In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models

Abstract

IntroductionDespite spectacular progress in cellular transplantology, there are still many concerns about the fate of transplanted cells. More preclinical studies are needed, especially on large animal models, to bridge the translational gap between basic research and the clinic. Herein, we propose a novel approach in analysis of cell transplantation effects in large animals explants using in vivo imaging system (IVIS®) or similar equipment.Material and methodsIn the in vitro experiment cells labeled with fluorescent membrane dyes: DID (far red) or PKH26 (orange) were visualized with IVIS®. The correlation between the fluorescence signal and cell number with or without addition of minced muscle tissue was calculated. In the ex vivo study urethras obtained from goats after intraurethral cells (n = 9) or PBS (n = 4) injections were divided into 0.5 cm cross-slices and analyzed by using IVIS®. Automatic algorithm followed or not by manual setup was used to separate specific dye signal from tissue autofluorescence. The results were verified by systematic microscopic analysis of standard 10 μm specimens prepared from slices before and after immunohistochemical staining. Comparison of obtained data was performed using diagnostic test function.ResultsFluorescence signal strength in IVIS® was directly proportional to the number of cells regardless of the dye used and detectable for minimum 0.25x106 of cells. DID-derived signal was much less affected by the background signal in comparison to PKH26 in in vitro test. Using the IVIS® to scan explants in defined arrangement resulted in precise localization of DID but not PKH26 positive spots. Microscopic analysis of histological specimens confirmed the specificity (89%) and sensitivity (80%) of IVIS® assessment relative to DID dye. The procedure enabled successful immunohistochemical staining of specimens derived from analyzed slices.ConclusionsThe IVIS® system under appropriate conditions of visualization and analysis can be used as a method for ex vivo evaluation of cell transplantation effects. Presented protocol allows for evaluation of cell delivery precision rate, enables semi-quantitative assessment of signal, preselects material for further analysis without interfering with the tissue properties. Far red dyes are appropriate fluorophores to cell labeling for this application.

Intraurethral co-transplantation of bone marrow mesenchymal stem cells and muscle-derived cells improves the urethral closure

“…The DID dye was used for staining cells in the MDC and MSC groups and for staining MDCs in the co-transplanted group. We have used the previously described protocol of ex vivo tissue analysis, which allows the localization of grafted spots within the tissue and enables semi-quantitative estimation of fluorescence strength in the whole organ [ 20 ]. The assessment revealed that the median TRE value normalized to injected DID-stained cell number in 28-day urethras was almost twice as high in the co-transplantation group than in the MDC group (6.4E + 09 vs 3.4E + 09).…”

“…We have exploited the in vivo imagining system (IVIS® Spectrum) for screening of explanted urethras in order to search for transplanted cell depots. The procedure for ex vivo imagining was recently optimized and described in detail by our group [ 20 ]. We have demonstrated that under certain conditions it is a suitable method for a preliminary assessment of transplanted cell survival and location in the entire isolated organ.…”

Limited accuracy of transurethral and periurethral intrasphincteric injections of cellular suspension

“…If a cell depot was unambiguously observed in microscopic cross‐sections, fulfilling the criteria of specific fluorescence described above, it was classified as depot even if there was no corresponding spot in IVIS image (see “spot 1” in Figure ). In PERI group, IVIS images had lower value as we previously demonstrated that PKH26 is not a suitable dye for analysis of transplanted cell presence . However, if PKH26 positive cell depot was big enough, it was recognized by IVIS properly.…”

“…Herein, we employed an IVIS imager to perform preliminary screening of the whole isolated organ in order to decrease the risk of depot omission. Previously we have demonstrated that such an ex vivo imaging is an adequate method to search for cell depots stained with far red fluorochromes . However, it must be stated that although we have used an advanced protocol, including the visualization of the whole urethra, the analysis of injection accuracy is fraught with relatively high risk of error.…”

Quantitative Impact of Cell Membrane Fluorescence Labeling on Phagocytosis Measurements in Confrontation Assays

“…In our study, the effect of FITC staining on the capability of MH-S cells to engulf the spores of two strains of L. corymbifera and one strain of A. fumigatus is studied thoroughly. DID [DiIC18 (5); 1, 1 -dioctadecyl-3, 3, 3 , 3 tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt] is a lipophilic dye that is widely used to determine the proliferation of tumor, phagocytosis and cell transplantation in vivo (Kraibooj et al, 2014;Yumoto et al, 2014;Zarychta-Wiśniewska et al, 2017). Even though there are several studies measuring the effect of DID on cell differentiation, apoptosis, proliferation, production of reactive oxygen species (ROS) (Mohtasebi et al, 2014), there is no study concerning whether DID staining of professional immune phagocytes has side effects on the value of the phagocytosis ratio of opportunistic fungi.…”