 Da), ${\text{Clear}}^{\mathrm{T}2}/8$ (PEG 8000 Da), and ${\text{Clear}}^{\mathrm{T}2}/10$ (PEG 10,000 Da).

Initially, whole PI-stained spheroids were immersed in a 25% formamide/10% PEG solution for 10 min. Afterward, spheroids were immersed in 50% formamide/20% PEG solution for 5 min and last in another 50% formamide/20% PEG solution for 1 h. PEG 4000, 8000, and 10,000 Da were used to prepare the solutions used in the ${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, and ${\text{Clear}}^{\mathrm{T}2}/10$ method, respectively. All ${\text{Clear}}^{\mathrm{T}2}$ methods were performed at room temperature using a plate shaker at 100 RPM.

For comparative purposes, some spheroids were only immersed in PBS instead of the clearing solutions (noncleared PI-stained spheroids). Moreover, nonstained spheroids were subjected to ${\text{Clear}}^{\mathrm{T}2}$ methods (cleared nonstained spheroids) to evaluate the influence of the method in the spheroids’ autofluorescence.

After the clearing of the intact spheroids, samples were transferred to $\mu$-slide 8-well imaging plates (Ibidi GmbH, Germany) for imaging experiments.

3.1.

${\text{Clear}}^{\mathrm{T}2}$ Clearings Do Not Influence Spheroids’ Size and Enhance their Transparency

A suitable clearing method must preserve samples’ size and structure in order to allow the study of its initial morphology.³⁵ Additionally, the clearing method should not increase samples’ dimensions, since larger samples require longer imaging times and they may also become unsuited for whole imaging through CLSM.⁶ Therefore, the effect of ${\text{Clear}}^{\mathrm{T}2}$ methods in the preservation of the spheroids’ morphology was evaluated by comparing the area of the cleared spheroids with that of the noncleared spheroids (Fig. 2).

Fig. 2

Influence of ${\text{Clear}}^{\mathrm{T}2}$ methods on HFIB spheroids size. (a) Area measurements and (b) optical microscopy images of noncleared spheroids, spheroids cleared using ${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, or ${\text{Clear}}^{\mathrm{T}2}/10$ methods, and spheroids after reversing the clearing process. Scale bars correspond to $200\mu \mathrm{m}$. Data are presented as $\text{mean}\pm \mathrm{s.d}$. ($n=8$), n.s. means nonsignificant.

The mean area of the spheroids cleared by ${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, and ${\text{Clear}}^{\mathrm{T}2}/10$ increased by 10.35%, 9.91%, and 10.11%, respectively, when compared to that of the noncleared spheroids [Fig. 2(a)]. The slight increase of spheroids’ size was not statistically significant, which demonstrates that all the ${\text{Clear}}^{\mathrm{T}2}$ methods did not induce any significant changes on the spheroids’ morphology and that PEG MW does not affect spheroids’ size during the clearing procedure (Fig. 2). Similar results were obtained in other studies, which demonstrated that the ${\text{Clear}}^{\mathrm{T}2}$ method performed with PEG 8000 Da did not induce significant volume changes on brain sections ²² or on spheroids.²⁶ In fact, morphological and volumes changes are mostly associated with organic solvent-based clearing methods, which use benzylalcohol/benzylbenzoate (e.g., BABB) or dichloromethane/dibenzylether (e.g., 3DISCO and iDISCO).⁶

The capacity of the ${\text{Clear}}^{\mathrm{T}2}$ clearing methods to enhance the transparency of spheroids was also observed in the optical microscopy images, as previously reported in the literature.^{21,22,26,35,36,41} The results demonstrated that, independently of the PEG MW used in the clearing process, all spheroids become gradually more transparent after each immersion step in the clearing solutions [Figs. 3(a1)–3(a4) and (9)]. In fact, the transparency of the spheroids did not seem to differ among the different ${\text{Clear}}^{\mathrm{T}2}$ methods variations. Such is explained by the fact that formamide is the main agent responsible for rendering transparency to the cells,^6,42 and that all the samples were immersed in solutions containing the same concentration of formamide.

