pharmaceutics
Article
Assessing the Potential of Molecular Imaging for Myelin
Quantification in Organotypic Cultures
Ander Egimendia 1,2,3 , Susana Carregal-Romero 1,4 , Iñaki Osorio-Querejeta 2,3 , Daniel Padro 1 ,
Jesús Ruiz-Cabello 1,4,5,6 , David Otaegui 2,3, * and Pedro Ramos-Cabrer 1,5, *
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Citation: Egimendia, A.;
Carregal-Romero, S.;
Osorio-Querejeta, I.; Padro, D.;
Ruiz-Cabello, J.; Otaegui, D.;
Ramos-Cabrer, P. Assessing the
Potential of Molecular Imaging for
Myelin Quantification in Organotypic
Cultures. Pharmaceutics 2021, 13, 975.
https://doi.org/10.3390/
pharmaceutics13070975
Academic Editor: Twan Lammers
Received: 14 May 2021
Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology
Alliance (BRTA), Paseo Miramón 182, 20014 Donostia-San Sebastián, Spain;
aegimendia@cicbiomagune.es (A.E.); scarregal.ciberes@cicbiomagune.es (S.C.-R.);
dpadro@cicbiomagune.es (D.P.); jruizcabello@cicbiomagune.es (J.R.-C.)
Multiple Sclerosis Unit, Biodonostia Health Institute, 20014 Donostia-San Sebastián, Spain;
inaki.osorio@biodonostia.org
Spanish Network of Multiple Sclerosis, 08028 Barcelona, Spain
CIBER de Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain
Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain
Departamento de Química en Ciencias Farmacéuticas, Universidad Complutense de Madrid,
28040 Madrid, Spain
Correspondence: david.otaegui@biodonostia.org (D.O.); pramos@cicbiomagune.es (P.R.-C.)
Abstract: Abstract: BackgroundEx vivo models for the noninvasive study of myelin-related diseases
represent an essential tool to understand the mechanisms of diseases and develop therapies against
them. Herein, we assessed the potential of multimodal imaging traceable myelin-targeting liposomes
to quantify myelin in organotypic cultures. Methods: MRI testing was used to image mouse
cerebellar tissue sections and organotypic cultures. Demyelination was induced by lysolecithin
treatment. Myelin-targeting liposomes were synthetized and characterized, and their capacity to
quantify myelin was tested by fluorescence imaging. Results: Imaging of freshly excised tissue
sections ranging from 300 µm to 1 mm in thickness was achieved with good contrast between white
(WM) and gray matter (GM) using T2w MRI. The typical loss of stiffness, WM structures, and
thickness of organotypic cultures required the use of diffusion-weighted methods. Designed myelintargeting liposomes allowed for semiquantitative detection by fluorescence, but the specificity for
myelin was not consistent between assays due to the unspecific binding of liposomes. Conclusions:
With respect to the sensitivity, imaging of brain tissue sections and organotypic cultures by MRI is
feasible, and myelin-targeting nanosystems are a promising solution to quantify myelin ex vivo. With
respect to specificity, fine tuning of the probe is required. Lipid-based systems may not be suitable
for this goal, due to unspecific binding to tissues.
Accepted: 22 June 2021
Published: 28 June 2021
Publisher’s Note: MDPI stays neutral
Keywords: organotypic brain cultures; myelin; demyelination; myelin-targeting liposomes; magnetic
resonance imaging; molecular imaging; imaging probes
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Copyright: © 2021 by the authors.
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4.0/).
1. Introduction
Myelin is an essential element of the central nervous system (CNS) and is implicated
in many neurological diseases. The study of myelin loss (demyelination) and regeneration
(remyelination) is of paramount importance for a better understanding of such processes, as
well as for the design of therapeutic strategies for myelin-related diseases. The development
of suitable in vitro models that allow for reliable myelin quantification in the study of deand remyelination processes is a very important and not yet fully resolved issue that may
help to boost the development of novel therapies for neurological diseases [1].
Indeed, the use of in vitro models may facilitate the obtaining of faster results, more
controllable and reproducible experimental conditions, and more statistically powerful
results than in vivo models. The possibility of isolating the target subject of study from
Pharmaceutics 2021, 13, 975. https://doi.org/10.3390/pharmaceutics13070975
https://www.mdpi.com/journal/pharmaceutics
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concomitant confounding effects present in the complexity of in vivo settings (e.g., avoiding
the effects of an immune response) is an additional important feature of in vitro models.
The development of an in vitro model is a complex task that requires not only the definition of a suitable system that mimics the relevant biological aspects that are expected to
be replicated, but the definition of proper experimental techniques that allow for observing
and, ideally, quantifying the phenomena under study.
