Cell Type-specific Adaptation of Cellular and Nuclear Volume in Micro-Engineered 3D Environments

8/17/2019 Cell Type-specific Adaptation of Cellular and Nuclear Volume in Micro-Engineered 3D Environments

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Cell type-specic adaptation of cellular and nuclear volume in

micro-engineered 3D environments

Alexandra M. Greiner a , Franziska Klein a , Tetyana Gudzenko b, Benjamin Richter a ,Thomas Striebel a, Bayu G. Wundari a , Tat jana J. Autenrieth c, Martin Wegener b , d,Clemens M. Franz b , *, Martin Bastmeyer a , b, c, **

a Karlsruhe Institute of Technology (KIT), Zoological Institute, Department of Cell- and Neurobiology, Haid-und-Neu-Straße 9, D-76131 Karlsruhe, Germanyb Karlsruhe Institute of Technology (KIT), DFG-Center for Functional Nanostructures (CFN), Wolfgang-Gaede-Straße 1a, D-76131 Karlsruhe, Germanyc Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces (IFG), 76344 Eggenstein-Leopoldshafen, Germanyd Karlsruhe Institute of Technology (KIT), Institute for Applied Physics, Wolfgang-Gaede-Straße 1, D-76131 Karlsruhe, Germany

a r t i c l e i n f o

Article history:

Received 5 August 2015

Accepted 7 August 2015

Available online 8 August 2015

Keywords:

Direct laser writing

3D cell culture substrates

Cell-matrix adhesions

Cell volume

Nuclear volume

a b s t r a c t

Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are

structurally and mechanically well-dened and ideal for systematically investigating the inuence of

three-dimensionality and substrate stiffness on cell behavior. Here, we show that different broblast-like

and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in

DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular compo-

sition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D

culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-

paxillin, phospho-focal adhesion kinase, vinculin, b1-integrin). However, broblast-like and epithelial

cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments.

While broblast-like cell lines display signicantly increased cell and nuclear volumes in 3D substrates

compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D envi-

ronments. Despite differential cell volume regulation between broblasts and epithelial cells in 3D en-

vironments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture

conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments

are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio

regardless of culture conditions.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Much of the current knowledge of adherent cell behavior has

been obtained by in vitro cell culture experiments using conven-

tional stiff and planar plastic or glass substrates. These two-dimensional (2D) cell culture substrates have been improved by

micro-contact printing techniques, resulting in uniform [1e3] or

graded patterns of adhesion molecules [4], or by micro-well

structuring methods providing topographic cues [5e8]. Neverthe-

less, 2D cell culture conditions do not reect the three-dimensional

(3D) environment of cells in vivo. In contrast to stiff 2D cell culture

substrates, the natural extracellular matrix (ECM) is a compliant,

complex and information-rich 3D environment [9,10]. A growingunderstanding of the shortcomings of 2D cell culture systems in

biomedical sciences has stimulatedthe development of novel, more

physiological 3D cell culture systems. These systems allow for

investigating factors that are operative only in 3D micro-

environments, for instance guiding cellular phenomena in devel-

opment, tissue homeostasis, and disease [10,11]. Depending on the

makeup of these scaffolds, cells show dramatic changes in cell

behavior compared to 2D culture [12]. For instance, cells cultured in

cell-derived 3D ECM scaffolds typically exhibit differences in

migration [13e15], proliferation behavior [16], or bronectin

brillogenesis [17]. Additionally, relatively little is known about the

* Corresponding author. Karlsruhe Institute of Technology (KIT), DFG-Center for

Functional Nanostructures (CFN), Wolfgang-Gaede-Straße 1a, D-76131 Karlsruhe,

Germany.

** Corresponding author. Karlsruhe Institute of Technology (KIT), Zoological

Institute, Department of Cell- and Neurobiology, Haid-und-Neu-Straße 9, D-76131

Karlsruhe, Germany.

E-mail addresses: [email protected] (C.M. Franz), [email protected]

(M. Bastmeyer).

Contents lists available at ScienceDirect

Biomaterials

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c om / l o c a t e / b i o m a t e r i a l s

http://dx.doi.org/10.1016/j.biomaterials.2015.08.016

0142-9612/©

2015 Elsevier Ltd. All rights reserved.

Biomaterials 69 (2015) 121e132

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structure and function of cell-matrix adhesions formed in 3D en-

vironments compared to the 2D situation. Cell-matrix adhesions

are important cellular sensors of matrix rigidity as they mechani-

cally link the ECM with the acto-myosin system [9,10,18]. Reports

about cell-matrix adhesions in 3D vary widely across the literature

and are often in apparent conict [10,11,16,19e23]. It has been re-

ported that cells differ in adhesion, adhesive signaling and overall

migration mode not only between 2D and 3D micro-environments,

but also between different 3D substrate types [11]. It has also been

shown that cells in soft cell-derived 3D matrices or 3D collagen

matrices develop so-called 3D cell-matrix adhesions, which differ

in molecular composition and localization, and morphology from

contacts formed on planar 2D substrates [16,19e26]. In addition,

the local ECM architecture, including the orientation and diameter

of ECM bers and consequently the functional area available for

cell-matrix adhesion formation, can inuence the appearance and

morphology of cell-matrix contacts [11,13,16,27].

Although naturally-derived 3D matrices retain important fea-

tures of in vivo environments, their complex molecular composi-

tion, variable architecture (pore size, connement, degree of cross-

linking) and variable stiffness complicates the systematic analysis

of their inuence on cell behavior [9]. To better understand cell

behavior in 3D environments, reductionist approaches are neededto correlate cellular responses to particular chemical and physical

properties of the 3D substrate. To this end, a number of articial 3D

systems, including micro-carriers [28,29], synthetic hydrogels

[30,31], and micro-well arrays [5e7] have been established. These

systems offer reproducible biochemical conditions and some of

them can even mimic ECM porosity, but they frequently lack the

architectural, mechanical, and biochemical versatility necessary for

comprehensive cell-biological studies.

To overcome some of the limitations of articial planar and rigid

cell culture substrate, we [32e36] and others [12,28,37e40] have

used the direct laser writing (DLW) photo-polymerization technique

to fabricate tailored 3D scaffolds for single cell cultivation. By using

polymers with different mechanical properties or by adjusting

scaffold feature sizes, the stiffness of DLW-produced cellular micro-environments can be accurately adjusted [35]. Furthermore, DLW

scaffolds can be selectively bio-functionalized by sequential photo-

activation [33,34]. Thus, DLW-produced 3D scaffolds can ll a gap

between the vast structural complexities of natural 3D cell envi-

ronments on the one hand and the oversimplied 2D situation of

planar cell culture substrates for in vitro studies on the other hand.

