Safety and toxicity of human limbus-derived stromal/mesenchymal stem cells with and without alginate encapsulation for clinical application

Background The purpose of this study was to assess the ocular and systemic toxicity of topically applied human limbus-derived stromal/mesenchymal stem cells (hLMSCs) with and without alginate encapsulation as per Indian regulatory guidelines for stem cell therapy. Methods The hLMSCs were obtained from cadaveric corneoscleral rims and expanded in a current good manufacturing practice compliant laboratory. The hLMSCs were checked for viability, chromosomal stability, growth kinetics, contamination, and endotoxin levels. Cells with (En+ hLMSCs) or without (En− hLMSCs) alginate encapsulation were used for the animal experiments. The study involved 3 groups of 6 New Zealand white rabbits each, which underwent corneal wounding followed by treatment with sham (G1), En− hLMSCs (G2), and En+ hLMSCs cells (G3). Ophthalmic assessment including intraocular pressure (IOP), blood investigations and inflammatory marker (IL-6, TNF-α, IgE) expression in serum and tears were assessed on days 1, 7, 14, 21, and day 28. At the end of 28 days, the animals were sacrificed, and the organs were subjected to histopathological examination. Results The hLMSCs had 88.33 ± 2.37% viability at the end of 6 hours and 78.21 ± 1.47% at the end of 24 hours. The cells showed positive expression for the stem-cell biomarkers (p63α, Pax6, and ABCG2), extracellular matrix marker (Col-III) and mesenchymal biomarkers (VIM, CD73, CD90 and CD105). No contamination by the Mycoplasma species was found in either of the En-/En + hLMSCs and the levels of bacterial endotoxins in the En- hLMSCs and En + hLMSCs cell suspension was found be within the permissible levels (≤ 0.12 EU/mL). Ophthalmic examination showed no significant difference in IOP, corneal clarity and conjunctival congestion between the three groups at every time point. Haematological parameters were comparable between the three groups. The inflammatory markers in tear and serum (TNF-α and IL-6) were not significantly elevated in the groups receiving En+/En− hLMSCs. Histological examination did not show any abnormality in the ocular or corneal tissue, and the viscera. Conclusions The results of the study show that hLMSCs do not cause any local or systemic toxicity in recipients, implying that these cells are safe for clinical use and their efficacy can be assessed in human clinical trials.

Evaluation of Anticancer Bioactive Compounds and Cytotoxicity of Kaffir Lime (Citrus hystrix Dc.) Callus Extract Post Preservation

Previous studies have shown that kaffir lime extract is toxic to breast cancer T47D cells. To increase the bioactive compound's production for traditional cancer medicine raw material, we induce kaffir lime callus in vitro. One strategy to continuously produce kaffir lime callus is by using callus preservation. Our preliminary study used two preservation methods of callus, which are stored in 4 0C with or without alginate encapsulation. However, low temperatures and alginate encapsulation can be stress factors for plants, affecting the production of bioactive compounds and their anticancer ability. This study's objective was to determine the bioactive compounds of callus with and without preservation and their effect on the cytotoxicity against cancer cells. GC-MS detected the bioactive compounds in the extract, and cytotoxicity of callus against breast cancer (T47D), and non-cancer cell (Vero) is tested using MTT method. The results showed that preservation in 4 0C with and without encapsulation caused a change in bioactive compounds' profile. The change mainly precursor compound in callus, after being preserved and recultured, then changes into intermediates or final compounds, indicating the difference in growth phases. The terpenoid compounds detected after preservation are squalene and geranyl linalool. However, Kaffir lime callus extract pre and post preservation are not cytotoxic to both cancer and non-cancer cells. Therefore, the preservation method did not act as an elicitor to callus.

Process standardization for alginate encapsulation of potentially probiotic Pediococcus pentosaceus DM101

The main objective of this study was to standardise the procedure for encapsulation of potentially probiotic breast milk isolate Pediococcus pentosaceus DM101 using sodium alginate by extrusion method. The encapsulation parameters were optimized by comparing the encapsulation efficiency obtained with varying concentration of sodium alginate (0.5, 1.0, 1.5 and 2.0%), calcium chloride (0.05, 0.1 and 0.2M) and gelling time (5, 10, 20 and 30 minutes). Encapsulation efficiency was ascertained by finding the survival percentage after exposing the encapsulated cells to acid stress (pH 2.0 for 3h). Encapsulation carried out using two per cent sodium alginate and 0.1M CaCl2 following 20 min gelling time was found to confer maximum protective effect. Encapsulation efficiency of alginate beads prepared under optimized conditions was found to be 81.37±2.44 per cent. The results endorse alginate encapsulation as a means to confer a protective shielding effect thereby facilitating effective probiotic delivery.

Conservation, Regeneration and Genetic Stability of Regenerants from Alginate-Encapsulated Shoot Explants of Gardenia jasminoides Ellis

The present study demonstrates the potential of the alginate encapsulation of shoot tips and nodal segments of Gardenia jasminoides Ellis, the short-term cold storage of artificial seeds and subsequent successful conversion to desirable, uniform and genetically stable plantlets. Shoot tips and first-node segments below them, derived from shoots of in vitro cultures, responded better than second-to-fourth-node segments on agar-solidified Murashige and Skoog (MS) nutrient medium and thus, they were used as explants for alginate encapsulation. Explant encapsulation in 2.5% sodium alginate in combination with 50 mM of calcium chloride resulted in the production of soft beads, while hardening in 100 mM of calcium chloride formed firm beads of uniform globular shape, suitable for handling. The addition of liquid MS nutrient medium in the sodium alginate solution doubled the subsequent germination response of the beads. The maintenance of alginate beads under light favored their germination response compared to maintenance in darkness. Encapsulated shoot tip explants of gardenia, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in their regeneration response (73.3, 68.9, 53.3%, respectively), whereas, non-encapsulated explants (naked), stored under the same time durations of cold conditions, exhibited a sharp decline in regeneration response up to entirely zeroing (48.9, 11.1, 0.0%, respectively). Shoots, derived from 12-week cold-stored encapsulated explants, were easily rooted in solid MS nutrient medium with the addition of 0.5 μM of Indole-3-acetic acid (IAA) and after transplantation of the rooted plantlets individually to pots containing a peat–perlite (3:1, v/v) substrate, they were successfully acclimatized in the greenhouse under the gradual reduction of 75 or 50% shading with survival rates of 95–100%. The genetic stability of the acclimatized plantlets was assessed and compared with the mother plant using inter simple sequence repeat (ISSR) markers. ISSR analysis confirmed that all regenerated plantlets were genetically identical to the mother plant. This procedure of artificial seed production could be useful for the short-term storage of germplasm and the production of genetically identical and stable plants as an alternative method of micropropagation in Gardenia jasminoides.

Alginate encapsulation of Trichoderma harzianum against brown spot disease on rice (Oryzae sativa) in vivo assays

Bipolaris oryzae causes brown spot disease which is one of the most devastating diseases in rice. Nowadays, biological methods are effective controls which need to be developed in order to use less chemical control. Therefore, the objectives of the present study were to characterize the B. oryzae as a biocontrol agent and to measure the performance of encapsulated T. harzianum in controlling brown spot disease. Bipolaris oryzae was characterized based on morphological characteristics and alginate encapsulation was produced from conidial suspension by adding sodium alginate and calcium chloride which results in small beads. Alginate formulation was applied to the rice seedling to find the result on this formulation. As a result, the application of encapsulation T. harzianum to control brown spot disease showed the inhibition of the disease. As a conclusion, this can be an alternative method to control brown spot disease which will render easier application to rice plantation in the future.