In general, in vitro models may be constructed with increasing degrees of complexity,
starting from specific cell-type monocultures and scaling to complex co-cultures (including
the physical separation of cells by permeable membranes, flowing fluids, etc.), organoids,
perfused excised organs or tissues, and 3D bioprinted structures. Despite multiple in vitro
models having been successfully reported in the literature for the study of the central
nervous system (CNS) [2], for myelin-related diseases, it is important to replicate the
complex multifaceted nature of the brain, with strong interactions among its components,
a task that is difficult to achieve using just cell cultures. In this case, ex vivo excised brain
sections transformed into organotypic brain cultures (OBCs) have been proposed as a more
realistic setting, in which glia closely interact with neuronal axons in a three-dimensional
space [3]. Thus, OBCs have become a simplified way to study de- and remyelination
processes and are generally considered a suitable tool for the evaluation of therapies
against myelin-related diseases [4].
However, the quantification of myelin in organotypic cultures—not a trivial issue—
has traditionally been tackled by invasive experimental methods such as RT-PCR, Western
blot, or immunofluorescence [5]. Each of the mentioned techniques has advantages and
disadvantages that encourage the search for robust; universal; affordable; and, above
all, noninvasive experimental techniques that allow for the quantification of myelin in a
longitudinal manner. Thus, the identified disadvantages include: (1) indirectness. For
example, RT-PCR enables the quantification of myelin-related genes but does not provide
a direct measure of myelin content. (2) Limited sensitivity. For example, Western blot
can provide a semiquantitative measure of myelin content but is not sensitive enough
to detect the myelin content of single tissue slices. (3) Limited quantifiability. For example, immunofluorescence is based on the separate staining of axons and myelin and
co-localization of imaging channels—a challenging way to reliably quantify myelin. Other
methods, such as long-term transgene-induced fluorescence live imaging, have also been
performed in organotypic cultures of mice [6], but no single experimental technique has
shown sufficient simplicity, accuracy, and reproducibility to assess myelin content in OBCs
in a robust manner.
In this context, we have explored the possibility of using functional biomaterials for
the quantitation of myelin content in OBCs by magnetic resonance imaging (MRI), which
has the great advantage of being noninvasive and therefore enabling longitudinal studies
in the same cultures. Despite MRI having been widely used for the quantification of myelin
in clinical and preclinical in vivo studies, with several MR imaging biomarkers of myelin,
such as T2-weighted MR signal or radial diffusivity [7], the use of this imaging technique
for the study of organotypic cultures represents a huge challenge, due to several factors
related to the changes that brain tissue samples experience when evolving into organotypic
cultures (including the loss of white matter or gray matter structuring, loss of stiffness, and
thinning of tissue samples, which spread out on the surface of the supporting membrane
over time) [8].
We believe that the use of molecular imaging approaches, including the use of targeting
imaging probes that help to increase the sensitivity of detection, may offer an opportunity
to overcome such challenges; thus, in this work, we describe our attempts to achieve
an experimental protocol for the MRI-based noninvasive imaging of tissue sections and
organotypic cultures, and to develop myelin-specific imaging nanoprobes that enable the
quantification of myelin content in organotypic cultures from the mouse cerebellum.
The development of nanomaterials that act as imaging probes for MRI is an active field
of research, and examples can be found in the literature [9–11]. In the past, we developed
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liposome-based imaging probes [12], which may be suitable for the detection of myelin
in OBCs.
The results demonstrate that, although it is feasible to image organotypic brain cultures
(OBCs) by MRI, and molecular imaging enables the quantification of myelin in ex vivo
models, the design of the functional imaging probe is an especially critical issue that
requires further development to ensure the sufficient sensitivity for myelin, avoiding
specificity-related issues.
2. Materials and Methods
All experiments involving the use of research animals have been performed according
to the appliable legislation (European Union Directive 2010/63/EU on the protection of
animals used for scientific purposes) and were approved by our Institutional Animal Care
and Use Committee (IACUC) and by the local authorities (Diputación Foral de Guipuzcoa
and our institutional ethics committee, with license number CEEA17_002). Studies were
conducted on three different types of samples.
2.1. Brain Tissue Sections
C57BL/6JRj mice (Janvier Labs, Le Genest-Saint-Isle, France) were sacrificed at postnatal day 7–12, and their cerebellums were sliced to different thicknesses (typically 1.9
mm, 0.8 mm, 0.5 mm, 0.3 mm, or 0.1 mm) and fixed in paraformaldehyde (10%) for 40 min.
Next, the brain sections were washed 2× (for around 10 min) with phosphate-buffered
saline (PBS) and immersed in 2% low melting point agarose (Ref: A9539; Sigma-Aldrich,
St. Louis, MO, USA) in a 50 mL Falcon tube for imaging. The possibility of performing
multiplanar MR imaging allows for scanning sets of several tissue sections in a single
imaging experiment by piling the sections inside the agar gels.