In this study we demonstrate that bronectin-coated 3D scaf-

folds fabricated by DLW display different degrees of stiffness and

geometries and support cell proliferation in a similar manner as

standard 2D cell culture techniques. Furthermore, using different

cell-matrix adhesion marker proteins we show that the composi-

tion of cell-matrix adhesions in the 3D scaffolds is similar to those

formed on planar 2D cell culture surfaces, even though some

morphological differences were detected. However, we nd strik-ing cell-type dependent differences in cell and nuclear volume

regulation between 2D and 3D culture conditions, but nuclear-to-

cell (N/C) volume ratios are maintained in 2D and 3D environ-

ments for all cell types. Tailored 3D environments produced by

DLW can therefore reveal cell type-specic changes in cell and

nuclear volume regulation, which may reect different tissue-

specic tasks of these cell types in vivo.

2. Materials and methods

2.1. Direct laser writing

Direct laser writing (DLW) is a twophoton polymerization (2 PP)

technique [35,36,41] in which a femto-second laser beam is focused

into a photo-sensitive liquid material consisting of a mixture of

monomeric matrix molecules and a photo-initiator. Simultaneous

absorption of two photons leads to the excitation of photo-initiator

molecules, which then trigger a highly localized chemical poly-

merization event that is conned to the focal volume of the laser

due to the non-linearity of the 2 PP process (Supplemental Fig S1)

[12,28,40,42]. The spot size for the 2 PP is diffraction limited and in

our setup has a size of approx. a xy ¼ 314 nm and az ¼ 767 nm (for

l ¼ 780 nm, NA ¼ 1.4, n ¼ 1.52) [40]. For scaffold writing, a single

droplet (~100 ml) of the photo-sensitive viscous liquid material is

rst drop-cast onto a planar supporting carrier substrate. Due to its

high surface tension, the viscous photo-sensitive liquid retains a

roughly spherical shape on the carrier substrate. In our study the

carrier substrates were plasma-cleaned high precision microscopy

glass cover slips (22 mm 22 mm, 170 ± 5 mm, No. 1.5H LH24.1

from Roth) functionalized with 3-(trimethoxysilyl) propyl meth-

acrylate (Sigma Aldrich) dissolved at a concentration of 1 mM in

toluene (Sigma Aldrich). However, other transparent or non-

transparent carrier substrates of different materials, e.g., poly-

mers or silicon, are also suitable for the DLW fabrication process.

The writing process starts with focusing the laser on the interface

between the glass substrate and the photo-resist to ensure a stable

and covalent attachment of the emerging 3D polymer scaffold tothe carrier substrate [12]. After the writing process the substrates

were developed in a 1:1 mixture of methyl isobutyl ketone and iso-

propanol, rinsed in iso-propanol and dried in a nitrogen ow.

2.1.1. Stiff 3D PETTA structures

We have previously shown that DLW-induced polymerization of

a mixture of petaerythriol tetraacrylate (PETTA, SigmaeAldrich)

monomer and an appropriate photo-initiator produces 3D struc-

tures with increased physical stability and high protein adsorbing

properties [36]. The monomeric PETTA solution also contains about

400 ppm monomethyl ether hydroquinone to inhibit pre-

polymerization, but no other solvents. For DLW, one weight

percent of the monomeric PETTA was mixed with three weight

percent of Irgacure 379 photo-initiator (BASF). Typical writing pa-

rameters of the DLW system for this resist were an average laser

power of 20 mW and a writing speed of 200 mm/s. The produced

wheel structures have a total diameter of 102 mm. The spokes have a

height of 15 mm; the two sets of arcs which form the outer wall of

the wheel structure are each approx. 5 mm highand 1 mm thick. The

spokes are arranged at 36 angles to each other. The dimensions of

the outer and inner sector areas of the wheel are 582.92 mm2 and

185.64 mm2, respectively.

2.1.2. Soft 3D Ormocomp structures

Ormocomp (Micro Resist Technology, Berlin, Germany) is a

member of the Ormocer family of inorganic (SieOeSi)eorganic

hybrid polymers. It has been previously used to fabricate DLW-produced elastic scaffolds for single cell force measurements [35]

and for controlled 3D cell culture applications [36]. Ormocomp

possess good protein binding properties in its polymerized state.

Ormocomp is a proprietary viscous liquid containing an undis-

closed photo-initiator (according to the manufacturer's informa-

tion) and requires no further supplements for photo-

polymerization. During DLW the Ormocomp photo-resist was

polymerized at a laser speed of 200 mm/s and a minimal laser po-

wer of 12.5 mW, which produced beams with a cross section of

approximately 1.5mm. The beams are arrangedat 90 angles to each

other. The height of the pillars supporting the net structures is

20 mm and the spacing between the pillars varies incrementally

between 20 mm and 30 mm. The area of the squares formed by the

beams of a net is 400 mm

2

and 900 mm

2

.

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2.1.3. 2D substrates

As 2D substrates we used conventional glass cover slips

homogenously coated with human plasma bronectin (Sigma-

eAldrich, see below for details about the substrate coating). In

control experiments we also produced 2D PETTA and 2D Ormo-

comp substrates on glass cover slips using the same DLW setup as

for the corresponding 3D scaffolds (see above). However, the

writing parameters were adjusted to produce homogenous PETTA

or Ormocomp polymer lms of approximately 1 mm thickness

which were then coated with bronectin.

2.2. Cell culture

Buffalo rat liver cells (BRL, broblasts), mouse embryonic -

broblasts (MEF), and normal rat kidney cells (NRK, epithelial) were

purchased from the American Type Tissue Culture Collection (ATCC,

Manassas, VA, USA). Spindle-shaped B16F1 (B16) (mouse mela-

noma) cells were a generous gift of Beat Imhof (CMU, Genova) and

epithelial A549 cells (human lung carcinoma cells) were a generous

gift of Harald Kruge (ITG, KIT, Karlsruhe). BRL, B16, MEF, and NRK

cells were cultured in Dulbecco's Modied Eagle Medium (DMEM,

Gibco) and A549 cells F-12K medium (Gibco). Both cell culture

media were supplemented with 10% fetal calf serum (FCS,HyClone). Cells were kept in an incubator at 37 C with 5% CO2 in a

humidied environment and passaged approximately two to three

times a week. Prior of experiments, cells were grown to 70e80%

conuence and detached from the cell culture container by trypsin/

EDTA (0.25% trypsin/1 mM ethylenediaminotetraacetate, Invi-

trogen). Cell detachment was stopped by adding DMEM medium

supplemented with 10% FCS. The cell suspension was centrifuged

and the resulting supernatant discarded. Cells were resuspended in

fresh medium and the cell numbers per ml were determined in a

Neubauer counting chamber. All cells were used for experiments up

to a passage number below 40.

