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In recent years, tissue engineering has emerged as a new and ambitious approach that aims to restore damaged organs or tissues by delivering functional cells, supporting scaffolds, and biologically active molecules 1. One of the challenges yet to be overcome in this field is to find a suitable biomaterial scaffold capable of creating enriched microenvironments to improve cell survival in vivo. In order to address this issue, we compared 3 different matrices—Matrigel (an extract of basement membrane proteins derived from the Engelbreth-Holm-Swarm (EHS) tumor), Collagen I (a fibrillar extracellular matrix protein which is also the most abundant collagen type), and Puramatrix (a self assembling peptide with sequence AcN-(RADA)4-CNH2) 2. These matrices were used in conjunction with bone marrow-derived mesenchymal stem cells isolated from L2G85 transgenic mice that constitutively express firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP) (BM-MSCsFluc+/eGFP+) under the constitutive β-actin promoter. Using longitudinal bioluminescence imaging (BLI), we demonstrate here that the combination of Matrigel with BM-MSCsFluc+/eGFP+ has the best long-term survival.
At present, two critical problems limit the field of cell-based tissue engineering: (1) poor survival of transplanted cells and (2) lack of a non-invasive modality to monitor cell fate. In this study, we hypothesize that biomaterial scaffolds can furnish the biomechanical and structural characteristics of the seeded cells until they are able to fully incorporate into their new environment and that BLI can be adapted to image the efficacy of different biomatrices in living subjects. We evaluated three common biomatrices, including Matrigel (BD Biosciences, San Jose, CA), Collagen I (BD Biosciences, San Jose, CA), and Puramatrix (BD Biosciences, San Jose, CA). We focused on BM-MSCsFluc+/eGFP+ because these cells have the potential to differentiate into many clinically relevant cell types such as muscle, liver, brain and epithelial lineages3,4.
BM-MSCsFluc+/eGFP+ were isolated from adult male L2G85 transgenic FVB mice (n=6) using a modified protocol 5, 6. Fluorescence microscopy showed typical mesenchymal spindle-like morphology of BM-MSCsFluc+/eGFP+ (Figure 1a). Ex vivo BLI of cultured cells demonstrated a robust correlation between cell numbers and Fluc signal activity (r2=0.98), suggesting that BLI could be used to track cell fate in vivo (Figure 1b). Cell characteristics were further analyzed by LSR Flow Cytometry (Becton Dickinson Immunocytometry Systems) and FlowJo analysis software (TreeStar, San Carlos, CA). These cells expressed high levels of MSC-specific markers such as CD44 and CD90, but were negative for CD34 and CD45 markers present on hematopoietic stem cells and white blood cells (Figure 1c). Overall, the patterns of cell surface markers seen in BM-MSCsFluc+/eGFP+ were consistent with those reported in other studies 4, 7. Furthermore, there is no significant changes of BM-MSCGFP+/Fluc+ morphology after culturing in the presence of different biomatrices for 48 hours in same culture condition (Figure 1d).
To assess stem cell viability in different biomatrices in vivo, the same numbers (5×105) of MSCsFluc+/eGFP+ were suspended with (i) PBS as a control, (ii) Matrigel alone, (iii) Collagen I alone, (iv) Puramatrix alone, or (v) with an equal mixture of Matrigel, Collagen I, and Puramatrix (20 μl total volume for all groups). Cells and biomatrices were implanted subcutaneously into the backs of adult athymic nude mice (n=10) at 5 different sites. After transplantation, mice were imaged repeatedly using the Xenogen In Vivo Imaging System (IVIS; Xenogen, Alameda, CA). The reporter probe was D-Luciferin (375 mg/kg body weight). BLI was acquired at 1-min intervals and activities expressed as photons per second per centimeter square per steridian (p/s/cm2/sr) as described before 8. BLI signals were normalized to day 0 and expressed as % activity of day 0. Within 2 hours (day 0) after cell transplantation, BLI signals (mean±SD) in all five groups were similar: 5.57±0.58×105 p/sec/cm2/sr in PBS control, 6.90±0.43×105 in Matrigel, 5.35±0.62×105 in Collagen I, 2.76±0.23×105 in Puramatrix, and 4.44±0.41×105 in the mixture group (P=NS) (Figure 2a). However, the BLI signals decreased progressively in all groups subsequently. By one month, the PBS control and Puramatrix groups had significantly lower activities (1.6 to 1.8%) compared to the others (5.1 to 8.2%) (P<0.05) as shown in Figure 2b. Interestingly, after 5 months of follow-up, cell signals in the Matrigel and mixture groups (5 to 9%) maintained significantly higher signal activities compared to the other groups (all <1%) (P<0.01). BLI data were also confirmed by postmortem histology. Immunostaining showed engraftment of MSCsFluc+/eGFP+ in the Matrigel and mixture groups. Fewer MSCsFluc+/eGFP+ were seen in the Collagen I, Puramatrix, and PBS control groups (Figure 2c).
In summary, we have demonstrated that Matrigel-supported stem cell engraftment is superior to cells alone (PBS control) or cells supported with other matrices (Collagen I and Puramatrix). This can be attributed to the special properties of the Matrigel basement membrane matrix, which is known to release various growth factors and to provide structural support for cell seeding and differentiation 9. These data support the notion that loss of transplanted cells occurring within the heterotopic sites is likely due to the lack of appropriate extracellular matrix components needed for the cells to attach and develop in their new environment. We succeeded in assessing the longitudinal survival course and proliferation of engrafted cells by performing high throughput in vivo BLI. This technology is superior to the traditional histology-based assessment of cell grafts, which cannot provide longitudinal information within the same animal 10. To fully realize the clinical potential of tissue engineering, careful in vivo evaluation of the viability of engrafted cells and/or tissues will be needed. Further optimization and validation of both the supporting biomatrices and imaging technology will advance the development of this burgeoning and exciting field.