2.2. Thick Organotypic Cultures
C57BL/6JRj mice (Janvier Labs, Le Genest-Saint-Isle, France) were sacrificed at postnatal day 10–12 and their cerebellums immediately extracted and placed in an organotypic
culture medium prepared with 24 mL 2-mercaptoethanol (BME) (Ref. 41010; Thermo Fisher
Scientific, Waltham, MA, USA), 24% Hanks’ balanced salt solution (HBSS) (REF. 24020091;
Thermo Fisher Scientific), 24% horse serum (Ref. 26050088; Thermo Fisher Scientific),
0.125% glutamine (Ref. 25030024; Invitrogen, Carlsbad, CA, USA), 1% antimycotic and
antibiotic (Ref. A5955; Sigma-Aldrich), and 3.5% glucose (Ref. A1422, Panreac Química
SLU, Barcelona, Spain), for a total solution volume of 50 mL.
Following this procedure, the cerebellums were sliced into 750–800 µm sagittal sections
using a McIlwain tissue chopper (World Precision Instruments Ltd., Hitchin, UK). Sections
were separated and placed on a Millicell Cell Culture Insert membrane (Ref. PCIM ORG
50, Millipore Corp., Burlington, MA, USA) on a P6 plate and incubated for seven days in
an organotypic culture medium (as described above) at 37 ◦ C and 5% CO2 (Figure 1).
For imaging studies, cultures were fixed with 4% paraformaldehyde for 40 min and
kept in PBS (0.05% sodium azide) at 4 ◦ C until use. Organotypic cultures were embedded
in 2% agarose before MRI scanning. For this purpose, the support membrane of the culture
insert was gently cropped around the organotypic tissue with a scalpel.
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Figure 1. Preparation of cerebellar organotypic brain cultures from tissue sections. Postnatal
(7–12 days) mouse brains were extracted and sliced into 300–350 µm sections. Slices were cultured for one week on a permeable membrane in contact with the medium. Optionally, lysolecithin
may be used to induce demyelination. Remyelination may take place spontaneously or may be
promoted by therapeutic intervention. (WM: white matter, GM: gray matter).
2.3. Thin Organotypic Cultures
Thin organotypic culture samples were prepared as described in the previous section,
but with slices of 350 µm thickness, which represents the desired target for our experimental
ex vivo assay (see Section 3).
When required, demyelination was induced in these cultures after one week of culturing by exposing OBCs for 15–17 h to a medium containing 0.5 mg/mL lysolecithin (Ref:
L4129; Sigma).
2.4. Myelin-Targeting Liposomes
MRI-traceable myelin-targeting liposomes were prepared by the lipid film hydration and extrusion method [12], using a mixture of lipids composed of 1,2-distearoyl-snglycero-3-phosphocholine (DSPC), at molar fraction x = 0.6; 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (PEGDSPE), at a molar fraction of x = 0.025; (diethylenetriaminepentaacetic acid)-bis(stearylamide)
(gadolinium salt) (Gd-DTPA-BSA), at a molar fraction of x = 0.017; and cholesterol (Chol),
at molar fraction x = 0.333. All liposome components (obtained from Avanti Polar Lipids,
Alabaster, AL, USA) were dissolved and mixed in a chloroform–methanol mixture (6:1).
After solution, a lipid film was formed by the evaporation of the organic solvent on a
rotavapor (high vacuum at 30 ◦ C) and drying under nitrogen flow for 2 h. The lipid film
was then rehydrated with 7 mL of HPLC-grade water (Ref. W/0106/17; Thermo Fisher
Scientific) at 65 ◦ C. Rehydrated liposomes were extruded 14 times at 65 ◦ C through polycarbonate membrane filters (Whatman PLC, Maidstone, UK) using consecutive decreasing
pore sizes of 400 nm (×2), 200 nm (×4), and 80 nm (×8). After extrusion, liposomes were
stored at 4 ◦ C and characterized for lipid content by the Rouser method [13], measuring
size, and z-potential by dynamic light scattering, using a Malvern z-Sizer system (Malvern
Panalytical, Malvern, UK), as described elsewhere [12].
Liposomes were designed as multimodal imaging probes detectable by magnetic
resonance imaging and fluorescence microscopy. Thus, gadolinium ions (Gd3+ ) responsible
for the generation of T1-weighted MRI contrast were complexed with the lipid DTPABSA(Gd) (Ref. 791268P, Avanti Polar Lipids), used as a constitutive element of the liposome
Pharmaceutics 2021, 13, 975
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membrane. It was possible to tailor the magnetic properties of liposomes to obtain the best
performance from the MRI by modifying the DTPA-BSA(Gd) percentage during the lipid
film formation. Four different molar fractions of Gd-BSA lipid were assayed (x = 0.017,
0.033, 0.100, 0.133) to achieve the highest T1-induced contrast. In parallel, the fluorescent
dye DiOC18(3) (3,3′ -dioctadecyloxacarbocyanine perchlorate, Ref. D275; Thermo Fisher
Scientific) was added (300 µL of a 1 mg/mL solution) to the formulation in the organic
phase before a lipid film was formed.
