Magnelle® Cell Tracking Solutions
Bell Biosystems, Inc. www.bellbiosystems.com Recent advances in regenerative medicine demonstrate the potential of cell
therapies to provide new, and in some cases curative solutions to a range of therapeutic
areas such as neurodegenerative disease, immune-oncology, heart disease, spinal cord
injury, tissue regeneration and others; however, few are clinically approved while R&D
efforts continue to explode. This backlog is due, in part, to the immense challenges in
precisely determining the location and fate of therapeutic cells post delivery.1,2,3
Information on biodistribution and persistence of cells post injection or transplantation is
essential to experimentally correlating their therapeutic benefit with the cell therapy.
Also of critical importance are discriminating issues in accurate delivery from
engraftment; and diagnosing and dealing with off-target effects, among other things.
These concerns are reflected in the FDA’s recommendation that all cell therapies
include non-invasive imaging.4 Numerous imaging methods exist, but none that were
specifically developed for regenerative medicines. We are developing novel living MRI
contrast agents tailored to therapeutic cell tracking needs following the endosymbiotic
process that created organelles like the mitochondria and chloroplast. Magnelles (or
magnetic organelles) are derived from non-pathogenic magnetotactic bacteria that
create lipid enclosed iron nanoparticles (producing dark MRI contrast) and are
phylogenetically related to the ancestors of endosymbiotically-derived organelles.
Discussed below, the Magnelle cell tracking solution has been validated in a number of
preclinical uses and, while not commercially available today, is being made available to
selected researchers through our beta early adopter program.
Current Imaging Modalities used in Cell Tracking
Table 1 summarizes parameters that should be considered when choosing
imaging modalities for specific experiments. The ideal imaging technique is noninvasive, provides high sensitivity and resolution, complete anatomical access, allows
longitudinal visualization and is specific for live cells. Optical reporters, such as
fluorescent and bioluminescent imaging (BLI) proteins are commonly used and powerful
for in vitro and small animals studies. However, use of optical cell tracking is limited in
larger animals due to tissue penetration restrictions and genetic engineering
requirements, thus is not translational. Positron emission tomography (PET) and singlephoton emission computed tomography (SPECT) are quite sensitive and do not have
penetration limitations. However, the requirement for radioactive probes raises safety
requirements, increases costs and has limited widespread use, especially for
longitudinal studies.1 MRI has been identified as the modality of choice for cell tracking
due to its non-invasive nature, 3D imaging capabilities and full anatomic access.2
However, contrast agents tailored to regenerative medicine need to be designed.
Importantly, contrast agents need to be specific for viable cells, therefore eliminating
false positives associated with a lack of live cell specificity.
Modality
Energy
used
Spatial Resolution
(mm)
Clinical
Animal
Acquisition
time/frame(s)
Penetration
Depth(mm)
Use in
humans
PET
β+ particle
3-8
1-3
1-300
>300
Dose limits
SPECT
γ-ray
5-12
1-4
60-2000
>300
Dose limits
CT
X-ray
0.5-1
0.03-0.4
1-300
>300
Yes
Optical
Vis and IR
−
3-10
10-2000
1-20
Limited
Ultrasound
Radio freq
0.1-1.0
0.05-0.1
0.1-100
1-200
Yes
MRI
Radio freq
0.2-1.0
0.025-0.1
50-3000
>300
Yes
Table 1. Summary of various considerations relevant to choice of imaging modality. MRI-based cell tracking requires the addition of a contrast agent prior to
transplantation to resolve implanted cells from background tissue. While highly valuable
for diagnostic imaging, passive contrast agents like iron oxides (Ferridex®, Resovist®,
etc.), 19F agents and transition metal complexes (gadolinium, etc.) have some critical
disadvantages for cell tracking. For example, in vivo these agents can be expelled into
the surrounding tissue by exocytosis or upon cell death generating false positive signals
(e.g. a lack of live cell specificity).5 In addition, passive contrast agents dilute out in
dividing cells limiting longitudinal studies.6
The Magnelle Cell Tracking Solution
Creation of Magnelles was inspired by the endosymbiotic theory of organelle
biogenesis. The Magnelle is a living contrast agent derived from magnetotactic bacteria
(MTB) and has the potential to overcome some limitations of passive contrast agents for
cell tracking.7 MTB are non-pathogenic bacteria that coordinate over 100 genes to
create lipid enclosed magnetic nanoparticles called magnetosomes that enable them to
use the earth’s magnetic field for navigation.8 Importantly, magnetosomes have been
shown to be highly sensitive MRI contrast agents.9
Several organelles like the mitochondria and chloroplast evolved through the
process of endosymbiosis, where free-living bacteria were stably integrated into the
eukaryotic cell, providing new functions like aerobic respiration and photosynthesis,
respectively.10 Through organelle compartmentalization, complex biochemical and
genetic processes are sequestered
subcellularly, reducing biocompatibility
issues with normal cell function. The strict
definition of an endosymbiotically derived
organelle involves gene transfer from the
prokaryote to the host DNA; by this criteria,
Magnelles should be considered pseudoorganelles since genetic modification of
the host is not required for Magnelle based
cell tracking.
