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Introduction:As scientists better understand the benefits of
growing cellsin three dimensions (3D) and routinely adopt 3D
culturetechniques, methods for visualising cells must also
beadapted and optimised.
The most common and routinely used technique for trackingtwo
dimensional (2D) cell cultures is light microscopy.Traditional 2D
monolayer cultures are highly transparent andwithin a single
optical plane. The minimal light diffraction anddiffusion presented
by the plastic surface allows thecollection of focussed microscopic
images. Cells cultured ingenuine 3D environments, such as in
AlvetexScaffoldpresent some of the same constraints as tissue
samples orbiopsies in that simple, live observation of cultures via
phasemicroscopy is not optimal.
There are however, other techniques that can beimplemented which
will allow the user to monitor cultureprogress easily and
effectively in 3D; Simple dyes can beused to identify culture
confluence and viability. The varietyof end-point visualisation
techniques available to thoseculturing cells in 3D is extensive.
Options include, but arenot limited to, live cell imaging,
fluorescent marker analysis,confocal analysis, histology using a
range of cytologicalstains and electron microscopy. All of these
techniques havebeen performed on cultures grown in AlvetexScaffold
withexcellent results. Here we review common imaging methodsand
outline their use and suitability for cultures grown in 3Dwithin
the AlvetexScaffold.
Cell visualisation options withinAlvetexScaffold:One of the
simplest methods to visualise cultured cells isphase contrast
microscopy. This technique is routinely usedto observe cells grown
in 2D. Cells do not have to besacrificed, therefore cultures can be
visualised throughoutthe experimental time course, allowing the
scientist togauge culture quality and progress. This method
ofvisualisation, however, is not optimum for detailed analysisof 3D
cultures or sections of tissue.
Why cant I easily see cells inAlvetexScaffold under the
lightmicroscope?AlvetexScaffold is a polystyrene scaffold in which
voidshave an average diameter of 40m and interconnects ofaverage
diameter 13m. AlvetexScaffold is supplied as a200m thick membrane.
It is this specific architecture thatallows extensive cell-cell
contacts throughout the scaffoldand the formation of tissue-like
structures. Cells withinAlvetexScaffold retain a more cuboidal
morphology and 3Dcell structure particularly in the z-plane (Figure
1) and this inturn has a significant impact on cell function.
A Review of Imaging Techniques Compatiblewith Three Dimensional
Culture of CellsGrown in AlvetexScaffold
AlvetexScaffoldtechnology represents anovel tool for the
scientistworking in the cell culturefield by offering anopportunity
for majoradvancements in cellularorganisation overtraditional 2D
cultures.Because cells inAlvetexScaffold formtissue-like
structures,sophisticated techniquestraditionally reserved forthe
analysis of tissuesbecome more appropriatefor cell
visualisation.
Setting The Standard For REAL 3D Cell Culture 01
Legend: Scanning electron microscopy image of the structure
AlvetexScaffold (main picture) and populated withTERA2.cl.SP12
cells as visualised by histological staining (right)
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AB
C
D
2D cell culture 3D cell culture
SEM image of thestructure ofAlvetexScaffold
(highmagnification).
SEM image showingcells growing throughAlvetexScaffold so thatthe
scaffold itself is nolonger clearly visible.
When viewing an unstained, unsectioned AlvetexScaffoldculture
under a standard light microscope, the combineddensity and
thickness of the scaffold and the 3D culturewithin it prevent the
clear visualisation of individual cells, dueto light diffraction
and its inability to penetrate into the 3Dstructure. This is to be
expected of true 3D cultures andpieces of tissue, and is a well
documented fact in recentpublished reviews, for example see
[2,3].
As a result, many groups are focussing on devisingalternative
methods for cell visualisation within 3D scaffoldcultures and
tissue-engineered materials (for a recent reviewsee [4]). Methods
vary, ranging from the detection offluorescent labelling (for
example see [5, 6]) orautofluorescence [7], to the use of magnetic
resonanceimaging to locate and track cells within the
scaffoldarchitecture [8]. Many of these methods require
expensiveequipment, although alternative methods exist to track
3Dculture progress easily and cheaply as outlined in thisdocument.
A quick comparison of common techniquesavailable for imaging cells
in 2D versus 3D cultures / tissuesis summarised in Table 1 (page
3).
To visualise cells cultured in AlvetexScaffold, a range
ofmethods are appropriate and available. These are discussedin
detail below.
Which techniques can be used tovisualise cells in
AlvetexScaffold?Choosing the most appropriate technique to
visualise cells inAlvetexScaffold depends on several factors. The
followingsections will help in deciding which methods are
bestadapted to the individual experimental situation.
