Morphological surface analysis
to study cell viability induced
by proliferative and toxic
treatments
The use of profilometry in determining changes
in cell morphology can be considered an exciting
tool for screening potential drugs and toxic
materials, distinguishing between the two
greatest models of cell death.”
During apoptosis and necrosis processes, dramatic cell volume changes occur
in the early stages of the treatment. Distinguishing between these processes
is crucial from a biophysical and therapeutical point of view. Apoptosis is a
programmed process of cellular death, while necrosis is an accidental death
resulting from environmental perturbations. When a treatment induces
apoptosis instead of necrosis, it is more likely to have a better outcome. Inaddition, cell proliferation processes cause changes in cell volume as a measureof cell growth.

This work was conceived in theframework of the Cooperation
Agreement between the Facultyof Pharmacy and Food Science(UB) - Institute for Chemistryof Condensed Matter andTechnologies for Energy (ICMATECNR) (Codi GREC 18407). The present
paper is a part of the research [1]carried out by Dr. Michele Ferrari andDr. Francesca Cirisano (ICMATE-CNR,Genova-Italy) and Dr. M. Carmen
Morán (Faculty of Pharmacy andFood Science, UB, Barcelona-Spain).

In this study, we show non-invasive morphological surface analysis techniquesmatching well-assessed biochemical methods to distinguish physiologicalfrom pathological cells clearly. Briefly, profilometry analysis allows variations
in cell volume induced by suitable nanoparticles (NPs) as precursors ofCASE STUDY MORPHOLOGICAL SURFACE ANALYSIS TO STUDY CELL VIABILITY INDUCED BY PROLIFERATIVE AND TOXIC TREATMENTS1proliferation and cell death compatible with cell viability responses measured
by conventional methods.
This project is intrinsically interdisciplinary since it requires the characterizationof different materials and their interaction with the biological entities ofinterest, as appreciated on the team members’ different backgrounds. The
group led by Dr. Ferrari aims at studying wettability processes, adsorption,
and aggregation of amphiphilic molecules at the liquid-solid interface, thepreparation of highly liquid-repellent coatings, and the characterization
using AFM and 3D-profilometry techniques. The group led by Dr. Moránfocuses on the development of controlled delivery systems and their in vitro
characterization under 2D (bulk and substrate) and 3D conditions.

■ Measurements
The S neox 3D profilometer was used due to its capability to analyze large
surfaces and obtain important surface parameters from the scans. The surface
characterization using profilometry was done according to the ISO 25178
standard, which provides the rules for the three-dimensional parametricassessment of surface textures.
The entire surface of the individual coverslips—containing cells under thestudied conditions—was analyzed using Confocal mode. Cells in selectedareas were chosen, and the corresponding profiles on height (H) and length(L) were analyzed with the SensoSCAN S neox software. The corresponding
shape factor (SP) was determined as a function of the H/L values.
The advantages of optical scanning profilometry against atomic force
microscopy (AFM) are multiple: non-invasive and non-destructive
characterization and the possibility of analyzing larger surfaces not being
limited to a few cm2. Moreover, measurement times are reduced comparedwith demonstrated experience of the AFM technique [2, 3], making opticalprofilometry a microscopic technique which offers more qualitative andquantitative information in terms of high resolution at nano-microscales.
Despite no fluorescent protein markers or optically active dyes being required
for this technique, 3D scanning profilometry in Confocal and Interferometricmodes can provide high accuracy in cell geometry parameters assessment(Fig.1a). Consequently, 3T3 fibroblasts under standard conditions show atypical bipolar or multipolar structure with elongated shapes. In addition,epithelial-like cells such as HaCaT, HeLa, and A431 cells are polygonal in shapewith more regular dimensions, growing in discrete patches. Due to the NPs
treatment, 3T3 fibroblasts showed an oval appearance with no filopodia.
Epithelial cells undergo a massive lack of boundaries between the cells andshape irregularity.
The analysis of the obtained profiles (Figure 1b) has allowed evaluating, froma quantitative point of view, several structural parameters, such as the heightand length of control cells, and those subjected to treatments for the inductionof cell proliferation and cell cytotoxicity.

CASE STUDY MORPHOLOGICAL SURFACE ANALYSIS TO STUDY CELL VIABILITY INDUCED BY PROLIFERATIVE AND TOXIC TREATMENTS

2abFig. 1. Representative 3D profilometry images in Confocal mode (magnification 20x) of fibroblasts(3T3 cell line), keratinocytes (HaCaT cell line), epithelial carcinoma (HeLa cell line) and squamouscell carcinoma (A431 cell line) under standard conditions (control cells -C) and after incubation
with bare NPs (blank NPs) and 5-FU containing NPs (5-FU NPs) (a) deduced by the corresponding
profile sections, of which an example is shown in (b). (“3D profilometry and cell viability studies for
drug response screening” by M. C. Morán, F, Cirisano and M. Ferrari is licensed under CCC).

