Isolation and Characterization of Human Conjunctival Mesenchymal Stromal Cells and Their Extracellular Vesicles.

Purpose: The purpose of this study was to isolate and culture human conjunctival mesenchymal stromal cells (Conj-MSCs) from cadaveric donor tissue, and to obtain and characterize their extracellular vesicles (EVs) and their effect on conjunctival epithelium. Methods: Stromal cells isolated from cadaveric donor conjunctival tissues were cultured and analyzed to determine whether they could be defined as MSCs. Expression of MSC markers was analyzed by flow cytometry. Cells were cultured in adipogenic, osteogenic, and chondrocyte differentiation media, and stained with Oil Red, Von Kossa, and Toluidine Blue, respectively, to determine multipotent capacity. EVs were isolated from cultured Conj-MSCs by differential ultracentrifugation. EV morphology was evaluated by atomic force microscopy, size distribution analyzed by dynamic light scattering, and EVs were individually characterized by nanoflow cytometry. The effect of EVs on oxidative stress and viability was analyzed in in vitro models using the conjunctival epithelial cell line IM-HConEpiC. Results: Cultured stromal cells fulfilled the criteria of MSCs: adherence to plastic; expression of CD90 (99.95 ± 0.03% positive cells), CD105 (99.04 ± 1.43%), CD73 (99.99 ± 0.19%), CD44 (99.93 ± 0.05%), and absence of CD34, CD11b, CD19, CD45 and HLA-DR (0.82 ± 0.91%); and in vitro differentiation into different lineages. Main Conj-MSC EV subpopulations were round, small EVs that expressed CD9, CD63, CD81, and CD147. Conj-MSC EVs significantly decreased the production of reactive oxygen species in IM-HConEpiCs exposed to H2O2 in similar levels than adipose tissue-MSC-derived EVs and ascorbic acid, used as controls. Conclusions: It is possible to isolate human Conj-MSCs from cadaveric tissue, and to use these cells as a source of small EVs with antioxidant activity on conjunctival epithelial cells.

Characterization and functional performance of a commercial human conjunctival epithelial cell line.

The conjunctiva is a complex tissue that covers the eye beginning at the corneal limbus and extending over the inner surfaces of the eyelids. Due to its important functions in maintaining the health of the ocular surface, adequate in vitro models of conjunctival structure and function are essential to understand its roll in different pathologies. Because there is scarcity of human conjunctival tissue that can be used in research, cell lines are often the only option for initial studies. An immortalized human conjunctival epithelial cell (IM-HConEpiC) line is now commercially available; however, it is not very well characterized. In this study, we have developed a new protocol to culture these cells without the use of collagen-coated culture surfaces, but with a defined cell culture medium. We characterized IM-HConEpiCs cultured under these conditions and corroborated that the cells maintained a conjunctival epithelial phenotype, including acidic and neutral mucins, junctional proteins E-cadherin and zonula occludens 1, and expression of CK8 and CK19, among others. In addition, we analyzed the response to oxidative stress and inflammatory stimuli and found that IM-HConEpiCs respond as expected for conjunctival epithelial tissue. For instance, cells exposed to oxidative stress increased the production of reactive oxygen species, and that increase was blocked in the presence of an antioxidant agent. In addition, after stimulation with TNF-α, IM-HConEpiCs significantly increased the production of IL-1ß, IL-6, IL-8, and IP-10. Therefore, with this study we conclude that IM-HConEpiCs can be a useful tool in functional studies to determine the response of the conjunctiva to pathological conditions and/or to test new therapeutic strategies.

Luminescent Citrate-Functionalized Terbium-Substituted Carbonated Apatite Nanomaterials: Structural Aspects, Sensitized Luminescence, Cytocompatibility, and Cell Uptake Imaging.