If the aim is to simply check for and monitor the presence
ofgrowing cells within AlvetexScaffold, then a number ofdifferent
techniques can be employed. Images obtained viastandard light
microscopy can be enhanced by the additionof a cellular dye which
increases the contrast of cells overthe background.
Neutral Red Staining of Cultures forLight MicroscopyNeutral Red
is a common histological dye used for stainingcell nuclei (Figure
2.) It is also used widely as a cell vitalitystain. Results can be
qualitative or quantitative dependingupon the method of analysis
implemented.
Figure 1. An example of how cells retain a more in-vivo-like
structure when cultured in 3D within AvetexScaffold.It is the
increased volume of cells in the z plane that hinders optimal
in-focus visualisation of 3D cultured cellsunder standard light
micrcoscope.conditions. Data generated as part of a collaborative
project betweenReinnervate and LGC Standards [1].
AlvetexScaffold is a 200m thickhighly porous, inert
polystyrenescaffold that provides culturedcells with an
optimalenvironment to grow in 3D. Thisallows for the formation of
3Dniche microenvironments inwhich cell-cell interaction
andcommunication networks occur.
The geometry and dimensions ofAlvetexScaffold have
beenspecifically designed to mimicthe in vivo cellular
environment:no cell is more than 100 m froma source of nutrients
and gasses.This compares favourably to thetypical in vivo
arrangementwhere cells are generally nomore than 150-200 m awayfrom
a capillary. Once seededonto the AlvetexScaffold,typically cells
easily invade thescaffold and start to producegenuine, homogeneous
3Dcellular structures that resemblemicro-slabs of tissue.
Figure 2. Cell cultures can be visualised on AlvetexScaffold by
staining with the non-toxic dye Neutral Red. CHO-K1 cells were
seeded onto AlvetexScaffold (AVP002) ata density of 0.5 million
cells per scaffold. After 2 days cells were exposed to 0.5 %Neutral
red dye solution (Sigma, N6264-50ML). For full experimental details
refer toNeutral red staining protocol located at
www.reinnervate.com/alvetex/protocols
Setting The Standard For REAL 3D Cell Culture 02
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Brightfield / PhaseContrast Microscopy
Standard FluorescenceMicroscopy
Confocal LaserScanning Microscopy
Histology
Transmission ElectronMicroscopy (TEM)
Scanning ElectronMicroscopy (SEM)
Commonly availableNo costEasy-to-useRoutineBest suited for 2D
culture and tissuesections
Generally availableExpensiveModerate training
requiredRoutineBest suited for 2D culture and tissuesections
Less availableExpensiveTraining requiredLess routineSuited for
both 2D and 3D cultures andtissues
Generally availableInexpensiveModerate training
requiredRoutineIdeally suited for 3D cultures andtissues
Less availableExpensiveSubstantial training requiredLess
routineSuited for both 2D and 3D cultures andtissues
Less availableExpensiveSubstantial training requiredLess
routineSuited for both 2D and 3D cultures andtissues
Unstained or stained samplesLive or fixed samples
Stained samples onlyLive or fixed samples
Stained samples onlyLive or fixed samples
Fixed and Stained samples onlyFixed samples only
Fixed samples only
Fixed samples only
Stained samples only (brightfield)Fixed and sectioned
samplespreferablyLive or un-sectioned samples maybe visible if the
cells are at arelatively low density (phase)
Stained samples onlyFixed and sectioned samplespreferablyNot
recommended for un-sectioned samples
Stained samples onlyFixed and sectioned samplesobtain higher
resolution imagesLive or un-sectioned samples canbe visible (using
reporters) Reconstruction of 3D architectureof thicker samples is
feasible
Stained samples onlyFixed and sectioned samples only
Fixed and sectioned samples only
Fixed and un-sectioned samplesonly
Method Comments 2D samples 3D & tissue samples
Setting The Standard For REAL 3D Cell Culture 03
Table 1. Comparison of common techniques available for imaging
cells in 2D versus 3D cultures/tissues.
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The benefit of using non-toxic dyes are that they can
beadministered at a range of time points throughout theexperiment,
washed off and the scaffolds re-incubated withculture media for
further cell growth. (Please note; it isrecommended that if using
dyes for the first time their effecton culture growth and cell
survival is checked for each celltype used. Reinnervate recommends
setting up extrascaffolds for dye analysis). This allows users to
monitor cellsurvival and proliferation within the AlvetexScaffold
over atime course.