2.3. Substrate coating and cell cultivation in 2D and 3D

The cell culture substrates (i.e. unmodied glass cover slips,

PETTA- or Ormocomp-covered glass cover slips, or glass cover slips

carrying 3D structures) were placed into a single well of a six-well

plate without further xation to the bottom of the well. All cover

slips were sterilized with 70% ethanol (Roth) and washed three

times with phosphate buffered saline (PBS, Gibco). The 3D struc-

tures and the 2D control substrates were coated with 10 mg/ml

bronectin at RT for 60 min and then rinsed with PBS twice to

remove unbound bronectin. A total of 50.000 cells in 3 ml of the

respective cell culture media were added per well and cultured in

serum-containing medium on the different 3D structures or planar

2D control substrates for three hours. For Matrigel© (BD Bioscience)

experiments cells suspended in PBS were carefully mixed with

Matrigel (nal Matrigel concentration 80%) and applied to a planarglass surfaces. The Matrigel was left to stiffen for 15 min at 37 C

before cells were cultured for three hours in serum-containing

medium under standard cell culture conditions.

2.4. Immuno- uorescence

Cells on 2D surfaces or in 3D structures were xed for 10 min

with 4% paraformaldehyde (SigmaeAldrich) in PBS. After per-

meabilization in 0.1% Triton/PBS (SigmaeAldrich), cells were

incubated with primary antibodies in 1% bovine serum albumin

(SigmaeAldrich) in PBS for 1 h at RT: (a) mouse monoclonal anti

phospho-tyrosine (Tyr99) (1:100) (Santa Cruz, Heidelberg); (b)

mouse monoclonal anti focal adhesion kinase (FAK) (1:100) (BD

Bioscience); (c) rabbit polyclonal anti phospho-FAK (Tyr397)

(1:300) (Biosource, Solingen); (d) mouse monoclonal anti paxillin

(1:500) (BD Bioscience); (e) rabbit polyclonal anti phospho-paxillin

(Tyr118) (1:400) (Cell Signaling, Danvers/MA, USA), (f) rabbit

polyclonal anti bronectin (1:400) (SigmaeAldrich). After thor-

ough rinsing with PBS, the samples were incubated with secondary

antibodies and dyes in 1% BSA/PBS for 1 h at RT: (i) goat anti mouse

Cy3 (1:500) (Dianova, Hamburg); (ii) goat anti rabbit Cy3 (1:500)

(Dianova); (iii) goat anti rabbit AlexaFluor488 (1:200, Invitrogen);

(iv) DAPI (1:1000) (SigmaeAldrich); (v) phalloidin AlexaFluor488

(1:200, Invitrogen). Samples were embedded in mounting media

(Mowiol, Hoechst) containing 1% N-propyl-gallate as antifade

(SigmaeAldrich). Matrigel-embedded cells were xed for 15 min

with 4% paraformaldehyde/PBS at 37 C. After permeabilization

with 0.1% Triton/PBS, cells were incubated with a mouse mono-

clonal paxillin antibody (1:500, BD Bioscience) in 1% BSA/PBS

overnight at RT. After thorough rinsing with PBS, samples were

incubated with the corresponding secondary antibodies and dyes

in 1% BSA/PBS overnight at RT (goat anti mouse Cy3 (1:500) (Dia-

nova); DAPI (1:500); phalloidin AlexaFluor488 (1:200)).

2.5. Microscopy and image analysis

Cells in 3D structures embedded in mounting media were

analyzed using a laser scanning microscope (LSM 510 Meta, Zeiss)

equipped with a Plan Apochromat 20/0.8 objective (Zeiss) and LCI

Plan Neouar 63/1.3 DIC ImKorr objective (Zeiss). Maximum in-

tensity projections were producedusing the LSM Zeiss software. 3D

reconstructions of the confocal image stacks were prepared with

the Volocity Software version 4.3.2 (Perkin Elmer). For atomic force

microscopy (AFM) imaging, cells on planar 2D control substrates

were transferred into 35 mm cell culture dishes and imaged in PBS

using a NanoWizard II AFM (JPK Instruments, Germany) mounted

on top of an inverted optical microscope (Carl Zeiss AxioObserver

A1) and a designated Petri dish holder. AFM imaging was per-

formed in contact mode at a typical scan frequency of 0.4 Hz with

V-shaped cantilevers with a spring constant of 0.03 N/m (MLCT,

Bruker). For scanning electron microscopy (SEM), scaffold samples

required no dehydration and were sputtered with gold (6e10 nm)

before they were imaged under vacuum at 5 kV using a Supra 55

scanning electron microscope (Zeiss).

2.6. Cell volume measurements in 2D and 3D micro-environments

Cell volumes in 2D were calculated from AFM height images

using two independent software systems: (1) Gwyddion (http://

gwyddion.net/) and (2) Matlab (Mathworks). When using the

Gwyddion software, the base level of the AFM height images was

determined by placing a plane through three ctive points posi-

tioned at the cells-substrate border. A height threshold was then

adjusted to isolate the cell from the substrate and to obtain the cellvolume data. Volume calculation using Matlab was performed us-

ing AFM height image in ASCII text le format. The cell volume was

then calculated using the following formula:

XdV ¼

X z dA ¼

X z dx dy (1)

where z is the height of a data point, dA the basal plane which is the

product of the distance of two pixels in horizontal and vertical

direction. The total volume corresponds to the sum of the single

volume elements. The zero level was set manually and the average,

minimum and maximum values were determined. The cell volume

was then calculated from the difference of the measured value to

the average, minimum and maximum value.

To obtain cell volumes in 2D and 3D by optical microscopy,

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samples were stained for F-actin and imaged by confocal laser

scanning microscopy (CLSM). Complete image z -stacks of single

cells were generated with the distance between two neighboring z -

slices set to 410 nm. For volume analysis, the cell cross section area

(mm2) in each z -slice of the confocal image stack was rst deter-

mined using morphometric segment analysis in Metamorph®

(Visitron). The cross section areas from all z -slice were then added,

and the sum of the single area elements multiplied with the z -

spacing value (here 410 nm) to obtain the volume (mm3) of a single

cell. In the case of the 3D structures, only cells that were fully

suspended in the third dimension were evaluated and only cells

that adhered to the outer segment of the 3D wheel structure were

analyzed.