Fig. 3

Influence of ${\text{Clear}}^{\mathrm{T}2}$ methods on HFIB spheroids transparency and PI fluorescence intensity. (a1)–(a4) Optical transparency of noncleared spheroids and cleared spheroids with ${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, and ${\text{Clear}}^{\mathrm{T}2}/10$. Spheroids were imaged on crosshatched backgrounds to show relative differences in transparency. (a5)–(a12) CLSM images of PI-stained noncleared and ${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, and ${\text{Clear}}^{\mathrm{T}2}/10$ cleared spheroids. Each image is a maximum projection of the $z$-stacks (thickness: 50 and $100\mu \mathrm{m}$). Analysis of the fluorescence intensity of the spheroids stained with PI for the maximum projections of (b) 50 and (c) $100\mu \mathrm{m}$ of thickness. Scale bars correspond to $200\mu \mathrm{m}$. Data are presented as $\text{mean}\pm \mathrm{s.d}$. ($n=5$), *$p<0.05$, and n.s. means nonsignificant.

3.2.

${\text{Clear}}^{\mathrm{T}2}$ Clearing Methods Are Reversible

The reversibility of the clearing methods, i.e., if the cleared samples can return to their original morphology and nontransparency, is also important for the application of spheroids in therapeutics screening.^22,23 According to Ke et al.,²³ the reversibility of the clearing method also enables the analysis of the same samples by other techniques (e.g., immunohistochemistry). Additionally, nonreversible clearing methods may be indicative that the sample is chemically modified during its treatment, leading to unrealistic interpretations.²³

Kuwajima et al.²² already demonstrated that the ${\text{Clear}}^{\mathrm{T}2}$ method that uses PEG 8000 Da can be reversed by immersing samples of mice embryos in PBS. Herein, it was evaluated if PEGs with other MW have any influence in the clearing reversal process of the spheroids. For this purpose, the size and transparency of the cleared spheroids were also evaluated after immersion in a PBS solution during 30 min. Independently of the ${\text{Clear}}^{\mathrm{T}2}$ method used, all spheroids maintained their original size [Fig. 2(a)] and were able to return to their nontransparent state (Fig. 9). Therefore, the results indicate that the PEG MW does not influence the ${\text{Clear}}^{\mathrm{T}2}$ reversibility and/or that it does not induce permanent modifications on spheroids’ general structure.

3.3.

${\text{Clear}}^{\mathrm{T}2}$ Methods Preserve the PI Fluorescence Intensity

The clearing process should not affect negatively the fluorescence intensity of the samples, i.e., clearing methods must preserve the fluorescence intensity in order to allow their imaging through fluorescence microscopy. In previous studies, it was demonstrated that some clearing methods, such as BABB⁴³ and Scale,⁴¹ lack in the preservation of the probes fluorescence intensity due to the fluorescence quenching effect. Such effect was also observed in tissues that were only cleared with formamide (${\text{Clear}}^{\mathrm{T}}$ method).²² This result may be explained by protein denaturation caused by formamide.⁶ To surpass this drawback, PEG was added to the formamide solutions to stabilize proteins’ structure and maintain their fluorescence. Kuwajima et al.²² reported that the addition of PEG 8000 Da to formamide resulted in the preservation of the fluorescence of proteins, immunohistochemistry labelings, and dye tracers (e.g., 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, cholera toxin subunit B). Still, in other studies, it was demonstrated that the use of PEG 8000 Da did not contribute to preserve the fluorescence intensity of PI within mice brain sections.³⁵