In this context, Neutral Red solution can be used as a veryquick
and simple staining technique to follow culture growthand survival
within AlvetexScaffold. Note with increased celldensities the
staining intensity also increased bothmacroscopically and
microscopically (Figure 3).
x20
x10
x4
disc
no cells 0.5 million cells 2 million cells
Visualisation of cellsgrowing in 3D isenhanced by reagentswhich
produce a colourcontrast between thecells and the scaffold:Light
Microscopy is thuscompatible withAlvetexScaffold bystaining the
cells. Cellstaining as the by-product of a colorimetricassay or
fixationprocedure can also beexploited for cellvisualisation
andmonitoring the progressof cultures.
Setting The Standard For REAL 3D Cell Culture 04
Figure 3. Cell cultures can be visualised on AlvetexScaffold by
staining with the non-toxic dye Neutral Red. CHO-K1cells were
seeded onto AlvetexScaffold (AVP002) at a density of 0, 0.5 million
and 2 million cells per scaffold. After 24hours cells were exposed
to 0.5 % Neutral Red dye solution (Sigma, N6264-50ML). Scaffolds
were then transferred to aglass microscopic slide, kept wet by
adding a drop of PBS and imaged under an ICC50HD Leica microscope
with LAS EZsoftware (brightfield setting). For full experimental
details refer to Simple staining methods for viewing cells
onAlvetexScaffold by light microscopy protocol located at
www.reinnervate.com/alvetex/protocols.
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Methylene Blue Staining of Cultures forLight MicroscopyMethylene
Blue is a heterocyclic aromatic chemicalcompound with the molecular
formula C16H18N3SCl.Following a very quick and easy staining
procedure, the dyestains cell nuclei a blue colour which are
visible understandard light microscopy (Figure 4). Methylene Blue
dye istoxic to cells, therefore once cells have been stained,
theculture is sacrificed.
Visualisation as a By-product of an Assay Cultures can also be
visualised as an extra step duringroutine chromogenic cell
viability assays such as MTT. Thisassay involves the conversion of
a yellow, water solublecompound, MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a
tetrazole, to a purple,insoluble formazan, which remains within the
cell until themembranes are lysed, releasing the dye for assay
detection.At the point before cell membranes are usually
lysed,images of the purple-stained cells can be obtained (Figure
5).While this method provides a means for imaging cells, theMTT
reagent is cytotoxic and therefore can only be used atthe
experiment end point, or on surplus scaffolds.
disc
no cells 0.5 million cells 2 million cells
Setting The Standard For REAL 3D Cell Culture 05
Figure 4. Visualisation of cells on AlvetexScaffold with
Methylene Blue solution. HepG2 cells were seeded
ontoAlvetexScaffold (AVP002) at a density of 0, 0.5 million and 2
million cells per scaffold. After 24 hours cells wereexposed to 0.5
% Methylene Blue dye solution (Sigma, 03978-250ML). For full
experimental details refer to Simplestaining methods for viewing
cells on AlvetexScaffold by light microscopy protocol located
atwww.reinnervate.com/alvetex/protocols.
Figure 5: The gross location of viable cells is clearly visible
on the AlvetexScaffold disc after stainingwith MTT cell viability
reagent. HaCat cells were cultured on AlvetexScaffold in the
12-well plate format(AVP002) for 4 days prior to analysis (for full
experimental details see separate MTT protocol available
atwww.reinnervate.com/alvetex/protocols.
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Live Cell Imaging in AlvetexScaffoldUsing Confocal MicroscopyFor
a more in-depth, single cell analysis, live cell imaging, and other
more involved techniques can be implemented.Traditionally, imaging
of live cells allows migrating cells to mimic wound healing or
substrate invasion in vitro. In more recentyears this technique has
been implemented to follow live cell cultures grown in 3D scaffolds
[9], and is providing informationregarding how cell cultures
interact within the 3D niche microenvironment.
Setting The Standard For REAL 3D Cell Culture 06
Figure 6. Cells can be visualised on AlvetexScaffold following
fixation with the yellow coloured Bouins fixative.SW480 colon
carcinoma cells were cultured on AlvetexScaffold and removed for
fixation at 4, 7, 11, 14, 18, and 21days after seeding. The
increasing number of cells over time is reflected in increasing
staining intensity. A. SW480cultures in AlvetexScaffold discs
following Bouins fixation and ethanol dehydration; B. Bouins fixed
and waxembedded SW480 AlvetexScaffold cultures; C. Slide mounted
cross sections (10m slices) of Bouins fixed and waxembedded SW480
AlvetexScaffold cultures stained with haematoxylin and eosin.