2.7. Nuclear volume measurements in 2D and 3D micro-

environments

The nuclear volume of DAPI-stained cells was determined from

CLSM image z -stacks using a custom-made Matlab routine. The

CLSM image intensity was normalized to a 0e1 range according to

the following equation:

I normalized ¼I input emin

I input

max

I input min

I input

(2)

where min(Iinput) and max(Iinput) denotes the minimum and

maximum intensity of the input image, respectively. Image

denoising based on wavelet (Bayes soft thresholding) and Wiener

lter was performed on the normalized CLSM image. This proce-

dure was followed by image deconvolution based on the scaled

gradient projection algorithm [43]. To determine the boundary of

the cell nucleus, level-set based image segmentation was per-

formed on the deconvoluted image [44]. The resulting binary image

was furthered processed by image dilation and erosion with one-

pixel-sized diamond structure element. The nucleus volume was

then computed from the binary image using Minkowski measures

[45]. Nuclear-to-cell volume ratios (N/C ratio) were obtained by

dividing the nuclear volume by the cell volume.

2.8. Statistics

OriginPro 8G software (OriginLab Cooperation, Northampton,

USA) was used for statistical analysis (ANOVA test). Differences

were considered as statistically signicant when the calculated p

value was less than 0.05 (*). A p value above 0.05 was considered

non-signicant (z, þ).

3. Results

3.1. Tailored bio-functionalized 3D cell culture substrates support cell proliferation

The majority of cell culture experiments are performed on rigid

and planar (2D) substrates, even though in vivo most cells reside in

pliable 3D environments. New technologies to fabricate dened 3D

culture substrates are becoming increasingly available, but the

impact of 3D cell culture conditions on single cell behavior is still

incompletely understood. To analyze the inuence of 3D culture

conditions on cell behavior systematically, we used DLW to produce

two types of 3D polymeric scaffolds with different mechanical

properties. The rst scaffold type is made of pentaerythritol tet-

raacrylate (PETTA) and containsattened beam structures arranged

into a wheel-like architecture (Fig.1A). This conguration results in

a mechanically stable scaffold that cannot be visibly deformed by

cellular contraction forces (“stiff ” scaffold). In the second scaffold

type made of Ormocomp, pillars are connected by thin, exible

beams (Fig. 1B), yielding a scaffold that can be easily deformed by

contractile forces exerted by single cells (“soft” scaffold). “Stiff ” and

“soft” thus refer to the ability (or inability) of a single cell to deform

parts of these scaffolds. Both PETTA and Ormocomp have compa-

rable E-moduli values in the high three-digit MPa or even GPa

range. Scaffold deformability thus depends primarily on its geom-

etry, rather than the E-modulus of the polymeric material.

We rst tested the biocompatibility of the produced 3D DLW-

scaffolds and analyzed whether they supported fundamental cell

functions, such as cell adhesion, growth, and proliferation. Both

scaffold types were homogeneously coated with bronectin to

support stable cell attachment and to provide identical surface

chemistry (Supplemental Fig. S2). Buffalo rat liver broblasts (BRL)

grown in these 3D micro-environments easily adapted their cell

shape to the complex scaffold geometries (Fig. 2), indicating ef -

cient attachment to both scaffold types. Proliferation rates of BRL

cells were similar in both 3D scaffold types and when compared to

2D control cell culture substrates (bronectin-coated glass cover

slips, Supplemental Fig. S3). This indicates that both 3D scaffold

types support normal cell proliferation rates.

3.2. Comparing cell-matrix adhesions between 2D and 3D micro-

environments

Previous studies have shown that cells cultured in cell-derived

3D matrices develop cell-matrix adhesions which differ in

morphology and molecular composition from those formed on

planar 2D substrates [16,23,24]. Totest whether the dimensionality

and mechanics of our scaffolds affect the formation of 3D cell-

matrix adhesions, we analyzed the appearance of prominent

adhesion markers by immuno-uorescence staining. For this, we

cultured BRL cells on bronectin-coated 2D control surfaces and in

stiff or soft 3D scaffolds and rst visualized paxillin-containing cell-

matrix adhesions (Fig. 3AeC). Paxillin localized into classical focal

adhesions on 2D substrates or into elongated patches along thebeams of the 3D scaffolds (Fig. 3B, C). In addition, the prominent

cell-matrix adhesion markers phospho-tyrosine (Tyr99), phospho-

focal adhesion kinase (pFAK) (Tyr397), phospho-paxillin (Tyr118)

were present in all cells cultured in the 3D scaffolds, similar to cells

growing on planar glass substrates (Supplemental Fig. S4). How-

ever, we noticed a slight reduction of FAK and phospho-FAK

staining intensity in both 3D scaffold types compared to 2D sub-

strates. Furthermore, cell-matrix adhesion sites also stained for

vinculin and b1-integrin in BRLs cultured in 3D scaffolds (data not

shown). In addition, BRLs cultured in DLW-fabricated 3D scaffold

formed pronounced actin stress bers primarily located along the

cell periphery (Fig. 3B, C). These results established that

bronectin-coated 3D scaffolds (independently of their mechanics

and architecture) ef ciently promote cell-matrix adhesion siteformation similar to conventional 2D culture conditions.

3.3. Cell and nuclear volume varies between 2D and 3D micro-

environments in a cell-type dependent manner

To investigate potential cell type-specic effects of 3D culture,

we analyzed overall cell morphology in several mesenchymal and

epithelial cell lines. As further examples of mesenchymal cells in

addition to BRL cells, we studied mouse embryonic broblasts

(MEF) and B16 cells from a mouse melanoma tumor. As examples

for epithelial cells we investigated A549 cells from human lung

carcinoma and normal rat kidney cells (NRK). The different cell

types were cultured on bronectin-coated 2D glass surfaces and in

stiff 3D scaffolds for three hours. We then analyzed the morphology


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of all cell types after immuno-staining (Fig. 4A for a merged pro-

jection, Fig. 4B for a 3D reconstruction). All cell types attached to

the 3D scaffolds, but they displayed marked morphological differ-

ences. Epithelial cells were often in close contact with the walls of the 3D structure and elongated and roughly planar in morphology.

In contrast, broblasts typically explored the full 3D environment,

spanning their cell body across different 3D segments, and dis-

played an angular and bulky morphology. The extensive spreading

behavior suggested that broblasts display increased cell volumes

in 3D environments.

