Mesenchymal stem cells culture and labeling
We have chosen hMSCs as they have been considered among the other types of stem cells the safest and promising for treating brain injury.
HMSCs were derived from human placenta and maintained in culture.
HMSCs were labeled with SPIO (Bangs Laboratories; diameter of 0.96 μm) consisting of a magnetic iron oxide core and a chemically inert shell containing the Dragon Green fluorescent dye (ex=480 nm,
em=520 nm).
The efficacy of labeling evaluated by flow cytometry exceeded 90% and remained quite high during the first four passages after labeling.
Spectrophotometry evaluated that the average amount of iron was 5.6 pg/cell if the microparticles were added at the maximum non-toxic concentration of 5 g/ml.
MSC labeling with used paramagnetic microparticles did not alter cell proliferation,
viability and differentiation potential.
In vitro hMSCs phantom
After labelling with SPIO hMSCs were prepared in different cell concentration in five Eppendorf tubes (one for each concentration,
the same as were also injected into the rat brain): 10,
100,
103,
104 and 105 accordingly in 20 µl.
All tubes were laid in a water-filled box for further MRI.
Besides,
the concentration of 10 hMSCs in 20 µl were injected into the 10 ml of biopolymer hydrogel (sphero®GEL).
Latter is made on the base of embryonic and postnatal animal collagen and is homogeneous substance that could simulate tissue-equivalent conditions.
Transplantation of magnetically labeled hMSCs
Labelled hMSCs were transplanted stereotaxically intracerebral to male 250-280g Wistar rats (n=16) under inhalation anesthesia (izoflurane 1-1,5%,
E-Z Anesthesia® Systems) into the right striatum of intact brain.
The doses of transplanted cells ranged from 10 to 105 cells in 20 µl.
All procedures were carried out in accordance with the international rules «Guide for the Care and Use of Laboratory Animals» and were approved by the local ethics committee.
Cellular and in vivo MRI
In vitro and in vivo stem cells visualization was performed using 7,0 T (300 MHz for 1H) MRI scanner for small laboratory animals ClinScan-Bruker BioSpin with the use of two-segment surface head coil.
Table 1 shows the in vitro MR-protocol for the study of the phantom with hMSCs; table 2 shows the in vivo MR-protocol performed in the study of experimental animals.
Normal 0 false false false RU X-NONE X-NONE
For obtaining T2*-WI four different protocols were used:
- The classical protocol,
which is common in clinical practice (often referred as T2_Hemo) with the use of 2D Spoiled Gradient Echo pulse sequence with the small deviation angle.
- 2D Spoiled Gradient Echo with multiple echoes,
when due to combination of several gradient refocused echo signals T2*-weighting increases and SNR grows.
- 3D Spoiled Gradient Echo as a part of susceptibility-weighted image sequence.
- Susceptibility Weighted Image sequence.
In vitro obtained MRI data were analyzed using ImageJ (National Institutes of Health).
In all studied pulse sequences SNR were measured as the ratio of the signal from the biopolymer hydrogel to the standard deviation of the noise signal.
The degree of signal reduction was measured in the areas with pronounced changes in the magnetic susceptibility by constructing slice of intensity through this area.
Hystology
MRI data were confirmed by histology.
One,
two and three weeks after hMSC transplantation animals were transcardially perfused with 4% paraformaldehyde 0.1 M phosphate buffer.
The brains were removed and postfixed overnight at 4 C,
and then transferred to 30% sucrose for 72 h.
The brain was sectioned on a microtome set at 40 µm thickness.
Sections were stained with DAPI (Sigma-Aldrich).
Transplanted hMSCs were detected by recording the green fluorescence mark of magnetic iron oxide microspheres using a fluorescence microscope BZ-9000E (Keyence).