Figure 7: GFP-transfected CHO-K1 cells were cultured on
AlvetexScaffold 6 well inserts (AVP004) for 6 days. Capturedimages
were obtained every 30 minutes using a Zeiss LSM 510 confocal
microscope with heated stage (for fullexperimental details refer to
live-cell imaging protocol located at www.reinnervate.com). In this
example, a series ofintegrated z-stacks is presented which shows
the behaviour of a single transfected CHO-K1 cell over a period of
3 hours.
Visualisation as a by-product of Cell FixationCell fixation
methods which employ coloured fixatives are also able to provide a
visual estimation of cell growth inAlvetexScaffold. Bouins reagent
colours areas of cell growth yellow during fixation, which enables
visual comparison of cellgrowth between samples and also cellular
distribution within a single sample (Figure 6A). The Bouins stain
remains visiblethrough wax embedding and is only lost during
subsequent histological staining, for example using haematoxylin
and eosin.
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Fixation of Cells Within AlvetexScaffoldand Subsequent
Downstream AnalysisAll of the techniques discussed below require
fixation ofcells within the AlvetexScaffold. Once cells are
fixednumerous downstream analytical techniques can beperformed. The
choice of the method will depend on thespecific data required.
Fixation is achieved either by chemicalmeans or rapid freezing with
the use of a tissue supportsolution such as the cryoprotective
embedding solution OCTto keep the membrane in place. For full
details ofAlvetexScaffold -compatible fixation protocols refer
toHistology Series Part 1. Choosing the Right Fixative toPreserve
3D Cell Cultures found atwww.reinnervate.com/alvetex/protocols.
Given therelatively thin nature of AlvetexScaffold compared
withtypical tissue samples, fixation of cells is rapid, uniform
andefficient, preserving the 3D culture in a life-like
condition.
Fluorescence Microscopy:Immunofluorescence uses the recognition
of cellular targetsby fluorescent dyes or antigen-specific
antibodies coupled tofluorophores. Depending on the antibody or dye
used,proteins, lipids and DNA can be visualised within
individualcells and tissues. AlvetexScaffold can easily be
processedlike a standard tissue sample, allowing
establishedimmunocytochemical methods to be followed with
excellentresults (Figure 8).
AlvetexScaffold cellcultures are amenable tohighly sophisticated
cellimaging techniques suchas confocal imaging.Confocal microscopy
canbe used to visualise fixedcells or to follow livingcultures in
real time.
AlvetexScaffold culturescan be regarded as in vitrominiature
tissues. As suchstandard histologicaltechniques can be appliedto
their analysis. Thesemethods
includeimmunohistochemistry,histological staining,confocal
microscopy, andelectron microscopy.
Setting The Standard For REAL 3D Cell Culture 07
Figure 8. Human keratinocyte cell line (HaCaT) grown in
AlvetexScaffold (7 days air exposure). Theculture was fixed and
processed for paraffin wax embedding and immunohistochemical
analysis byfluorescent microscopy. The three images from the same
region show; phase contrast (top), bluefluorescent Hoescht 33258
nuclei stain (middle) and Ki67 staining (bottom). For the
fullexperimental details refer to Immunocytochemistry protocol
located atwww.reinnervate.com/alvetex/protocols.
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Confocal Laser Scanning Microscopy:Confocal microscopy relies on
the combination of pointillumination and a pinhole to eliminate
most of the out-of-focus light signal and allows for reconstruction
of 3Dvolumes, making it ideal to image cultures grown in
full-thickness AlvetexScaffold. It should be noted that
lipophilicdyes, such as Nile Red (Figure 9), will bind strongly to
theAlvetexScaffold. However, this feature can be used
toconveniently visualise the scaffold within the cell culture.
Ashigh-density cell cultures grown in AlvetexScaffoldapproximate
the complexity and structure of in vivo tissues,fluorophores
specifically developed for in vivo deep imagingcan be used to
improve performance if needed.
Setting The Standard For REAL 3D Cell Culture 08
Figure 9. Depth colour-coded Z stack of cell-freeAlvetexScaffold
stained with Nile Red. Picture taken ona Zeiss LSM 510 confocal
microscope. Note the depth ofthe Z-stack exceeds 150m. Scale bar
50m.
Figure 10. HepG2 cells grown for 3 days in AlvetexScaffold
12-well plate format (AVP002). Cells were stained withHoechst 33342
(blue), cytokeratin 8 (green) and Nile Red (red). Pictures were
taken on a Zeiss LSM 510 confocalmicroscope. Note the background
signal from AlvetexScaffold in the blue and green channels is very
low. For fullexperimental details refer to Confocal protocol
located at www.reinnervate.com/alvetex/protocols.
