Table 4 Comparison of contemporary cell tracking methods
Tracking method – mechanism | Disadvantages | Advantages |
|---|---|---|
GFP, luciferase – DNA transfer can be mediated virally (transduction), via liposomes (lipofection), or by electrical parameters (electroporation/transfection) | - Genetic manipulation of cells may alter their function | - Human ASCs with GFP or luciferase resume proliferation normally |
- Since they are cytosolic in location; the vector (mRNA/DNA) and/or the protein (GFP) could be transmitted to host cells via fusion elements and/or microvesicle secretion, resulting in contamination. Hence, there is concern for false positivity and their detection may not equate to viability of donor cells | - Detection is sensitive to in vivo non-invasive bioluminescence imaging | |
- Technique requires serial passages, not suitable for use with fresh uncultured cells | Â | |
- Many mammalian tissues have endogenous fluorescence | Â | |
BrdU – this nuclear marker is a thymidine analog that replaces (3H) thymidine and can penetrate cell membranes to incorporate into newly synthesized DNA strands of actively proliferating cells | - Optimal labeling requires longer incubation time | - BrdU labeling has no effects on the ASC differentiation/proliferation and is not cytotoxic |
- Not suitable for non-invasive methods of detection | ||
- Does not indicate viability | ||
- Cells lose BrdU rapidly with serial passages | ||
Lipophilic dyes (DiI, DiR) –long-chain carbocyanine dyes with long aliphatic tails that incorporate into the lipid regions of the cell membranes | - Rapidly contaminates neighboring cells by macrophage-mediated phagocytosis, exchange of membrane microdomains, microvesicle and/or exosome transfer | - Easy technique for labeling and identification of cells |
- May be cytotoxic to cells | ||
- The dye fades with serial passages | ||
Amine reactive probes (CFSE) – these diffuse into cells and react with cytosolic amine-containing residues to form dye–protein adducts that are retained | - The dye is toxic to ASCs | - Good staining efficiency |
- Not suitable for in vivo non-invasive imaging | ||
- Does not correlate with viability | ||
- Contamination of neighboring cells can occur via macrophage-mediated phagocytosis, microvesicle and/or exosome transfer | ||
Nanoparticles – small crystals made up of inorganic molecules; for example, iron oxide, cadmium | - Can be toxic to cells in high concentration and detection is difficult with low concentrations | - Photostable, remain resistant for long periods of time |
- Contamination of neighboring cells can occur via phagocytosis, microvesicles and/or exosome transfer | - Can be used for in vivo non-invasive imaging | |
- Does not correlate with viability | ||
Real-time PCR for endogenous retroviral sequence (ERV-3) – the gene is present as a single copy in the human genome and so can be used to detect the presence of transplanted human cells in animal models | - Not suitable for non-invasive methods of detection | - Gives a quantitative estimate of number of cells |
- False positives can occur by macrophage-mediated phagocytosis but this is very low | - Very sensitive and specific | |
| Â | - The gene is already present in the human cells, so there is no need to stain the cells | |
FISH detection of human-specific cell surface markers or Alu sequences | - Alu sequences occur in large numbers in the primate genome, which makes higher likelihood of a false positive detection by transmission to host cells/macrophages via microvesicles/exosomes | - The gene is already present in the human cells, so there is no need to stain the cells |
- Not suitable for non-invasive methods of detection | Â | |
- Does not correlate with viability | Â |