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

Introduction: Despite 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 methods: In 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. Results: Fluorescence 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. Conclusions: The 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

Abstract Background Cell therapy constitutes an attractive alternative to treat stress urinary incontinence. Although promising results have been demonstrated in this field, the procedure requires further optimization. The most commonly proposed cell types for intraurethral injections are muscle derived cells (MDCs) and mesenchymal stem/stromal cell (MSCs). The aim of this study was to evaluate the effects of MDC-MSC co-transplantation into the urethra. Methods Autologous transplantation of labeled MDCs, bone marrow MSCs or co-transplantation of MDC-MSC were performed in aged multiparous female goats (n = 6 in each group). The mean number of cells injected per animal was 29.6 × 106(± 4.3 × 106). PBS-injected animals constituted the control group (n = 5). Each animal underwent urethral pressure profile (UPP) measurements before and after the injection procedure. The maximal urethral closure pressure (MUCP) and functional area (FA) of UPPs were calculated. The urethras were collected at the 28th or the 84th day after transplantation. The marker fluorochrome (DID) was visualized and quantified using in vivo imaging system in whole explants. Myogenic differentiation of the graft was immunohistochemically evaluated. Results The grafted cells were identified in all urethras collected at day 28 regardless of injected cell type. At this time point the strongest DID-derived signal (normalized to the number of injected cells) was noted in the co-transplanted group. There was a distinct decline in signal intensity between day 28 and day 84 in all types of transplantation. Both MSCs and MDCs contributed to striated muscle formation if transplanted directly to the external urethral sphincter. In the MSC group those events were rare. If cells were injected into the submucosal region they remained undifferentiated usually packed in clearly distinguishable depots. The mean increase in MUCP after transplantation in comparison to the pre-transplantation state in the MDC, MSC and MDC-MSC groups was 12.3% (± 11.2%, not significant (ns)), 8.2% (± 9.6%, ns) and 24.1% (± 3.1%, p = 0.02), respectively. The mean increase in FA after transplantation in the MDC, MSC and MDC-MSC groups amounted to 17.8% (± 15.4%, ns), 15.2% (± 12.9%, ns) and 17.8% (± 2.5%, p = 0.04), respectively. Conclusions The results suggest that MDC-MSC co-transplantation provides a greater chance of improvement in urethral closure than transplantation of each population alone

Contingency table.

<p>Scheme.</p

Preparation of posthumously collected urethra for analysis in <i>ex vivo</i> experiment.

<p>A) Photograph of freshly isolated urethra with bladder and a method of length and diameter measuring; B) Slices of whole urethra for analysis with IVIS®; C) Schematic arrangement of tissue slices for IVIS® analysis. caudal–top, dorsal–upwards; D) Photograph of the microscopic preparation with drawn model "map" after fluorescence microscopy analysis. Lines shows the outline of the preparation (black), urethra light (red), location of spots showing specific fluorescence (blue).</p

The presence of specific DID fluorescence in urethras from study group after manual alignment to the control in IVIS®.

<p>The presence of specific DID fluorescence in urethras from study group after manual alignment to the control in IVIS®.</p

Contingency table for DID dye.

<p>Contingency table for DID dye.</p

Evaluation of the diagnostic test reliability based on the contingency tables.

<p>Microscopic examination was assumed as a reference method. Method using IVIS® constituted a diagnostic test. The comparison of data obtained from the IVIS® and microscopy was performed using diagnostic test function (PQStat software v.1.6.2).</p

The comparison of fluorescence signal originating from transplanted and control urethras read by IVIS®.

<p>The bar graphs indicating maximal strength of fluorescence signal from DID (red, A) and PKH26 (yellow, B) transplanted urethras compared to the control group (green). Data obtained after spectral unmixing algorithm application. The ranges of the min-max fluorescence intensity, were selected automatically by the program.</p

Contingency table for PKH26 dye obtained after applying manual settings in IVIS®.

<p>Contingency table for PKH26 dye obtained after applying manual settings in IVIS®.</p