Therefore, we investigated the influence of the PEG MW on the ${\text{Clear}}^{\mathrm{T}2}$ capacity to preserve the fluorescence intensity of labeled spheroids. For this purpose, the fluorescence of the PI-stained noncleared and ${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, and ${\text{Clear}}^{\mathrm{T}2}/10$ cleared spheroids was determined as previously described elsewhere (see Appendix for further details).³⁵ From the analysis of the maximum projection images (thickness equal to 50 and $100\mu \mathrm{m}$) [Figs. 3(a5)–3(a12)], it was possible to verify that all the ${\text{Clear}}^{\mathrm{T}2}$ methods under investigation were able to preserve the fluorescence of the PI [Figs. 3(b) and 3(c)]. However, among the ${\text{Clear}}^{\mathrm{T}2}$ methods variations, ${\text{Clear}}^{\mathrm{T}2}/4$ demonstrated a significantly higher capacity to preserve the PI fluorescence, even at deep penetration depths [Figs. 3(b) and 3(c)]. Importantly, this improved fluorescence intensity was not prompted by the autofluorescence of the clearing solutions constituents, since all the solutions used in the various ${\text{Clear}}^{\mathrm{T}2}$ methods have a transmittance of $\approx 100\%$ from 400 to 800 nm (the absorbance peaks of the PEG polymers-formamide solutions occur below 400 nm and according to literature PEG has absorbance below 400 nm^44,45) (Fig. 10). In other words, the clearing solutions are permissive to the PI excitation laser light (514 nm), and the emitted fluorescence (550 to 750 nm) is not absorbed by the solutions (Fig. 10). Moreover, nonstained cleared spheroids did not present fluorescence signals when analyzed in the same conditions used for PI-stained spheroids (Fig. 11).

3.4.

PI Imaging Depth and Cross-Section Imaging Are Significantly Improved Using the ${\text{Clear}}^{\mathrm{T}2}/4$ Method

The fluorescence signal depth in CLSM is limited by the equipment (e.g., the working distance of the commonly used objectives) and also by the scattering phenomena between the specimen and the excitation and emission photons.^4,6 Consequently, CLSM is more prone to be used for the imaging of thin samples [e.g., two-dimensional (2-D) cultures or sliced tissues]. Nevertheless, to improve the imaging of whole thick samples (e.g., spheroids) by CLSM and avoid the handicaps associated with samples sectioning,^4,19,20 the reduction of the light scattering using optical clearing methods has been investigated.¹⁶ To accomplish that, some clearing methods (e.g., Scale) remove the cellular lipids that are the main source of light scattering.^6,21 On the other hand, clearing methods, such as the ${\text{Clear}}^{\mathrm{T}2}$, reduce the inhomogeneity of the light scatter by equilibrating the RI throughout the sample.⁶ In the ${\text{Clear}}^{\mathrm{T}2}$ method, the formamide and PEG aqueous solutions have an $\mathrm{RI}\approx 1.44$ that will match with the overall RI of the fixed tissue ($\mathrm{RI}\approx 1.50$).^6,29,46

To study the possible improvement in the imaging depth after spheroids clearing by ${\text{Clear}}^{\mathrm{T}2}$ methods and the PEG MW influence on this process, the penetration depth of the PI fluorescence signal was measured and presented in Fig. 4 (see Appendix for details). The results show that the ${\text{Clear}}^{\mathrm{T}2}/4$ improved significantly the penetration depth of the PI signal [Figs. 4(b) and 4(c)]. In fact, the use of PEG 4000 Da allowed to detect PI signal up to $211.67\pm 16.81\mu \mathrm{m}$, whereas the PEG 8000 and 10,000 Da only enabled the acquisition of a signal up to $183.89\pm 14.13$ and $176.43\pm 11.44\mu \mathrm{m}$, respectively.

Fig. 4

Quantitative representation of PI imaging depth in the HFIB spheroids. (a) Schematic representation of the penetration depth measured in the spheroids. (b) Penetration depth of PI fluorescence signal and (c) depth coding CLSM images of (c1) noncleared, (c2) ${\text{Clear}}^{\mathrm{T}2}/4$, (c3) ${\text{Clear}}^{\mathrm{T}2}/8$, and (c4) ${\text{Clear}}^{\mathrm{T}2}/10$ cleared spheroids. Scale bars correspond to $200\mu \mathrm{m}$. Data are presented as $\text{mean}\pm \mathrm{s.d}$. ($n=5$) and *$p<0.05$.