Liposomes were designed to specifically target myelin. Thus, the maleimide groups
present in the surface of liposomes were conjugated with the antimyelin basic protein
antibody (Ref. Ab62631; Abcam, Cambridge, UK), substituted by anti-IgG protein antibody
in control liposomes (Ref. ab18447; Abcam). For conjugation, the antibody was preactivated
by mixing it in SATA solution (1:80 mol/mol). Afterwards, the SATA–antibody solution was
added to the liposome solution in a vial (50 µg of protein per 1 µmol of lipids), and the mix
was kept overnight at 4 ◦ C under N2 atmosphere. Uncoupled protein was removed from
the liposome solution by centrifugation (65,000 rpm, 45 min) and resuspension of the pellet
containing the liposomes in HEPES-buffered saline (HBS, pH 7.4). Transmission electron
cryomicroscopy (CryoTEM), dynamic light scattering (DLS), and inductively coupled
plasma mass spectrometry (ICP-MS) were used for the physicochemical characterization.
A Bradford Assay (Ref. B6916; Sigma-Aldrich) was used to verify that antibodies were
attached to the surface of the liposomes. Relaxometry properties were measured in a Bruker
Minispec MQ60 (Bruker Biospin GmbH, Ettlingen, Germany) contrast agent analyzer at
1.5 T and 37 ◦ C.
2.5. Molecular Recognition of Myelin by Targeting Liposomes
Cell culture inserts were prepared, each of them with three thin organotypic slices
lying on the surface of the membrane (Figure 1). After a one-week incubation, cultures
were demyelinated by adding lysolecithin (Ref. L4129; Sigma-Aldrich) at a concentration
of 0.5 mg/mL to the culture medium, for a period of 15–17 h (Figure 1). Removing this
chemical from the organotypic culture media triggers the spontaneous remyelination of
organotypic cultures, a process that may take from days to weeks, and can be enhanced by
therapeutic interventions (Figure 1). Control cultures were also prepared in the same way
but without exposing them to lysolecithin.
For myelin recognition by liposomes, cultures (control, demyelinated, and remyelinated) were fixed with 4% paraformaldehyde for 40 min and washed with DPBS. Tissue
was blocked with a solution composed of DPBS, 0.5% Triton (Ref. T8787; Sigma-Aldrich),
and 10% goat serum (Ref. G9023; Sigma-Aldrich) for 1 h at room temperature. A volume
of 350 µL of liposomes was added and samples were incubated overnight at 4 ◦ C. Then,
samples were washed with 0.1% Triton in DPBS and stained with Hoechst (Ref: B2261;
Sigma) 10% in DPBS for 10 min.
2.6. Imaging Studies
Fluorescence images were acquired using a Nikon Eclipse 80i digital microscope
(Nikon Instruments, Melville, NY, USA) and processed using NIS elements AR 3.2 software (Nikon).
MRI studies were conducted at 7 Tesla using a Bruker Biospec 70/30 USR MRI system
(Bruker Biospin GmbH, Ettlingen, Germany). Images of brain tissue slices and thin organotypic cultures were acquired with spatial resolutions ranging from 25 × 25 × 25 µm3
to 100 × 100 × 500 µm3 . Different T2-weighted imaging sequences were tested (RARE,
MSME, and FSE) with echo times ranging from 10 to 70 ms and repetition times ranging
from 2000 to 7000 ms.
For thin organotypic cultures, diffusion-weighted imaging was also conducted with
a spin-echo diffusion-weighted imaging (DWI) sequence, using the following parameters: b-value = 1500 s/mm2 ; gradient pulse durations δ = 6 ms and spacing ∆ = 14 ms;
TR = 2820 ms; slice thickness = 250 µm.
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Imaging of thick organotypic slices was performed with a turboRARE sequence with
the following parameters: TR = 2000 ms; RARE factor = 10; effective echo time TE = 70 ms;
field-of-view (FOV) = 10 mm × 10 mm; image matrix = 400 × 400 points; 24 slices with a
thickness of 0.3 mm.