Changes in the morphology of the cells and their shape factor as a functionof the H/L ratio values are summarized in figures 2a and 2b, respectively.
By using a model of spherical cap as a geometric approximation to the cell[5], the cellular volumes were estimated (Fig. 2c). The obtained values are inagreement with those achieved directly with the SensoSCAN S neox software,for which the cellular edges have been considered (differences lower than 5%in all cases).

CASE STUDY MORPHOLOGICAL SURFACE ANALYSIS TO STUDY CELL VIABILITY INDUCED BY PROLIFERATIVE AND TOXIC TREATMENTS

3

abcFig. 2. Representative images of profilometry on Confocal mode (magnification 20x) andmorphological parameters ( L: length and H: height) (a) shape factor (H/L ratio) values (b) andestimation of cell volume from the data obtained from optical profilometry profiles and using amodel of the spherical cap as a cellular geometrical approximation. Results are expressed as theaverage of ten independent cells ± standard deviation. *p<0.001 indicates significant differences
against control cells, *p<0.001 indicates significant differences between treatments for the same
cell line, and ≠p<0.001 indicates significant differences between cell lines for the same treatment.
(“3D profilometry and cell viability studies for drug response screening” by M. C. Morán, F, Cirisanoand M. Ferrari is licensed under CCC).

■ Conclusions
Using optical profilometry, analyses of cell morphology with response times
compatible with conventional methods, like the MTT cell viability assay, wereperformed [6]. However, these analyses avoid some contradictory results due
to changes in the metabolic activity that may appear by MTT action without
alteration of the viable cell number. Besides, the measurement of cell viability
results from the arithmetic means of the metabolic activity of 104-105 cells/mL. This large number of cells may hinder the determination of morphologicalchanges in very early stages or small portions of the cell population.

CASE STUDY MORPHOLOGICAL SURFACE ANALYSIS TO STUDY CELL VIABILITY INDUCED BY PROLIFERATIVE AND TOXIC TREATMENTS

4The determination of single-cell volume through 3D profilometry is an added
value in establishing patterns or mechanisms of cell death, otherwise notdetectable by a simple cell viability assay even on surfaces well over the limitsof other techniques (cm2). This technique can be regarded as an interesting
tool for observing changes in cell morphology during the screening ofpotential drugs and toxic materials, discriminating between the two greatest
models of cell death, especially in very short times in treatment procedure foran early prediction of success expectations of a therapeutical agent.
The success of research in biomedicine lies in the application of nanomaterials
in the treatment of diseases and in the development of tools for their diagnosis.
In this work, 3D optical profilometry, setting qualitative and quantitative
analyses, meets the requirements for a screening tool of potential therapeuticor toxic drug delivery systems, stimulating cell growth or inducing cellulardeath. This work is the research team’s first publication combining bothdisciplines (treatment and diagnosis).

■ References
1. M. C. Morán, F. Cirisano, M. Ferrari. 3D profilometry and cell viability
studies for drug response screening. Mat. Sci. Eng. C. 115(2020) 111142.
2. E. Guzmán, L. Liggieri, E. Santini, M.Ferrari, F. Ravera, F. Mixed DPPCcholesterol Langmuir monolayers in presence of hydrophilic silica
nanoparticles. Colloids Surf B. Biointerfaces. 105 (2013) 284-293.
3. J.L. Rinn, C. Bondre, H.B. Gladstone, P.O. Brown, H.Y. Chang. Anatomic
demarcation by positional variation in fibroblast gene expression
programs. PLoS Genet. 2 (2006) e119.
4. M.C. Morán, J. Carazo, M.A. Busquets.Dual responsive gelatin-based
nanoparticles for enhanced 5-ﬂuorouracil eﬃciency. Colloids andSurfaces B: Biointerfaces 172 (2018) 646-654.
5. J.A. Hessler, A. Budor, K. Putchakayala, A. Mecke, D. Rieger, M.M B. Holl,
B.G.Orr,A. Bielinska, J. Beals, J.J. Baker. Atomic force microscopy studyof early morphological changes during apoptosis. Langmuir 21 (2005)
9280-9286.
6. T. Mosmann. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods
65 (1983) 55-63.

CASE STUDY MORPHOLOGICAL SURFACE ANALYSIS TO STUDY CELL VIABILITY INDUCED BY PROLIFERATIVE AND TOXIC TREATMENTS

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