To quantify cell volume in both 2D and 3D, we developed a dual

approach using AFM and CLSM imaging. Cell volume measure-

ments of thin, well-spread cells on 2D substrates were performed

by AFM height imaging in liquid (Fig. 4C). AFM imaging generates

high resolution topographic images of surface-attached cells and

provides the highest volume accuracy for 2D samples, but is typi-

cally limited to samples displaying comparatively low height

changes. Since cells spreading on 2D surfaces are usually at(~1e3 mm), they could be easily imaged by AFM (Supplemental

Fig. S5). In contrast, CLSM imaging is diffraction-limited and can

only offer a realistic resolution of ~500 nm in z-direction. In addi-

tion, given the low height of well-spread cells, only a few confocal

slices will intersect the cell, potentially decreasing the accuracy of

the volume data further. CLSM is therefore less suited for deter-

mining cellular volumes in 2D. To nevertheless determine the

correlation between AFM and CLSM 2D volume data given the

specic pros and cons of each technique, we cultured BRL cells on

bronectin-coated glass cover slips and rst determined the vol-

umes of individual cells by AFM imaging (Supplemental Fig. S5).

Subsequently, we stained the samples for F-actin and imaged the

respective cells by CLSM. Thus, we generated both AFM and CLSM

volume data for each individual cell. The cell volumes determinedby AFM and CLSM correlated well for individual cells and were not

signicantly different when averaged over all analyzed cells,

demonstrating the general feasibility of both techniques for

determining cellular volumes. However, cell volumes determined

by CLSM were systematically higher by 10e15% compared to cell

volumes determined by AFM. We interpreted the slightly higher

CLSM volumes to reect optical diffraction effects, leading to a

slight broadening of the cell contour and overestimation of the true

cellular volume from confocal stacks. We considered the AFM data

to provide a more accurate account of cell volumes and subse-

quently used AFM volume data in the 2D system in our analysis.

However, AFM is a surface scanning technique incompatible with

complex 3D samples. In these cases, analysis of CLSM z -stacks

provided a good estimate of cellular volumes, especially since the

often greatly increased cellular extension in z -direction could be

sampled more accurately with a larger number of confocal slices.

The complementary AFM and CLSM analysis conrmed that all

broblast-like cell lines display signicantly larger cell volumeswhen cultured in stiff 3D scaffolds compared to planar 2D surfaces

(Fig. 4D, BRL: single cell volume in 2D 878 mm3, ~55% increase in

volume in 3D; B16: 1586 mm3 in 2D, ~27% increase in volume in 3D;

MEF: 591 mm3 in 2D, ~63% increase in volume in 3D; ANOVA,

*p < 0.05). In contrast to broblast-like cell lines, the epithelial cell

lines A549 and NRK displayed similar cell volumes when growing

in bronectin-coated 3D scaffolds or on planar 2D surfaces (Fig. 4D,

A549 cells 2D: 1675 mm3, 3D: 1519 mm3; NRK cells 2D: 681 mm3 and

3D: 831 mm3; ANOVA, z, p > 0.05). These results thus revealed a

striking difference in cell volume regulation between broblast-

like and epithelial cells in 3D micro-environments. Additional 2D

control experiments demonstrated that cell volume regulation is

independent of the underlying polymer chemistry of the cell cul-

ture substrates. The volume of BRL broblasts cultured at 2D con-ditions was similar on bronectin-coated glass cover slips or thin

bronectin-coated PETTA or Ormocomp lms (Supplemental

Fig. S5). Hence, BRL cell volumes were generally smaller on all 2D

substrates compared to 3D cell culture conditions (compare Fig. 4D

with Supplemental Fig. S5).

We furthermore assessed whether BRL cell volume differs in 3D

micro-environments of different mechanics and geometries.

However, BRL cells cultured in any 3D micro-environment fabri-

cated by DLW displayed similar cell volumes (Figs. 5 and 3D stiff:

1942 mm3, 3Dsoft:2066 mm3; ANOVA, z, p > 0.05). Scaffold stiffness

and geometry therefore appear to be irrelevant for cell volume

regulation. However, natural ECMs have a heterogeneous compo-

sition, while our 3D scaffolds were only functionalized with a single

ECM component (

bronectin). We therefore also used Matrigel, asoft, gel-like, non-brillar cell-derived heterogeneous protein

mixture primarily composed of laminin and collagen IV and con-

taining different growth factors to introduce an alternative, more

complex 3D environment mimicking the in vivo ECM-environment

of cells. After culturing BRLs for three hours in pure Matrigel, the

cell volume was similar to BRLs growing in either “stiff ” and “soft”

DLW-fabricated 3D scaffold types (Fig. 5, Matrigel: 1977 mm3;

ANOVA, z , p > 0.05; Supplemental Fig. S6). Thus, BRL cell volume

was signicantly larger in all three 3D culture conditions (wheels,

nets, Matrigel) - regardless of scaffold geometry and stiffness e

then on 2D control substrates (Figs. 5 and 2D: 878 mm3; ANOVA,

*p < 0.05).

We also assessed the nuclear volume of broblast and epithelial

cell lines on 2D substrates and in stiff 3D scaffolds based on CLSM-

Fig. 1. Tailored 3D scaffolds fabricated by direct laser writing (DLW). DLW is a single step technique where a femto-second laser beam is focused into a photo-sensitive liquid

material. In the focal volume of the focused laser beam photo-initiator molecules are excited and cause a highly localized chemical polymerization event. (A) Scanning electron

microscopy (SEM) image of a 3D stiff scaffold made of pentaerythritol tetraacrylate (PETTA). (B) SEM image of a 3D soft scaffold made of Ormocomp and composed of pillars

interconnected with beams.


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imaging of DAPI-stained interphase nuclei. A constant nucleus-to-

cell volume ratio (N/C ratio) is known to be important for main-

taining cell integrity and tissue- and cell-specic functions,

whereas deviating N/C ratios have been observed in metastatic

cancer cells [46]. Mirroring the cell volumeresults, nuclear volumes

were increased in all broblast-like cell lines (BRL, B16, MEF)

residing in 3D scaffolds compared to a 2D surface (Fig. 6A, ANOVA,

*p < 0.05). Overall, nuclear volumes in 3D (~620e720 mm3) were

about two- to three-times higher than in 2D (~260 mm3) for all

broblast-like cell lines. In contrast, the nuclear volume of

epithelial cells was not signicantly different between 3D

(~420 mm3) and 2D (~380 mm3) culture conditions (Fig. 6A, ANOVA,

z, p > 0.05), again mirroring the cell volume results (Fig. 4D).