Image analytics were conducted using the NIH software FIJI [14]. For the analysis
of the images, signal-to-noise ratios (SNR) were calculated for white and gray matter
at different tissue thicknesses and both spatial resolutions (Table 1). SNR was defined
as the mean signal intensity in a region of interest (ROI) of the tissue under observation, divided by the standard deviation of the noise, obtained from a ROI at the background, outside of the object of interest (SNR = 0.66 × meantissue /SDbackground ). The
contrast -to-noise ratio (CNR) between gray and white matter was obtained as the difference of the mean of each tissue divided by the standard deviation of the background
(CNR = 0.66 × [meangm − meanwm ]/SDbackground ).
Table 1. Signal-to-noise and contrast-to-noise values for MRI images of thick tissue slices. SNR:
signal-to-noise ratio; CNR: contrast-to-noise ratio; GM: gray matter; WM: white matter.
25 × 25 µm2 Resolution
Tissue Thickness (mm)
1.0
0.8
0.5
0.3
SNRGM
SNRWM
CNRGM-WM
6.7
7.4
6.0
5.6
4.0
4.4
4.0
3.5
2.7
2.9
2.0
2.1
3. Results and Discussion
As discussed in the introduction, the development of remyelination therapies requires
experimental models and techniques that enable the evaluation of the effect of a given therapy. Both in vitro and in vivo models have been proposed in the literature, with growing
interest in mixed models such as the use of ex vivo organotypic brain cultures (OBCs),
in which the advantages of in vitro systems such as reproducibility, homogeneity, highthroughput capability, etc. can be applied to brain tissue samples with a more complex
structure than a culture. Thus, OBCs are being exploited for the study of remyelination
therapies. However, a reliable and noninvasive technique thar allows for the longitudinal
assessment of myelin content in organotypic cultures is still an unresolved issue [4,15]
that we have attempted to tackle by following a sequential reductionistic approach, described below.
3.1. Imaging Thick Tissue Sections
First, we tested the capability of MRI to image thick and large portions of cerebellar
tissue, fixated in formalin briefly after excision from mouse brains. This does not represent
a proper model of organotypic cultures per se but allowed us to establish a starting point
from manageable samples in the MRI scanner, assess the limits of the achievable sensitivity
of detection (signal-to-noise and contrast-to-noise ratios), and find the working limits of
image slice thickness and spatial resolution affordable for our MRI scanner.
Thus, several tissue sections of different thicknesses (0.3, 0.5, 0.8, and 1 mm) were
prepared, imbibed in agar gels, and scanned with multiple acquisition sequences, in order
to image such thick tissue slices, optimizing the balance between imaging time, SNR, CNR,
spatial resolution, and image quality (Figure 2).
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Figure 2. MR imaging of thick tissue slices: (A) mouse cerebellum tissue slices excised at different
thicknesses and stacked in agar (sagittal MR image); (B–E) coronal MRI 2D images (300 µm slice
thickness) acquired at two in-plane resolutions.
White matter structures (hypointense on T2w
.
imaging) are clearly distinguishable from gray matter.
The best results were achieved using a RARE (Rapid Acquisition with Relaxation
Enhancement, also referred to as Fast Spin Echo or Turbo Spin Echo) sequence with a
RARE factor of 8, an echo time of TE = 16 ms (effective echo time of TEeffective = 64 ms);
FOV = 1.28 × 1.28 cm2 ; image matrix = 512 × 512 (in-plane resolution: 25 × 25 µm2 ), slice
thickness = 300 µm; N = 20 averages; total scanning time = 2 h 4 min.
The images presented in Figure 2, where white matter appears as hypointense and
gray matter as hyperintense due to the T2 contrast, were used to calculate SNR values,
calculated as the ratio between the mean signal in a region of interest including 75% of the
tissue and the standard deviation of the noise, collected from a ROI of the background, far
from the object and avoiding potential influences of readout and phase encoding ghosting,
multiplying this ratio by a factor of 0.66, as described in Goerner et al. [16]. The results of
these calculations are presented in Table 1.
As shown in Table 1, for this set of samples, the tissue thickness has little influence
on the SNR and CNR values, presenting no significant differences for acquisitions in
which the imaging slice thickness is the same, and completely covers the tissue section,
in this way avoiding partial volume effects. In fact, slightly inferior SNR values were
≈ slice thickness,
observed for a tissue sample of 300 µm in thickness, matching the imaging
representing the limit at which partial volume effects start to show up, since it is difficult
to have a completely flat tissue section in the agar perfectly aligned with the imaging slice
to completely avoid those effects. Imaging of thinner tissue slices will be discussed in the
next section.
3.2. Imaging Thick Organotypic Cultures
The tissue samples described in Section 3.1 were fixed and embedded in agar just
after excision from the mice cerebellums. In a second set of experiments, we went one
step forward by culturing brain slices for a period of 7–12 days (as is usually done when
preparing OBCs), the period during which a loss of tissue thickness and stiffness is expected.
Thus, we cultured a series of 750 µm thick brain slices, as described in Section 2 and
represented in Figure 1.