A B
C D
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Histology:Histology is seen as the gold standard of cell
visualisation intissues, and therefore is very suitable for 3D cell
culture.Histology is essentially the art of observing a thin
section offixed material under either a light microscope or
electronmicroscope. The ability to specifically identify
cellularcomponents can be enhanced by the addition of
histologicalstains. Common stains include: Haematoxylin and
Eosin,which are generally used together to visualise gross
cellarchitecture; Toluidine Blue stain, which is also a general
cellstain, staining most proteins; and Massons trichrome which
is a three-colour staining protocol producing red keratin
andmuscle fibers, blue or green collagen and bone, light red orpink
cytoplasm, and dark brown to black cell nuclei.
Unlike other 3D cell culture supports, AlvetexScaffold caneasily
be processed like a standard tissue sample, allowingestablished
histology protocols to be followed with excellentresults as seen in
Figure 11.
Setting The Standard For REAL 3D Cell Culture 09
Figure 11: Cells grown in AlvetexScaffold can be fixed and
processed for histologicalanalysis using standard methods. Both
staining examples are carried out on humankeratinocytes (HaCaT)
grown in AlvetexScaffold for 7 days. The sample on the topshows a
culture that was fixed and processed for resin embedding (L R White
resin).Resin sections (1 m) were stained with Toluidene Blue for
structural analysis by lightmicroscopy. The sample on the bottom
shows a culture that was fixed and processed forparaffin embedding.
Sections (10 m) were stained with Haematoxylin and Eosin
formorphological analysis by light microscopy. For full
experimental details refer to theHistology Series of protocols
located at www.reinnervate.com/alvetex/protocols.
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Electron Microscopy:Scanning electron microscopy (SEM) is
becoming a popular method of visualisation of cultures grown in 3D.
SEM is a form ofelectron microscopy where images are obtained by
scanning samples using a high-energy beam of electrons. As the
samplesare scanned the electrons interact with the sample surface,
and these interactions are detected and processed, leading
tohigh-resolution images depicting the sample topography and
composition.
AlvetexScaffold can easily be processed like a standard tissue
sample, allowing established methods to be followed withexcellent
results (Figure 12).
Setting The Standard For REAL 3D Cell Culture 10
Figure 12. Detailed structure of 3D cultures can be visualised
using scanning electron microscopy. Inspection of piecesof
AlvetexScaffold at low magnification shows homogeneous coverage by
cultured cells (A). Higher magnificationimaging in this transverse
section reveals cells growing throughout the scaffold (B).
Increasingly higher magnificationmicrographs reveal how cells
interact with each-other and the AlvetexScaffold (C & D). See
scale bar inserts. For fullexperimental details refer to SEM
protocol located at www.reinnervate.com/alvetex/protocols.
A B
C D
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Transmission electron microscopy (TEM) can also beperformed on
cell cultures grown in AlvetexScaffold (Figure13). TEM allows the
ultrastructure of cells to be visualiseddue to the extreme high
magnification achieved. Samplesare processed in a similar way to
SEM, however, instead ofbeing sputter coated in gold the samples
are embedded in
resin and sectioned into ultrathin sections (1 m).Samples are
loaded into the TEM and a beam of electronsis transmitted through
the sections. The electron beaminteracts with the sample
architecture and it is theseinteractions which are detected,
processed and imagesare obtained.
Setting The Standard For REAL 3D Cell Culture 11
Figure 13. (A) TEM image of keratinocytes cultured at air-liquid
interface without collagen or fibroblasts for 14 days. Thescaffold
is indicated by a black arrow. (B)TEM image showing cells
progressively flattening towards the upper surface ofthe culture
after 14 days at air-liquid interface. (C) High magnification image
showing desmosomes, indicated with whitearrows. (D) High
magnification image showing bundling of keratin filaments
underneath cell membrane, indicated withwhite arrows. (Scale bar
500 m) For full experimental details refer to TEM protocol located
atwww.reinnervate.com/alvetex/protocols.
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Conclusions:
As outlined throughout the document, there are many imaging
techniques which canbe implemented to visualise cultures and cells
grown within AlvetexScaffold in . Forfollowing culture progress,
dyes stain cells contrasting them against the scaffoldbackground
allowing visualisation via light microscopy. For more in depth
cultureanalysis, a range of more complex techniques can be
implemented similar to thoseperformed on tissue samples with
excellent results.
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