Calculating the N/C ratio under 2D and 3D culture conditions for

Fig. 2. Growth of cells in stiff and soft 3D scaffolds. Buffalo rat liver cells (BRL) were cultivated on bronectin-coated 2D at glass surfaces or in different bronectin-coated 3D

scaffolds of different stiffness and architecture, xed and stained for F-actin (green) and nucleus (DAPI, blue). The rst column shows the maximum projection of a confocal image

stack with the DIC image and uorescence images merged. The second column gives the maximum projection of DIC only and the third column presents a 3D reconstruction. (A) A

BRL cell adherent on a 2D at glass surface. (B) A BRL cell growing in a segment of a stiff 3D scaffold made of PETTA. (C) BRL cells attached to a soft 3D scaffold made of Ormocomp

where the bending of the beams by the cells is clearly visible in the DIC image. The white arrows indicate the direction of view. (For interpretation of the references to color in this

gure legend, the reader is referred to the web version of this article.)



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broblast-like cells (BRL, B16, MEF) revealed no signicant changes

(Fig. 6B, ANOVA, z , p > 0.05). This is in agreement with the obser-

vation that both cell and nuclear volume of broblast-like cells

increase to a similar degree upon culturing in 3D environments

compared to 2D surfaces (compare 4D with Fig. 6A). The N/C ratio

was also maintained in epithelial cells (A549, NRK) for 3D and 2D

culture conditions (Fig. 6B, ANOVA, z, p > 0.05). This in turn is

consistent with the fact that these epithelial cells do not change

Fig. 3. Cell-matrix adhesions of cells on 2D substrates and in tailored 3D scaffolds. Buffalo rat liver cells (BRL) were cultivated on bronectin-coated 2D glass surfaces or bronectin-

coated 3D scaffolds of different mechanics and geometry, xed and stained for F-actin (green), paxillin-marked cell-matrix adhesions (red or white), and nucleus (DAPI, blue). The

rst column shows the maximum projection of a confocal image stack with the DIC image and uorescence images merged. The second column gives the maximum projection of

paxillin-stained cell-matrix adhesions only and the third column presents a 3D reconstruction of the cellular actin cytoskeleton. (A) A BRL cell adhering to a 2D glass surface. The

paxillin-containing cell-matrix adhesions are randomly distributed within the cells and most actin stress bers terminate in cell-matrix adhesion sites. (B) A BRL cell growing in a

stiff 3D scaffold made of PETTA. The paxillin staining is mainly localized along the beams of the scaffold. (C) BRL cells attached to a soft 3D scaffold made of Ormocomp. Paxillin-

positive adhesion sites are predominantly located along the beams of the 3D structure. The white arrows indicate the direction of view. (For interpretation of the references to color

in this gure legend, the reader is referred to the web version of this article.)



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Fig. 4. The cell volume varies between 2D and 3D micro-environments in a cell-type dependent manner. (A) Maximum projection of a confocal image stacks of different cells types

(buffalo rat liver cells (BRL), spindle-shaped subpopulation of mouse melanoma cells (B16), mouse embryonic broblasts (MEF), human lung carcinoma cells (A549), normal rat

kidney cells (NRK)) adherent to stiff PETTA 3D scaffold. DIC image and uorescence images (F-actin ¼ green; nucleus ¼ DAPI, blue) are merged. (B) 3D reconstruction of the

according image in (A). The white asterisks indicate the angle of view between the maximum projection image and the 3D reconstruction. (C) 3D reconstruction of atomic force

microscopy (AFM) height images of cells on 2D glass surfaces in top view. White color in the look-up-table indicates a height of one mm or more. (D) The volume (mm3) of the

different cells types in the 3D structure and on a 2D surface was determined using the actin channel of the confocal image stacks (for 3D) or AFM measurements (for 2D). Fibroblast-

like cells (BRL, B16, MEF) revealed a larger cell volume in 3D substrates compared to 2D surfaces. Epithelial cells (A549, NRK) showed no difference concerning their cell volume

between being cultured in 2D and 3D (ANOVA, *, p < 0.05; z, p > 0.05). The data were obtained from at least four independent experiments; the number of analyzed cells is indicated

in the according bars. The error bars are given as standard deviation of the mean. (For interpretation of the references to color in this gure legend, the reader is referred to the web

version of this article.)



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their cell or nuclear volume during the transition from 2D to 3D

environments (compare 4D with Fig. 6A). However, we observed

differences in the proportion of nucleus to overall cell volume be-

tween the various cell types. While the nucleus in NRK cells con-

stitutes 50% of the total cell volume, it takes up only 30e40% in the

other cell types (BRL, B16, MEF, A549; Fig. 6B). Overall, these results

indicate that changes in cell and nuclear volume in 3D vs. 2D en-

vironments is cell type-dependent, but that the N/C ratio is unaf-

fected by the substrate dimension.

4. Discussion

The in vivo ECM is not only characterized by the composition of

numerous adherent and soluble molecules, but also by its 3D-ge-

ometry and mechanical properties. Here, we demonstrate that

DLW-fabricated 3D scaffolds can be used to systematically study

the impact of these various factors on cell-type specic behavior.

Fibroblast-like cell lines exhibit increased cell and nuclear volumes

in all investigated 3D scaffold types compared to 2D substrates,

independently of 3D substrate mechanics and architecture. In

contrast, epithelial cells preserve similar cell and nuclear volumes

in 2D and 3D environments. However, N/C ratios remain roughly

constant for all cell types and culture conditions. Cell-matrix ad-

hesions forming in stiff and soft 3D micro-environments feature a

similar composition of prominent adhesion marker proteins as

adhesions in 2D environments.

Adherent cells require stable anchorage to their micro-

environment and the generation of intracellular tension to divide

[47e50]. Mechanical matrix properties also have a signicant in-

uence on cell proliferation [51e53]. Additionally, is has been

demonstrated that the proliferation rate depends on matrix

composition and substrate dimensionality [16,22]. Since we found

similar cell proliferation rates of cells on 2D and in 3D micro-

environments, we conclude that 3D DLW-structures provide suf -

cient support for maintaining cell growth and proliferation rates.