As expected for tissue samples evolving into organotypic cultures, incubation affected
the tissue thickness, resulting in approximately half-thickness slices (≈300 µm, which
corresponds to the minimum tissue sample thickness studied in Section 3.1). Incubation
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also affected the tissue integrity, showing more diffuse limits between white matter and
gray matter, appearing somewhat blurred in MR images (Figure 3).
Figure 3. MR imaging of thick organotypic cultures: (A) sagittal MR image of five mouse cerebellum
organotypic cultures stacked in agar (sagittal MR image); (B–F) coronal MRI 2D images (300 µm
slice thickness) of the organotypic slices. The white matter–gray matter limit blurred as the cultured
tissues evolved.
Only the results for the optimized MR imaging sequence and target spatial resolution
are presented here. The mean calculated SNR for this cultured tissue was 62.7 for gray
matter and 38.9 for white matter, with a CNR of 31.9 between both tissues. From these
results, we can conclude that good-quality images can be obtained from these cultured
tissues, where white matter is still clearly identifiable. However, the lines defining the limit
between white and gray matter, as well as the external limits of the culture sections, started
to look blurry and less defined when compared to those observed in the prior study with
tissue sections.
This experimental setup is closer but still does not represent proper organotypic
cultures. Typical organotypic cultures
were prepared from tissue sections with a maximum
μ
thickness of 300–350 µm. When thicker tissue sections are cultured using a setup such as
the one shown in Figure 1 (like the 750 µm sections used here), the permeation of nutrients
and oxygen to the inner parts of the tissues is compromised, usually leading to cell loss and
tissue necrosis [3]. Even though attempts have been carried out to culture organotypic thick
slices [17], they have not been successfully adapted for the study of de- and remyelination
processes thus far.
3.3. Imaging Thin Organotypic Cultures
In a final set of experiments, tissue sections of 350 µm were excised and cultured
for 7–12 days, obtaining in this way conventional organotypic cultures that are typically
prepared from slices of such thickness, and reaching values of around 100 µm at the end
of the incubation period. It should be borne in mind that the flattening and increased
transparency of the tissue sections over time (Figure 4A) are actually indicators of tissue
health and survival [3]. In this sense, using mice at postnatal day 12 resulted in a firmer
structure compared to younger cultures, facilitating MRI imaging and, at the same time,
enabling tissue survival.
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Figure 4. MR imaging of thin organotypic slices. (A) A photograph of organotypic slices in culture.
(B) Diffusion-weighted vs. T2-weighted imaging of an organotypic slice. (C–E) Different imaging
artifacts observed when imaging thin tissue slices ((C) air bubble; (D) misalignment of the supporting
membrane with imaging plane; (E) loss of CNR due to partial volume effects).
Imaging of thin organotypic slices is highly challenging and not satisfactory. Many
problems are encountered when imaging these cultures. Firstly, the excised tissues evolve
into a sort of slick mass after culturing, hardly attached to the supporting membrane,
which is a rigid structure. Positioning of this rigid structure in agar gels is hard to perform
while avoiding the formation of small air bubbles, which generate strongly susceptible
artifacts that spoil the image (Figure 4C). On the other hand, the porous film supporting
the culture can hardly be kept flat in the agar, complicating the orientation of the imaging
planes parallel to the thin tissue sample, creating in this way large “black shadows” on the
image due to the lack of signal of the supporting membrane, which contains no protons to
image (Figure 4D). Of course, one can consider removing the tissue from the membrane for
MR imaging, but this will affect OBCs’ viability and hamper the recovery of the tissue for
repeated imaging if one likes to follow a longitudinal approach.
In addition, even when the aforementioned distorting effects can be avoided, it is hard
to distinguish tissues from the surrounding agar on T1, T2, or T2 * weighted images, since
the contrast between the tissue and the background agar is seriously affected by partial
volume effects as a consequence of the reduced thickness of the tissues at the moment
of imaging (tissues spread on the surface of the membrane during culturing, from the
original 350 µm to <100 µm on the imaging day). The minimal imaging slice thickness is
determined by hardware constrictions (gradient strengths and SNR of the radiofrequency
coils) and the imaging time (the thinner the slice, the higher number of averages required
to achieve a high enough SNR). Our experimental conditions allowed us to acquire a
minimum imaging slice thickness of 300 µm, in practice,
and at this condition <1/3 of the
μ
voxel was filled with tissue, with the rest being occupied by agar. When ideal conditions
were met, it was possible to obtain images from OBCs (Figure 4B,E), but with very low
SNR, and with it no longer being possible to distinguish between white and gray matter.
