Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Recombinant human collagen for tissue engineered corneal substitutes.

We successfully fabricated transparent, robust hydrogels as corneal substitutes from concentrated recombinant human type I and type III collagen solutions crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). White light transmission through these gels is comparable or superior to that of human corneas. Hydrogels from both type I and type III collagens supported in vitro epithelium and nerve over-growth. While both these biocompatible hydrogels have adequate tensile strength and elasticity for surgical manipulation, type III collagen hydrogels tended to be mechanically superior. Twelve-month post-implantation results of type I recombinant collagen-based corneal substitutes into mini-pigs showed retention of optical clarity, along with regeneration of corneal cells, nerves and tear film. For clinical use, implants based on fully characterized, recombinant human collagen eliminate the risk of pathogen transfer or xenogeneic immuno-responses posed by animal collagens.

Geldanamycin-inspired compounds induce direct trans-differentiation of human mesenchymal stem cells to neurons.

Inspired from geldanamycin, the synthesis of a new series of 20-membered macrocyclic compounds is developed. The key features in our design are (i) retention of the fragment having the precise chiral functional groups of geldanamycin at C10, C11, C12 and C14, and (ii) replacement of an olefin moiety with the ester group, and the quinoid sub-structure with the triazole ring. The southern fragment needed for the macrocyclic ring formation was obtained from Evans' syn aldol as the key reaction and with the use of D-mannitol as the cheap source of a chiral starting material. For the synthesis of the northern fragment, we utilized l-ascorbic acid, which provided the desired chiral functional groups at C6 and C7. Further, the chain extension completed the synthesis of the northern fragment. In our approach, the crucial 20 membered macrocyclic ring was formed employing the click chemistry. When tested for their ability to directly trans-differentiate human mesenchymal stem cells to neurons, two novel compounds (20a and 7) from this series were identified and this was further validated by the presence of specific neuronal biomarkers (i.e. nestin, agrin and RTN4).

The transdifferentiation potential of limbal fibroblast-like cells.

We report the identification and isolation of limbal fibroblast-like cells from adult corneo-limbal tissue possessing self-renewing capacity and multilineage differentiation potential. The cells form cell aggregates or clusters, which express molecular markers, specific for ectoderm, mesoderm and endoderm lineages in vitro. Further, these cells mature into a myriad of cell types including neurons, corneal cells, osteoblasts, chondrocytes, adipocytes, cardiomyocytes, hepatocytes and pancreatic islet cells. Despite originating from a non-embryonic source, they express ESC and other stem cell markers important for maintaining an undifferentiated state. This multipotential capability, relatively easy isolation and high rate of ex vivo proliferation capacity make these cells a promising therapeutic tool.

A biomimetic scaffold for culturing limbal stem cells: a promising alternative for clinical transplantation.

Limbal tissues can be cultured on various types of scaffolds to create a sheet of limbal-corneal epithelium for research as well as clinical transplantation. An optically clear, biocompatible, biomimetic scaffold would be an ideal replacement graft for transplanting limbal stem cells. In this study, we evaluated the physical and culture characteristics of the recombinant human cross-linked collagen scaffold (RHC-III scaffold) and compared it with denuded human amniotic membrane (HAM). Optical/mechanical properties and microbial susceptibility were measured for the scaffolds. With the approval of the institutional review board, 2 mm fresh human limbal tissues were cultured on 2.5 x 2.5 cm(2) scaffolds in a medium containing autologous serum in a feeder cell-free submerged system. The cultured cell systems were characterized by morphology and immunohistochemistry for putative stem cells and differentiated cell markers. The refractive index (RI) and tensile strength of the RHC-III scaffold were comparable to human cornea, with delayed in vitro degradation compared to HAM. RHC-III scaffolds were 10-fold less susceptible to microbial growth. Cultures were initiated on day 1, expanded to form a monolayer by day 3 and covered the entire growth surface in 10 days. Stratified epithelium on the scaffolds was visualized by transmission electron microscopy. The cultured cells showed p63 and ABCG2 positivity in the basal layer and were immunoreactive for cytokeratin K3 and K12 in the suprabasal layers. RHC-III scaffold supports and retains the growth and stemness of limbal stem cells, in addition to resembling human cornea; thus, it could be a good replacement scaffold for growing cells for clinical transplantation.

Tissue-engineered recombinant human collagen-based corneal substitutes for implantation: performance of type I versus type III collagen.

PURPOSE: To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. METHODS: Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. RESULTS: Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. CONCLUSIONS: Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.