The cross-section imaging depth was also analyzed to demonstrate the influence of the ${\text{Clear}}^{\mathrm{T}2}$ clearing method, as well as the PEG MW in the PI-stained spheroids observation by CLSM (see Appendix for details). This evaluation was performed to determine which method is more suitable for acquiring fluorescence from the interior of the spheroid. In comparison to the noncleared spheroids, spheroids cleared with ${\text{Clear}}^{\mathrm{T}2}$ methods allowed the acquisition of images from PI-stained cells that were in the interior of the spheroid (Fig. 5), such is even more evident at the penetration depth of $100\mu \mathrm{m}$ [Fig. 5(b)].

Fig. 5

HFIB spheroids PI cross-section imaging depth analysis. (a) Schematic illustration of the spheroids PI cross-section imaging depth measurements. (b) Spheroids PI cross-section imaging depth analysis. (c) Cross-section CLSM images of (c1) noncleared, (c2) ${\text{Clear}}^{\mathrm{T}2}/4$, (c3) ${\text{Clear}}^{\mathrm{T}2}/8$, and (c4) ${\text{Clear}}^{\mathrm{T}2}/10$ cleared spheroids. Plot profiles of CLSM images of the (c5) noncleared, (c6) ${\text{Clear}}^{\mathrm{T}2}/4$, (c7) ${\text{Clear}}^{\mathrm{T}2}/8$, and (c8) ${\text{Clear}}^{\mathrm{T}2}/10$ cleared spheroids. CLSM images correspond to a $z$-tack at a penetration depth of $75\mu \mathrm{m}$. Scale bars correspond to $200\mu \mathrm{m}$. Data are presented as $\text{mean}\pm \mathrm{s.d}$. ($n=5$), n.s. means nonsignificant.

At this penetration depth, the spheroids fluorescence signal in the cross-section improved $24.95\%\pm 7.50\%$, $24.95\%\pm 15.07\%$, and $6.31\%\pm 29.11\%$ when cleared with ${\text{Clear}}^{\mathrm{T}2}/4$, ${\text{Clear}}^{\mathrm{T}2}/8$, and ${\text{Clear}}^{\mathrm{T}2}/10$, respectively. To corroborate these observations, plot profiles of the noncleared and cleared spheroids CLSM cross-section images at $75\mu \mathrm{m}$ of penetration depth ($Z$-axis) were performed [Fig. 5(c)] (see Appendix for details). These graphs display the color intensity of the pixels throughout the spheroid cross section. As observed in Figs. 5(c1)–5(c4), without the use of a clearing method, the fluorescence observed within spheroids’ core is limited. On the other side, the ${\text{Clear}}^{\mathrm{T}2}/4$ clearing method allowed a better observation of the spheroid’s interior and also the attainment of a higher PI fluorescence intensity, when compared to the other ${\text{Clear}}^{\mathrm{T}2}$ methods variations [Figs. 5(c5)–5(c8)].

This improved imaging capacity of the ${\text{Clear}}^{\mathrm{T}2}/4$ method may be linked to the smaller size of the polymer chain of PEG 4000. Such may facilitate the PEG distribution and penetration throughout the spheroid and, consequently, the stabilization of the fluorescence probe in deeper regions, thus allowing a better imaging. In fact, large molecules penetrate slowly into the tissues⁴⁷ and lower MWs are generally associated with higher diffusion coefficients.⁴⁸ Moreover, the use of PEG with a low MW may further contribute to a quicker establishment of the water balance between the clearing solution and the spheroids, leading to an improved penetration of the clearing agent through the spheroid caused by the osmotic pressure.^49–52 In future works, it may be interesting to use PEG with smaller MW (e.g., PEG 400 Da) to assess if they further improve the clearing efficacy of the ${\text{Clear}}^{\mathrm{T}2}$ method.