In light of these results, we attempted a different approach for conducting diffusion
weighted imaging, whereby the signal arising from water molecules in the agar gel is
suppressed by diffusional gradients (Figure 4B). In this case, it was possible to identify
organotypic cultures at high SNR (SNR > 45). However, the evolution of the tissue into
a smooth blob comes with a loss of internal structures, and white matter is no longer
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distinguishable from gray matter (Figure 4B). In this sense, it is not possible to quantify
myelin content, which was our original objective. Therefore, we concluded that conventional MRI, in any of its modalities, is not an ideal experimental technique to perform
the longitudinal quantification of myelin in organotypic cultures, and thus a different
experimental approach was developed.
3.4. Molecular Imaging Recognition of Myelin by Targeting Liposomes
As we have mentioned, sensing technology uses sensors to detect a physical, chemical,
or biological property with an experimental technique, and convert it into a readable signal
that allows for quantification. The development of nanomaterials acting as imaging probes
for MRI is an active field of research [9–11]. In the past, we developed liposome-based
imaging probes [12] that may be suitable for the detection of myelin in OBCs.
Liposomes doped with imaging probes (gadolinium for MRI detection and a fluorescence probe for microscopy) and decorated with antimyelin basic protein antibody
(anti-MBP) were prepared (as well as liposomes decorated with unspecific anti-IgG protein as a control) and incubated with thin organotypic tissues (Figure 5), as described in
Section 2.
Figure 5. Molecular imaging strategy for the quantification of myelin in organotypic cultures.
Antimyelin stealth liposomes doped with gadolinium and Dioc18 were incubated with the cultured
tissue for later detection with imaging techniques (see Section 2 for the full names of the abbreviated
chemicals in the figure).
Different liposomal compositions were prepared to optimize the load of MR contrast
agent, in order to achieve higher sensitivity of detection. Thus, up to four formulations of
liposomes were synthesized, with increasing amounts of gadolinium (7.1 × 109 , 40.2 × 109 ,
61.7 × 109 , and 112 × 109 Gd atoms per liposome unit, as measured by inductively coupled
plasma mass spectrometry (ICP-MS), and characterized by measuring their longitudinal
relaxivity (r1), calculated as the slope of plots of the relaxation rate (R1 = 1/T1, where T1 is
the longitudinal relaxation time) of solutions of different concentration of those liposomes
versus the concentration of liposomes (Figure 6).
Figure 6. Characterization of liposomes. (A) Relaxation rate vs. concentration plots. (B) Longitudinal
relaxivity vs. load of gadolinium plot, of prepared liposomes. (C) Hydrodynamic diameters of
liposomes, as determined by DLS.
An important aspect of the development of imaging sensors is the optimization of
the payload of imaging probes (gadolinium content per liposome) since, in the case of
MRI, it has been demonstrated that an excessive payload of this lanthanide in the probe
Pharmaceutics 2021, 13, 975
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could quench the T1 effect and reduce the sensitivity of detection and image contrast [18].
Thus, several formulations were prepared, containing different amounts of gadolinium
per liposome unit. Then, the longitudinal magnetic relaxivity of each formulation was
calculated as the slope of plots of measured relaxation rates (the inverse of the relaxation
times: R1 = 1/T1) versus the concentration of gadolinium (Figure 6A), as determined by
mass spectrometry [18]. The optimized load of gadolinium in liposomes (in terms of higher
longitudinal relaxivity) was achieved for a load of 7.1 × 109 atoms of gadolinium atoms
per liposome (corresponding to a molar fraction of 0.017 Gd lipids in the formulation),
resulting in a longitudinal relaxivity of 2.17 mM−1 s−1 , with respect to the concentration of
liposomes. As we anticipated, higher loads of gadolinium resulted in a partial quenching
of T1 contrast (Figure 6B).
The decoration of the surface of the naïve liposomes with antimyelin protein or
IgG (control) protein attached to polyethylene (PEG) chains has no significant effects on
liposomes’ sizes, as determined by dynamic light-scattering studies (Figure 6C; the mean
liposome hydrodynamic diameters were naïve: 124.0 ± 1.5 nm; anti-IgG: 133.9 ± 1.5 nm;
anti-myelin: 124.7 ± 1.5 nm), which is an important feature to prevent alterations in the
behavior of liposomes as sensing devices.
Myelin-targeting liposomes were incubated with both myelinated and demyelinated
organotypic cultures and imaged by fluorescence microscopy after washing unbound
liposomes from the culture media. In panels A and B of Figure 7, we present fluorescence
images of myelinated and demyelinated tissues, on which cell nuclei are stained blue,
incubated with antimyelin green fluorescent liposomes, showing that sensing of myelin
sheaths is feasible using our experimental approach.
Figure 7. Fluorescence imaging of tissue sections (blue for cell nuclei and green from DIOC18
fluorescent liposomes) incubated with targeting liposomes. (A) Healthy and (B) demyelinated
tissues, incubated with fluorescent anti-MBP liposomes. (C) Healthy tissues incubated with control
anti-IgG and (D) with antimyelin basic protein liposomes.