To date, approximately two dozen
mammalian cell types (mouse, rat and
human) have been tested for Magnelle Figure 1. Representative ICC of Magnelle-­‐labeled cells. Cells compatibility with little to no adverse effect were fixed post Magnelle labeling, stained with anti-­‐
on cell function. These include both Magnelle antibody (red), Phalloidin 488 (green) to visualize cell b orders and DAPI (blue) nuclear stain. primary, stem and cancer cells. We do
observe cell-to-cell variability but the tagging protocol (discussed below) can be
modified to, most often, obtain acceptable results. Figure 1 presents
immunocytochemistry (ICC) images for representative cell types. Magnelles are stained
red, appear punctate and perinuclear in each case. The induced pluripotent stem cells
(iPSCs), top right, maintained pluripotency post labeling and iPSC-derived
cardiomyoctyes (iCMs), bottom left, maintained spontaneous contractility and exhibited
positive staining for sarcomeric protein (i.e., cardiac troponin T) showing preservation of
the short-term cardiac properties (data not shown). Additionally iCMs have been
successfully engrafted into a mouse heart injury model, shown below. Figure 2 shows
human fetal derived neural progenitor
cells (NPCs) pre- and post-Magnelle
labeling stained with a neuronal
marker nestin and the glial marker
GFAP, showing that differentiation
capacity is preserved. A key
parameter in the tagging procedure is
the Magnelle per cell (MPC) ratio,
which, in cases where adverse
biocompatibility may be detected, can
be lowered to find suitable conditions.
Figure 2. Fetal derived human NPCs pre-­‐ and post-­‐Magnelle The tradeoff is detection limit as the
labeling. Cells were fixed and stained with anti-­‐Magnelle antibody MRI
contrast
enhancement
is
(red, left column) and the glial marker GFAP (green, middle column) to the number of
and the neural marker nestin (yellow, right column) and all counter-­‐ proportional
stained with DAPI to visualize nuclei.
Magnelles in each cell.
MRI-based In vivo Cell Tracking
Similar to iron
based MRI contrast
agents, Magnelles
create
dark
(T2)
contrast in labeled
cells enabling in vivo
Figure 3. MRI images for A) NPCs in rat spine, B) iCMs engrafted in mouse heart and c) visualization with MRI 231BR metastases in ex vivo mouse brain. White arrows indicate Magnelle-­‐labeled cells. instruments
(cell Various cell types were tagged with Magnelles using the Magtag device and either in situ as in A and B or injected using an ultrasound-­‐guided intracardiac tracking has been transplanted injection as in C. In C. MRI scans detected single breast cancer cells that migrated to the achieved with magnet brain. strengths from 1.4T to 11.7T). From these studies, T2w and T2*w pulse sequences
seem ideal for imaging, with T2*w images offering a higher sensitivity. Figure 3 shows
representative images using a preclinical 7T animal MRI scanner for: A) NPCs
transplanted into a live rat spinal cord injury model;11 B) iCMs engrafted in a live mouse
cardiac injury model;12 and C) single cell metastatic cancer cells seeding in an ex vivo
mouse brain.13 As indicated by the arrows, Magnelle-labeled cells are readily resolved
from background tissue, appearing as dark spots. MRI signal from transplanted cells
allows non-invasive monitoring of cell location post transplantation in a variety of tissues
with high temporal resolution and sensitivity.
The ex vivo brain image in panel C of Figure 3 illustrates the power of Magnellebased cell tracking to detect single
cells.
MDA-MB-231BR
breast
cancer cells (231BR) are known to
selectively
proliferate
in
the
14,15
brain.
Post
intracardiac
injection of 231BR cells in nude
mice, brains were fixed and single
cells seeding into the brain tissue
were detected. In addition to
illustrating the detection ability of
Magnelle-based cell tracking, these
results demonstrate the possibility
of visualizing the earliest stages of
tumorigenesis.