Cell-matrix adhesions are important cellular sensors of matrix

rigidity, as they mechanically link the ECM with the acto-myosin

system [9,10,18]. Compared to the 2D situation, relatively little is

known about the structure and function of cell-matrix adhesions

formed in 3D environments. Based on the markers investigated in

our study, we did not observe major differences in the composition

of cell-matrix adhesions of cells grown in 3D scaffolds in compar-

ison to 2D substrates. Nevertheless, we noticed small morpholog-ical differences of cell-matrix adhesionsbetween 2D and 3D culture

conditions. In 2D culture cell-matrix adhesions were usually ellip-

tical, whereas in 3D culture adhesions were either highly elongated

when localized on features of the DLW structures. Our observations

are consistent with several studies that describe the general exis-

tence of cell-matrix adhesions in 3D culture substrates, but reports

vary depending on the adhesion markers and 3D matrix used. For

example, it has been shown that cell-derived matrices are more

effective in mediating broblast adhesion than 2D substrates or 3D

collagen gels, and that cells in soft 3D cell-derived matrices develop

so-called 3D cell-matrix adhesions, which differ in their molecular

composition from those formed on planar 2D substrates

[16,22e25]. Additionally, it was revealed that multiple cancer and

mesenchymal cell lines produce cell-matrix adhesions in 3Dcollagen matrices depending on the cells' localization within the

matrix (close to the edges or to the center of a 3D collagen matrix)

[19e21,54]. We noticed in our study a slight reduction of FAK and

phospho-FAK staining intensity in both 3D scaffold types compared

to 2D substrates. Other studies showed increased FAK phosphory-

lation in dense and stiff collagen gels in comparison to softer gels in

Fig. 5. The volume of BRL cells is increased upon culture in 3D micro-environments.

The cell volume (in mm3) of buffalo rat liver cells (BRL) was determined using the

actin channel of confocal image stacks (for 3D scaffolds) or AFM measurements (for 2D

substrates). The cell volume of BRL cells was signicantly increased in stiff and soft 3D

micro-environments (dark gray and light gray bars) and in pure Matrigel (white bar),

in comparison to the 2D surface (black bar, ANOVA, *, p < 0.05). The data were obtained

from at least four independent experiments; the number of analyzed cells is indicated

in the according bars. The error bars are given as standard deviation of the mean.

Fig. 6. The nuclear volume increases in a cell-type dependent manner in 3D scaffolds while the nucleus-to-cell volume ratio is maintained. The cell volume and the nuclear

interphase volume (mm3) of different cells types (buffalo rat liver cells (BRL), spindle-shaped subpopulation of mouse melanoma cells (B16), mouse embryonic broblasts (MEF),

human lung carcinoma cells (A549), normal rat kidney cells (NRK)) on 2D glass surfaces and in stiff 3D PETTA scaffolds was determined. (A) The nuclear volume of cells residing in

3D substrates increased about two- to three-fold in broblast-like cells compared to the nuclear volume on 2D surfaces. Epithelial-like cells revealed no nuclear volume change

upon culturing in 3D structures. (B) The nucleus-to-cell volume ratio (N/C ratio) was maintained for all cell types (broblast-like vs. epithelial-like cells) under all culture conditions

(2D vs. 3D). While the nucleus occupied more than 50% of the cell volume in NRK cells, it was only about 20%e40% for BRL cells, B16 cells, MEF, and A549 cells (ANOVA, *, p < 0.05; z,

p > 0.05). The data were obtained from at least four independent experiments; the number of analyzed cells is indicated in the according bars. The error bars are given as standard

deviation of the mean.



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epithelial cells [55] and reduced FAK phosphorylation in 3D

collagen gels in smooth muscle cells [56]. Nevertheless, a numberof

studies are in apparent conict regarding the role of substrate

dimensionality and mechanics on the existence of cell-matrix

adhesion formation, composition, morphology, and function

[10,11,13,16,19e21].

Cell shape changes rely on the spatial and temporal coordina-

tion of biochemical and physical processes at the molecular, cellular

and tissue scale, and the homeostasis of cell volume is a major

challenge for eukaryotic cells and tissues [57e60]. Cell volume and

morphology may vary considerably under different environmental

conditions. For example, (i) cells regulate their volume in response

to changes in osmolarity of both extracellular and intracellular

environments [57,61]; (ii) cells show cell-cycle related volume

changes and in particular dividing cells dramatically change in

volume [59,60,62e64]; (iii) the volume of immune cells [65] or

metastasizing cells [13,14,64,66] changes when these cells squeeze

through narrow connements; and (iv) cell volume changes are

observed during epithelialemesenchymal transition in embryonic

development [67]. Changes in cell volumeare also associated with a

broad spectrum of patho-physiological conditions. For cancer it is

known that trans-epithelial and cell volume regulatory ion trans-

porters and channels are dys-regulated [58,61,64,66]. Using struc-turally dened DLW-fabricated scaffolds, we now demonstrate an

additional cell-type dependent effect on cell and nuclear volume

regulation in 2D and 3D environments. While broblast-like cell

lines exhibit increased cell and nuclear volumes in stiff 3D scaffolds

compared to 2D substrates, epithelial cells display similar volumes

in both (3D and 2D) environments. Volume measurements have

been mainly performed on cells cultured on planar and rigid (ECM-

coated) substrates. In general, the cell volumes we obtained in our

study werein the range of volumes measuredfor various cellsin 2D

studies (e.g., osteosarcoma cells: 1900 mm3 [68]; human osteo-

blasts: ~5000 mm3 [68]; human dermal broblast: 2400 mm3 [69];

rapidly proliferating mesenchymal stem cells: 1200e2100 mm3

[68]) and for further cell types [61,68e71]. However, so far only few

studies have analyzed single cell volumes in articial 3D environ-ments [72,73]. Forexample, Farooque et al. investigated the volume

of human bone marrow stromal cells (hBMSCs) cultured in 3D gels

made of collagen or poly(ethylene glycol)tetramethacrylate and in

3D scaffolds of different geometries fabricated from poly(D,L -lactic

acid) or poly-(ε-caprolactone) [72]. Strikingly, hBMSCs volumes

obtained in this study were on average two to ve times (poly(D,L -

lactic acid scaffolds)) or even up to 20-times (free-form fabricated

poly-ε-caprolactone scaffolds) larger than the cell volumes we

determined in our study; despite a similar approach for the

calculation of single cell volumes. These results point to large cell-

type specic differences in 3D volume regulation between stem

cells and broblast-like and epithelial cells. Furthermore, Farooque

et al. also obtained large cell-to-cell deviations in hBMSC volumeon

the same 3D substrate [72], while we observed a more narrowspread of cellular volume on a given substrate. Further differences

in 3D cell volume regulation could arise from the fact that different

materials and architectures were used compared to our study,

resulting in different chemical and physical signals received by the

cells. Additionally, Farooque et al. used culture times of up to

24 hours, while we analyzed cell volumes after three hours of

culture. In our 3D scaffold systems cells were well-spread after

three hours and we did not extend culture times further to avoid a

potential removal of the bronectin coating from the scaffold.

