Cellular labeling and quantification for nuclear magnetic resonance techniques
The disclosure provides, in part, a method for quantifying cell numbers in vivo. The disclosure comprises a method of quantifying labeled cells by nuclear magnetic resonance techniques and a computer method for the same.
1. A method for quantifying cell number in vivo, the method comprising:
a. administering to a subject cells that are labeled with a fluorocarbon imaging reagent, wherein at least a portion of the fluorocarbon imaging reagent was internalized into the cell;
b. examining at least a portion of the subject by 19 F magnetic resonance imaging (MRI), thereby detecting the cells that are labeled with the fluorocarbon imaging reagent in the subject; and
c. quantifying the number of labeled cells in a region of interest (ROI).
2. The method of claim 1 , wherein the fluorocarbon imaging reagent is a perfluoropolyether.
3. The method of claim 1 , wherein the fluorocarbon imaging reagent is a perfluoro-crown ether.
4. The method of claim 1 , wherein the imaging reagent is a perfluoro-15-crown-5-ether.
5. The method of claim 1 , wherein the fluorocarbon is a perfluorinated polyether having an average formula:
XO(Y—O) n Z
wherein Y is selected from the group comprising:
wherein n is an integer from 8 to 20; wherein X and Z are the same and are selected from the group comprising: perfluoroalkyls, perfluoroethers, fluoroalkyls terminated with fluoroacyl, carboxyl, amide or ester, methylols, acid chlorides, amides, amidines, acrylates and esters.
6. The method of claim 1 , wherein the fluorocarbon imaging reagent is selected from the group comprising: a linear perfluoropolyether, a cyclic perfluoropolyether and a mixture thereof.
7. The method of claim 1 , wherein prior to administration to the subject, the cells are contacted with a composition comprising the fluorocarbon imaging reagent, whereby the cells are labeled with the fluorocarbon imaging reagent.
8. The method of claim 7 , wherein the composition comprising the fluorocarbon imaging reagent further comprises an uptake enhancing reagent.
9. The method of claim 8 , wherein the uptake enhancing reagent comprises a compound selected from the group consisting of:
a. cationic lipid; and
b. cationic polypeptide.
10. The method of claim 9 , wherein the cationic peptide is a protamine.
11. The method of claim 7 , wherein the composition comprising the fluorocarbon imaging reagent further comprises:
a. a surfactant; and
b. a cationic lipid.
12. The method of claim 7 , wherein the composition comprising the fluorocarbon imaging reagent further comprises Pluronic L-35.
13. The method of claim 7 , wherein the fluorocarbon imaging reagent is formulated as an emulsion.
14. The method of claim 7 , wherein the emulsion comprises particles having a mean diameter of between 30 and 500 nm.
15. The method of claim 1 , wherein prior to administration to the subject, the cells are labeled with perfluorocarbon emulsion particles using electroporation.
16. The method of claim 1 , wherein the cell is a mammalian cell.
17. The method of claim 1 , wherein the cell is a cell of the immune system.
18. The method of claim 1 , wherein the cell is a T cell.
19. The method of claim 1 , wherein the cell is a dendritic cell.
20. The method of claim 1 , wherein the cell is a stem cell.
21. The method of claim 1 , further comprising collecting a 1 H data set.
22. The method of claim 1 , further comprising generating and comparing a 19 F image and a 1 H image.
23. The method of claim 1 , wherein the cell is administered to the subject as part of a cellular therapeutic regimen.
24. The method of claim 1 , wherein quantifying comprises using a calibrated 19 F signal in the ROI.
25. The method of claim 24 , wherein the cellular dose of fluorocarbon imaging reagent is calculated prior to administration of cells to the subject.
26. The method of claim 1 , wherein examining comprises detecting a pre-calibrated 19 F signal from which one can deduce a relationship between the 19 F signal in the ROI and the representative number of 19 F molecules or cell quantity.
27. The method of claim 1 , wherein examining comprises detecting a contemporaneous signal in the ROI from which one can deduce a relationship between the signal and the representative number of 19 F molecules or cell quantity.
28. The method of claim 1 , wherein examining comprises detecting a post-calibrated 19 F signal in the ROI from which one can deduce a relationship between the signal and the representative number of 19 F molecules or cell quantity.
29. The method of claim 1 , wherein quantifying is done by comparison to a calibrated external 19 F reference during the 19 F MRI scan of the subject.
30. The method of claim 29 , wherein the reference is a cell free reference.
31. The method of claim 1 , wherein quantifying comprises calculating ratios of the intensity of 19 F signal and the volume of labeled cells in a ROI compared to a reference.
32. The method of claim 1 , wherein quantifying the number of labeled cells comprises using at least one of the parameters from the group consisting of: (i) the cellular dose of labeling agent (i.e., F c ) measured in vitro; (ii) in vivo 19 F MRI data set taken in the subject at one or more time points following labeled cell administration; (iii) the voxel volume; (iv) the in-plane voxel area (i.e., area of the image pixel); (v) the MRI data set from the 19 F reference standard; (vi) the measured Johnson noise of the 19 F MRI data in the subject material; (vii) the measured signal-to-noise ratio (SNR) of one or more voxels of the 19 F MRI data set in the subject material; (viii) the measured SNR of one or more voxels of the 19 F MRI data set from the reference standard; (ix) the 19 F NMR relaxation times (T1, T2, and T2*) of the subject material; and (x) the 19 F NMR relaxation times (T1, T2, and T2*) of the reference standard.
33. The method of claim 1 , wherein quantifying the number of labeled cells comprises using the formula:
N
c
=
[
F
R
]
v
I
R
1
F
c
∑
i
=
1
N
ROI
I
c
(
i
)
where: N c =total number of labeled cells in the ROI; [F R ]=concentration of 19 F in the calibrated 19 F reference solution (or gel); v=voxel volume; I R =mean intensity of the calibrated 19 F reference taken with the MRI scan, averaged over one or more voxels; F c =average 19 F cellular dose of the labeling agent measured in vitro; N ROI =number of voxels in the ROI containing labeled cells; I c (i) =image intensity of the i th voxel in the ROI containing labeled cells; i=unitless index for voxels in the ROI containing labeled cells.