Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications.

BACKGROUND: Mesenchymal stem cells (MSCs) have been proven to prevent and clear corneal scarring and limbal stem cell deficiency. However, using animal-derived serum in a culture medium raises the ethical and regulatory bar. This study aims to expand and characterize human limbus-derived stromal/mesenchymal stem cells (hLMSCs) for the first time in vitro in the xeno-free medium. METHODS: Limbal tissue was obtained from therapeutic grade corneoscleral rims and subjected to explant culture till tertiary passage in media with and without serum (STEM MACS XF; SM), to obtain pure hLMSCs. Population doubling time, cell proliferation, expression of phenotypic markers, tri-lineage differentiation, colony-forming potential and gene expression analysis were carried out to assess the retention of phenotypic and genotypic characteristics of hLMSCs. RESULTS: The serum-free medium supported the growth of hLMSCs, retaining similar morphology but a significantly lower doubling time of 23 h (*p < 0.01) compared to the control medium. FACS analysis demonstrated ≥ 90% hLMSCs were positive for CD90+, CD73+, CD105+, and ≤ 6% were positive for CD45-, CD34- and HLA-DR-. Immunofluorescence analysis confirmed similar expression of Pax6+, COL IV+, ABCG2+, ABCB5+, VIM+, CD90+, CD105+, CD73+, HLA-DR- and CD45-, αSMA- in both the media. Tri-lineage differentiation potential and gene expression of hLMSCs were retained similarly to that of the control medium. CONCLUSION: The findings of this study demonstrate successful isolation, characterization and culture optimization of hLMSCs for the first time in vitro in a serum-free environment. This will help in the future pre-clinical and clinical applications of MSCs in translational research.

Pre-Clinical Evaluation of Efficacy and Safety of Human Limbus-Derived Stromal/Mesenchymal Stem Cells with and without Alginate Encapsulation for Future Clinical Applications.

Corneal opacification or scarring is one of the leading causes of blindness worldwide. Human limbus-derived stromal/mesenchymal stem cells (hLMSCs) have the potential of clearing corneal scarring. In the current preclinical studies, we aimed to determine their ability to heal the scarred corneas, in a murine model of corneal scar, and examined their ocular and systemic toxicity after topical administration to rabbit eyes. The hLMSCs were derived from human donor corneas and were cultivated in a clean room facility in compliance with the current good manufacturing practices (cGMP). Before the administration, the hLMSCs were analyzed for their characteristic properties including immunostaining, and were further subjected to sterility and stability analysis. The corneas (right eye) of C57BL/6 mice (n = 56) were stripped of their central epithelium and superficial anterior stroma using a rotary burr (Alger Brush® II). Few mice were left untreated (n = 8), while few (n = 24) were treated immediately with hLMSCs after debridement (prophylaxis group). The rest (n = 24, scar group) were allowed to develop corneal scarring for 2 weeks and then treated with hLMSCs. In both groups, the treatment modalities included encapsulated (En+) and non-encapsulated (En-) hLMSCs and sham (vehicle) treatment. The follow-up (4 weeks) after the treatment or debridement included clinical photography, fluorescein staining, and optical coherence tomography at regular intervals. All the images and scans were analyzed using ImageJ software to assess the changes in corneal haze, scar area, and the reflectivity ratio of the epithelium to the stroma. The scar area and the scar intensity were found to be decreased in the groups that received hLMSCs. The reflectivity of the stroma was found to be normalized to the baseline levels before the debridement in the eyes that were treated with hLMSCs, relative to the untreated. In the safety study, the central corneas of the left eye of 18 New Zealand rabbits were scraped with a needle and then treated with En+ hLMSCs, En- hLMSCs, and the sham (n = 6 each). Rabbits were then followed up for 4 weeks, during which blood and tear samples were collected at regular intervals. These rabbits were then assessed for changes in the quantities of inflammatory markers (TNF-α, IL-6, and IgE) in the sera and tears, changes in the ocular surface observations such as intraocular pressure (IOP), and the hematological and clinical chemistry parameters. Four weeks later, the rabbits were euthanized and examined histopathologically. No significant changes in conjunctival congestion, corneal clarity, or IOP were noticed during the ophthalmic examination. The level of inflammatory molecules (TNF-α and IL-6 TNF-α) and the hematological parameters were similar in all groups without any significant changes. Histological examination of the internal organs and ocular tissues did not reveal any abnormalities. The results of these studies summarize that the En+ and En- hLMSCs are not harmful to the recipient and potentially restore the transparency of debrided or scarred corneas, indicating that hLMSCs can be assessed for clinical use in humans.

Image-Guided Measurement of Radiation Force Induced by Focused Ultrasound Beams.

The radiation force balance (RFB) is a widely used method for measuring acoustic power output of ultrasonic transducers. The reflecting cone target is attractive due to its simplicity and long-term stability, at a reasonable cost. However, accurate measurements using this method depend on the alignment between the ultrasound beam and cone axes, especially for highly focused beams utilized in therapeutic applications. With the advent of dual-mode ultrasound arrays (DMUAs) for imaging and therapy, image-guided measurements of acoustic output using the RFB method can be used to improve measurement accuracy. In this article, we describe an image-guided RFB measurement of focused DMUA beams using a widely used commercial instrument. DMUA imaging is used to optimize the alignment between the acoustic beam and reflecting cone axes. In addition to image-guided alignment, DMUA echo data is used to track the displacement of the cone, which provides an auxiliary measurement of acoustic power. Experimental results using a DMUA prototype with [Formula: see text] shows that 1-2 mm of misalignment can result in 5%-14% error in the measured acoustic power. In addition to the use of B-mode image guidance for improving measurement accuracy, we present preliminary results demonstrating the benefit of displacement tracking using real-time DMUA imaging during the application of (sub)therapeutic focused beams. Displacement tracking provides a direct measurement of the radiation force with high sensitivity and follows the expected dependence on changes in amplitude and duty cycle (DC) of the focused ultrasound (FUS) beam. This could lead to simpler, more reliable methods for measuring acoustic power based on the radiation force principle. Combined with appropriate computational modeling, the direct measurement of acoustic radiation force could lead to reliable dosimetry in situ in emerging applications such as transcranial FUS (tFUS) therapies.