Figure 4. Combined BLI and MRI cell tracking of iCMs in mice Returning to the live cell myocardial infarct model. Left panel show results at week one, when specificity feature that is critical for iCMs are alive while right panel shows results at week two when cells have been cleared by the immune system. Bottom two lines regenerative medicine cell tracking summarize the results. applications, Figure 4 shows longitudinal cell tracking of iCMs labeled with Magnelles
and a commercially available superparamagnetic iron oxide (SPIO) contrast agent. In
both cases iCMs were co-labeled with luciferase, a common BLI reporter that tracks cell
viability, as the internal control. At week one (left panel), when cells are live the BLI and
MRI signals positively correlate providing anatomic and viability information. At week
two (right panel), however when cells are dead and removed by the immune system,16
this correlation breaks down for the SPIO as MRI signal persists in the heart.
Conversely, the MRI signal from Magnelles decays proving the live cell specificity
feature. SPIOs’ lack of live cell specificity has been observed by others and is
suggested to be from the inability of macrophages to digest or remove the synthetic
particles from the tissue.17,18 We believe that the prokaryotic origin of Magnelles coupled
with macrophages innate ability to digest and/or clear bacteria from tissue is the
underlining mechanism for the unique live cell specificity of Magnelles. Live cell
specificity is required for cell tracking in a regenerative medicine setting because
occurrence of false positives precludes extracting actionable insight.
Toxicological Considerations
Magnelles are living
contrast agents derived
from bacteria, thus safety
and toxicity considerations
must
be
critically
evaluated.
Presently,
Magnelles
are
only
available for preclinical
research-use. However, we
have
conducted
some
preliminary studies. To
model
the
worst-case Figure 5. Blood serum levels of vital markers of tissue and immunotoxicity were scenario of all Magnelle- obtained at day 5 from i9mmune competent SD rats that were subjected to a Magnelle dose of (of 10 Magnelles, 10 working dose) delivered via an IV injection. containing cells releasing The data shows no significant differences in levels of key blood markers of vital Magnelles into the animal organ toxicity. More importantly, the leucocyte counts remained unaltered at similar concentrations suggesting minimal immunotoxic effects.
simultaneously, we injected
pure Magnelles into immunocompetent and immune compromised small animal models.
These studies show that Magnelle reagents do not cause acute toxicity when
administered intravenously (up to 4x109), intrathecally (up to 5x107), or intramuscularly
(up to 5x108). Figure 5 displays serum analysis for the IV study assessing function of
vital organs such as the liver, kidney, spleen, heart and pancreas. All blood markers
remained in normal ranges indicating no organ malfunction. Also, key markers of
infection such as the white blood cells, red blood cells and platelet counts were not
significantly altered. Pathological evaluation of tissue samples did not show significant
differences in histopathology scores for the brain, kidney and heart. Furthermore, no
morbidity was observed in 100% of the animals, but slightly enlarged livers and spleens
were detected in some animals consistent with removal of bacteria by the immune
system. Magnelle Cell Labeling Protocol
For lab-scale labeling of cells,
we have developed MagTagTM devices
for ex vivo labeling, Figure 6. This
device
employs
a
form
of
magnetofection and current protocols
have been optimized for adherent cell
types. In step 1, cells are grown using
standard conditions, but in the absence
of antibiotic. In step 2, cells to be
labeled are layered with Magnelles at
the appropriate MPC and incubated on
the MagTag system for 3 to 24 hrs,
depending
on
experimental
considerations. Optimal conditions for
most cell types are obtained with 5%
CO2 and 5% O2. The following day,
6. Magnelle labeling work flow. Each step is described cells are washed and treated with Figure briefly in the text and detailed protocols are available on the web. antibiotic to remove extracellular
Magnelles. The tagging efficiency and a number of other factors can be evaluated, in
vitro (step 3) using quality control assays that we’ve developed, prior to transplantation
(step 4). Finally, in step 5, cells can be visualized in vivo using the appropriate pulse
sequences and instrument parameters.
Beta Early Adopter Program
Since launching our Beta Program in mid 2015, we have selectively engaged
with world-renowned opinion leaders in both academia and industry. Through
collaborative efforts, these researchers use the Magnelle Cell Tracking Solution to
advance their research in ways not possible without the Magnelle technology. As part of
the Beta Program, we provide our current version of the Magnelle Cell Tracking Kit,
which includes the Magnelle reagents, MagTag device, companion assays, all
necessary protocols and paperwork, and ready access to technical support from our
scientific and business teams. In addition to providing valuable feedback that helps us
to optimize our products for customer needs, we encourage all Beta partners to publish
their results in peer-reviewed publications. If you are interested in participating in our
Beta Program, or have questions, please contact us at [email protected]. Frequently Asked Questions:
Magnelles are modified bacteria, so are there concerns or considerations for
bringing these into my TC hood?