Nevertheless, we performed control experiments for broblasts

cultured in 3D scaffolds for 22 hours and observed that our 3D

scaffolds are suitable for longer cell culture times. However, we

detected not signicant differences in cell volume after 22 hours

compare to three hour of culture duration (data not shown).

Accurately measuring the volume of cells in complex environ-

ments presents an ongoing challenge in many areas of experi-

mental and diagnostic science. Many different techniques have

been applied to provide estimates of single cell volumes, including

electrophysiological methods, interferometry, optical sectioning by

confocal microscopy, light scattering detection, quantitative uo-

rescence or phase contrast microscopy, and AFM measurements

[68]. Unfortunately, often only relative changes in volume are

given, rather than absolute values which complicate the compari-

son between different studies [57,74,75]. We also realized that most

studies only investigate the volume of single cells that are cultured

eitherat 2D conditions or in 3D environments, while changes in cell

and nuclear volume during the transition from 2D to 3D cell culture

environments have rarely been reported. Our results therefore

identify an important but often disregarded aspect of cell type

specic behavior that is subject to cell volume regulation. Scaffolds

produced by DLW can be used to investigate such cell type-specic

differences in volume regulation in dened 3D environments.

Generally, cell volume and pressure regulation incorporates the

spatio-temporal synchronized function of water permeation, ion

channels, and transporters [57e59,64,74]. Although the precise

mechanisms have yet to be elucidated, early signals of volume

perturbation have also been demonstrated to involve the clusteringof integrin adhesion receptors into focal adhesion complexes and

the initiation of downstream signaling events such as the activation

of e.g., Rho family GTPases, phospholipases, lipid kinases, and

tyrosine- and serine/threonine protein kinases [58,64,76]. Adhe-

sion receptor-mediated processes may therefore play a role in cell

volume regulation. However, we did not observe a signicant

change in the molecular composition of cell-matrix adhesions with

the markers tested here under the different 2D and 3D culture

conditions. Nevertheless, cell-matrix adhesions sites contain a large

number of adhesion proteins, and some variations in the compo-

sition of adhesion sites between 2D and 3D environments might be

expected inuencing cell volume regulation [10,11,16,19e21,23].

We also assessed the cell volume of BRL cells in pure Matrigel

lacking a DLW-fabricated 3D scaffold. Here, cells were signicantlylarger than on 2D substrates, similar as in both 3D scaffold types

(stiff and soft). BRL cells in pure Matrigel were mainly spherical

with some elongated protrusions, resulting sometimes in an

amoeboid to star-like cell morphology. An amoeboid cell

morphology and formation of cell-matrix adhesions were reported

in previous studies on different broblast cells cultivated in other

3D gel-like substrates [9e11,13,16,19,22]. In contrast, we did not

detect distinct paxillin-positive cell matrix adhesions in BRL cells

cultured in Matrigel. However, this is likely due to the short culti-

vation time of three hours because we observed dot-like paxillin-

positive cell-matrix adhesions of BRL cells after Matrigel culture for

22 hours (data not shown).

Our study also revealed that both nuclear and total cell volume

of broblast-like cells increased in 3D vs. 2D micro-environments.Interestingly, the N/C ratio remained unchanged for each cell line

under all culture conditions. A constant N/C ratio appears impor-

tant for maintaining cell integrity and normal function. For

example, a decreased N/C ratio is an indication for metastatic cells

and is often used as a metric in cancer detection [46]. In breast

cancer cells the nuclear volume increases from normal to meta-

static cell stages by a factor of about 1.6 for the cell volume and a

factor of two for the nuclear volume. Since we and others observed

that increased nuclear volume correlates with an increase in cell

volume [77e79], the question arises how total cell and nuclear

volumes could be co-controlled. Potential scenarios include water

inux through nuclear pores, signaling downstream of ECM and

growth factor receptors, and mechanical coupling between the

nucleus and the cytoskeleton [80e

82]. For instance, nuclear



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volume could be regulated by mechanical tension, organization of

the actin network or other cytoskeleton elements, and by how the

nucleus is connected to cytoskeletal laments. In fact it has been

demonstrated that reducing mechanical tension in the cytoskel-

eton leads to a decrease in nuclear size of endothelial cells [83].

In contrast to broblast-like cells, epithelial cells displayed

similar cell and nuclear volume in 2D and 3D micro-environments.

The constant cell volume of epithelial cells in tailored 3D structures

and on 2D substrate might be linked to the in vivo environment of

these cells: the ECM of different tissues is known to vary consid-

erably in structure and composition, as does the morphology and

size of the respective matrix-embedded cells. For instance, polar-

ized epithelial cells reside in vivo on a planar basal lamina con-

taining a large amount of densely packed ECM proteins. In contrast,

broblasts reside in a 3D environment of loosely organized con-

nective tissue.

5. Conclusion

Dimensionality-dependent effects on total cell and nuclear

volume expand our understanding of cell behavior in 3D environ-

ments. Cell and nuclear volume regulation seem to reect the

tissue-specic arrangement of cells in vivo such as loose connectivetissues (broblasts) or connement into at layers (epithelial cells).

In future, it will be of interest to investigate volume-sensitive

signaling pathways to elucidate in more detail the inuence of

substrate architecture on cell volume regulation processes. Poten-

tially interesting pathways include second messenger release (e.g.,

Ca2þ signaling), phosphorylation levels of proteins involved in

volume regulation (e.g., caveolin-1, cortactin, FAK), signaling via

integrins and receptor tyrosine kinases, and cytoskeletal reorga-

nization [58,59,64,76]. Of interest willalso be experiments studying

cell growth and cell volume regulation in response to different

ECM-ligand composition and the ligand's density. This will allow us

to progress beyond investigating only effects of altered dimen-

sionality and mechanics on cell behavior. Overall, this will lead to a

more detailed understanding of cell behavior in engineered 3Dmicro-environments that more accurately emulate in vivo situa-

tions. Generally, studying cells in 3D environments will be helpful

elucidating physiological and pathological aspects such as the im-

mune response and cancer progression.

Acknowledgments

We thank Tanja Landmann for her help with the cell culture

routine and for her assistance performing cell experiments. C.M.

Franz acknowledges nancial support from the Deutsche For-

schungsgemeinschaft (DFG) and the State of Baden-Württemberg

through the DFG-Center for Functional Nanostructures (CFN)

within subprojects E2.4.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.biomaterials.2015.08.016 .

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Cell Type-specific Adaptation of Cellular and Nuclear Volume in Micro-Engineered 3D Environments

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