Pharmaceutics 2021, 13, 975
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Imaging studies showed that green fluorescent MBP-targeting liposomes can specifically bind to myelin sheaths in healthy organotypic cultures (Figure 7A), while no visible
attachment was observed when demyelinated cultures were cultured with the liposomes
(Figure 7B). Unfortunately, both nontargeted (IgG control) and myelin-targeted liposomes
experienced nonspecific binding (Figure 7C,D, respectively) to tissue in OBCs. Despite the
different incubation strategies followed (varying incubation times and liposome concentration in the media), we were not able to avoid this phenomenon.
Certainly, it has been described that liposomes have an affinity for biological tissues,
in general, due to electrostatic and nonspecific hydrophobic forces [19]. In the case of
myelin sheaths, their lipidic nature increases even further their affinity for liposomes,
presenting slightly increased fluorescence compared to the surrounding tissue, but the
general unspecific binding of liposomes to all tissue, irrespective to the presence of targeting
antibodies, discourages their use as sensing probes for the molecular recognition of myelin
in OBCs.
Overall, our results have provided proof-of-concept that molecular recognition via
imaging-driven sensing studies is feasible and a good approach for the detection and
quantification of myelin in organotypic cultures. However, liposomes do not seem to be
an ideal molecular platform to perform this task. The development of a different sort of
nanomaterial with no affinity for biological tissues is a must, but is a complex task that
requires fine-tuning of the composition, structural and physicochemical properties, as well
as the study of the biological compatibility (cytotoxicity) and optimization of imaging
properties, which is outside of the scope of this work. Work along this line is currently in
progress in our laboratories.
A limitation of this study is that we do not report the MRI detection of myelin in OBCs
through the use of prepared liposomes. Due to the much higher sensitivity of detection of
fluorescence imaging versus MRI, we performed tests of specificity with this technique.
Since multimodal imaging liposomes were nonspecific for myelin sensing, we decided not
to continue MRI studies with them and instead will wait until an optimized nanomaterial
is available. We will continue with the search for alternative materials that may be effective
for the specific labeling of myelin in organotypic brain cultures.
4. Conclusions
The development of experimental protocols for the noninvasive in vitro characterization of myelin in cultures may represent a breakthrough for the performance of highthroughput studies of treatments, in highly reproducible experimental conditions. In this
work, we have shown that the MRI imaging of freshly excised and fixed brain slices is
feasible with high SNR and CNR, demonstrating the potential of this technique for the
quantification of myelin, but such systems are inadequate as ex vivo models, since the
preservation of cells in the interior of thick sections in culture is highly compromised by
the limited diffusion of nutrients and oxygen through them.
We have also shown that thin organotypic cultures are inherently problematic for
imaging and quantification by MRI and that, alternatively, sensing of myelin by molecular
recognition with multimodal imaging nanomaterials can be a suitable experimental strategy,
providing proof-of-concept for this approach, but we have not yet succeeded in finding a
suitable molecular platform to achieve the required level of specificity required for this task,
since liposomes have high unspecific affinity for biological tissues in general. Ongoing
research in this line will tell us if this is a good approach for obtaining experimental ex
vivo tools for the longitudinal study of demyelination and remyelination processes.
Author Contributions: Conceptualization, P.R.-C., A.E. and D.O.; methodology, A.E., P.R.-C., J.R.-C.
and D.P.; investigation, A.E., S.C.-R., I.O.-Q. and P.R.-C.; writing—original draft preparation, A.E.,
D.O. and P.R.-C.; writing—review and editing, D.P., I.O.-Q., J.R.-C. and S.C.-R.; supervision, P.R.-C.
and D.O.; funding acquisition, J.R.-C., P.R.-C. and D.O. All authors have read and agreed to the
published version of the manuscript.
Pharmaceutics 2021, 13, 975
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Funding: This work was performed under the Maria de Maeztu Units of Excellence Program, grant
no. MDM-2017-0720 from the Ministry of Science, Innovation and Universities, and was funded by
the Spanish Research Agency (grant number: SAF2017-87670-R), and the Basque Government (grant
no. 2018222025). P.R.-C. and J.R.-C. are financed by Ikerbasque, the Basque Foundation for Science.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Ethics Committee of Biodonostia (protocol number:
CEEA17_002, approved by the local authorities (Diputación Foral de Guipuzcoa, protocol code
PRO-AE-SS108, approved 13 February 2017)).
Informed Consent Statement: Not applicable.
Data Availability Statement: Raw imaging data will be available from the corresponding author
P.R.-C. on reasonable demand.
Conflicts of Interest: The authors declare no conflict of interest.
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