This work explores the preparation of luminescent and biomimetic Tb3+-doped citrate-functionalized carbonated apatite nanoparticles. These nanoparticles were synthesized employing a citrate-based thermal decomplexing precipitation method, testing a nominal Tb3+ doping concentration between 0.001 M to 0.020 M, and a maturation time from 4 h to 7 days. This approach allowed to prepare apatite nanoparticles as a single hydroxyapatite phase when the used Tb3+ concentrations were (i) ≤ 0.005 M at all maturation times or (ii) = 0.010 M with 4 h of maturation. At higher Tb3+ concentrations, amorphous TbPO4·nH2O formed at short maturation times, while materials consisting of a mixture of carbonated apatite prisms, TbPO4·H2O (rhabdophane) nanocrystals, and an amorphous phase formed at longer times. The Tb3+ content of the samples reached a maximum of 21.71 wt%. The relative luminescence intensity revealed an almost linear dependence with Tb3+ up to a maximum of 850 units. Neither pH, nor ionic strength, nor temperature significantly affected the luminescence properties. All precipitates were cytocompatible against A375, MCF7, and HeLa carcinogenic cells, and also against healthy fibroblast cells. Moreover, the luminescence properties of these nanoparticles allowed to visualize their intracellular cytoplasmic uptake at 12 h of treatment through flow cytometry and fluorescence confocal microscopy (green fluorescence) when incubated with A375 cells. This demonstrates for the first time the potential of these materials as nanophosphors for living cell imaging compatible with flow cytometry and fluorescence confocal microscopy without the need to introduce an additional fluorescence dye. Overall, our results demonstrated that Tb3+-doped citrate-functionalized apatite nanoparticles are excellent candidates for bioimaging applications.

Crystallization, Luminescence and Cytocompatibility of Hexagonal Calcium Doped Terbium Phosphate Hydrate Nanoparticles.

Luminescent lanthanide-containing biocompatible nanosystems represent promising candidates as nanoplatforms for bioimaging applications. Herein, citrate-functionalized calcium-doped terbium phosphate hydrate nanophosphors of the rhabdophane type were prepared at different synthesis times and different Ca2+/Tb3+ ratios by a bioinspired crystallization method consisting of thermal decomplexing of Ca2+/Tb3+/citrate/phosphate/carbonate solutions. Nanoparticles were characterized by XRD, TEM, SEM, HR-TEM, FTIR, Raman, Thermogravimetry, inductively coupled plasma spectroscopy, thermoanalysis, dynamic light scattering, electrophoretic mobility, and fluorescence spectroscopy. They displayed ill-defined isometric morphologies with sizes ≤50 nm, hydration number n ~ 0.9, tailored Ca2+ content (0.42-8.11 wt%), and long luminescent lifetimes (800-2600 µs). Their relative luminescence intensities in solid state are neither affected by Ca2+, citrate content, nor by maturation time for Ca2+ doping concentration in solution below 0.07 M Ca2+. Only at this doping concentration does the maturation time strongly affect this property, decreasing it. In aqueous suspensions, neither pH nor ionic strength nor temperature affect their luminescence properties. All the nanoparticles displayed high cytocompatibility on two human carcinoma cell lines and cell viability correlated positively with the amount of doping Ca2+. Thus, these nanocrystals represent promising new luminescent nanoprobes for potential biomedical applications and, if coupled with targeting and therapeutic moieties, they could be effective tools for theranostics.

Self-Assembled Type I Collagen-Apatite Fibers with Varying Mineralization Extent and Luminescent Terbium Promote Osteogenic Differentiation of Mesenchymal Stem Cells.

This work explores in depth the simultaneous self-assembly and mineralization of type I collagen by a base-acid neutralization technique to prepare biomimetic collagen-apatite fibrils with varying mineralization extent and doped with luminescent bactericidal Tb3+ ions. Two variants of the method are tested: base-acid titration, a solution of Ca(OH)2 is added dropwise to a stirred solution containing type I collagen dispersed in H3 PO4 ; and direct mixing, the Ca(OH)2 solution is added by fast dripping onto the acidic solution. Only the direct mixing variant yielded an effective control of calcium phosphate polymorphism. Luminescence spectroscopy reveals the long luminescence lifetime and high relative luminescence intensity of the Tb3+ -doped materials, while two-photon confocal fluorescence microscopy shows the characteristic green fluorescence light when using excitation wavelength of 458 nm, which is not harmful to bone tissue. Cytotoxicity/viability tests reveal that direct mixing samples show higher cell proliferation than titration samples. Additionally, osteogenic differentiation essays show that all mineralized fibrils promote the osteogenic differentiation, but the effect is more pronounced when using samples prepared by direct mixing, and more notably when using the Tb3+ -doped mineralized fibrils. Based on these findings it is concluded that the new nanocomposite is an ideal candidate for bone regenerative therapy.