Magnelles are modified bacteria, but contamination risks are low. They were
created from BSL1, non-pathogenic, microaerophiles that cannot tolerate extended
aerobic conditions. Magnelle growth media is different from that used for mammalian
cells and the TC media that we’ve tested to date does not support growth. Magnelles
are highly sensitive to a range of antibiotics except kanamycin and ampicillin. That said,
aseptic techniques should always be used.
Does Magnelle-labeling change the function of my cells?
In the handful of the cell types we have studied to date, cell function is not
significantly altered. Discussed above are a few of these results. Biocompatibility should
be evaluated with each new cell type and optimizations may be required.
Are Magnelle reagents toxic?
Magnelles were derived from non-pathogenic BSL1 bacteria. Preliminary toxicity
studies are discussed above that show no acute toxic effect at concentrations well
above the doses used for cell tracking.
How sensitive are Magnelle reagents?
Fundamentally, MRI sensitivity is determined by the concentration of contrast
agents in the voxel, magnet strength, pulse sequence and scan time. Practically,
sensitivity depends on biological factors such as the number of Magnelle reagents per
cell, cell volume, how tightly cells are clustered in the tissue and native tissue contrast.
Magnelle reagents are about 1-2 µm by 0.5 µm (similar to a mitochondria) and thus the
number per cell is tightly dependent on cell volume. Figure 3 shows that single cell
detection is feasible, but that required optimization of many factors including a scan time
of >1hour.
How long does the Magnelle signal persist?
Magnelle signal persistence will depend on cell type and animal model, but users
can expect the signal to last several weeks, in vivo. Interestingly, in vivo persistence
exceeds in vitro in all cases we’ve tested. As with biocompatibility this should be
reevaluated for unique cases.
References: 1. Naumova A, et al., Clinical imaging in regenerative medicine. Nature Biotech. (2014) 32(8).
2. Bulte JW. In vivo MRI cell tracking: Clinical studies. AJR Am J Radiology. (2009) 193:314-25
3. Bauer SR. Assuring safety and Efficacy of Stem-Cell based products. (2010) FDA Office of
Cellular, Tissue and Gene Therapies
4. Guidance for Industry. Preclinical Assessment of Investigational Cellular and Gene Therapy
Products. (2013) FDA, Center for Biologics Evaluation and Research.
5. Svendsen CN, et al., In vivo tracking of human neural progenitor cells in the rat brain using
bioluminescence imaging. Journal of Neuroscience Methods. (2014) 288:67-78.
6. Agdeppa E & Spilker M. A Review of Imaging Agent Development. The AAPS, (2009) 11(2).
7. Brewer K, et. al. Relaxometry of Bacterially Derived Organelles: A Novel Class of MRI
Contrast Agent for Cell Labeling and Tracking. (2014) Joint Annual Meeting ISMRM-ESMRMB.
8. Komeili A. Molecular mechanisms of compartmentalization and biomineralization in
magnetotactic bacteria. FEMS microbio rev (2012) 36:232-55.
9. Matin A, et al., Visualizing implanted tumors in mice with magnetic resonance imaging using
magnetotactic bacteria.Clinical Cancer Research (2009) 15(16):5170-7
10. Dyall SD, Brown MT, Johnson PJ. Ancient invasions: from endosymbionts to organelles.
Science (2004) 304:253-7.
11. Unpublished results, Svendsen CN, et al., Cedars-Sinai Regenerative Medicine Institute.
12. Unpublished results, Yang PC, et al., Department of Medicine, Stanford University.
13. Unpublished results, Rutt BK, et al., Department of Radiology, Stanford University.
14. Heyn C, et al., In vivo magnetic resonance imaging of single cells in mouse brain with
optical validation. Magn Reson Med, (2006) 55(1): p. 23-9.
15. Rutt BK, Cell Tracking and Single Cell Imaging by MRI, World Congress on Medical Physics
and Biomedical Engineering, (2009) Volume 25/13 pp 218-220.
16. Yang, PC, et al. Direct Evaluation of Myocardial Viability and Stem Cell Engraftment
Demonstrates Salvage of the Injured Myocardium. (2015) Circulation research 116: e40-e50
17. Berman SC, et al., Long-term MR cell tracking of neural stem cells grafted in
immunocompetent versus immunodeficient mice reveals distinct differences in contrast between
live and dead cells. Magn Reson Med, (2011) 65(2): 564 -74
18. Amsalem Y, et al., Iron-Oxide Labeling and Outcome of Transplanted Mesenchymal Stem
Cells in the Infarcted Myocardium. Circulation, (2007) 116: I-38-I-45