Cryopreservation of mesenchymal stem cell and tissue engineered constructs using non-toxic cryoprotective agents

The thesis work deals with the development of cryopreservation strategy for long term storage of MSCs and MSCs seeded tissue engineered constructs using non toxic cryoprotective agents as freezing medium. In the first phase, different freezing medium consisting of a combination of natural extracellular cryoprotectants namely trehalose, hydroxyl ethyl starch, polyvinyl pyrolidine and intracellular CPAs like erythritol, taurine and ectoin were used for cryopreservation of MNCs following the Taguchi Orthogonal Array method. Among the various combinations, freezing medium consisting of trehalose (0.05mM), ectoin (0.10mM) and catalase (100μg/ml) has shown maximum MNCs viability. These CPAs were further investigated individually as well as in combination to see their effectiveness towards long term preservation of MSCs. Among the freezing solutions, solution prepared using trehalose (0.3mM), ectoin (0.3mM), and catalase (100μg/ml) was found to be the most effective in preserving MSCs in long term basis. The viability of MSCs (73%) is found to be higher than the viability achieved with 10% (v/v) Me2SO (61%) used as control. The apoptotic study has indicated that the addition of general caspase and calpain inhibitors can reduce the apoptosis rate upto 10-15% thereby achieving increased cell viability of 80%. The optimum condition for the controlled rate freezing of MSCs was established as prenucleation cooling rate -1oC/min, nucleation temperature -7.5oC, cold spike -80oC/min, post nucleation holding time 5min, post nucleation cooling rate -1oC/min, cell density (3×106/ml/cell) and storage temperature (-150°C ) using the most effective freezing medium achieving cell viability of 85%. The developed freezing medium has also shown its ability to preserve MSCs seeded tissue engineered construct. The maximum viability of 80% achieved at optimum controlled rate freezing of TECs was established as cooling rate -1oC/min, nucleation temperature -7.5OC and freezing medium consisting of trehalose (0.3mM), ectoin (0.3mM), catalase (100μg/ml) in presence of caspase (50μg) and calpain (50μg) inhibitors. Overall, it is demonstrated that the developed freezing medium may pave the way for long term preservation of MSCs and also MSCs seeded scaffold

Design and Synthesis of H3 Receptor Inverse Agonists with AchE Inhibitor Activity and QSAR Study of H3 Receptor Antagonists

Currently, acetyl cholinesterase and N-methyl-D-aspartate antagonists are commercially available for the treatment of Alzheimer's disease (AD). Approach of using multifunctional inhibitors to reduce the side effects of available drugs is the main objective of this work. Presently, Histamine-3 (H3) receptor antagonists are used for the treatment of several neurodegenerative disorders such as Epilepsy, Alzheimer‘s and Parkinson‘s diseases. Both H3 and AchE inhibitors cure the symptoms of Alzheimer by enhancing the acetylcholine levels in the brain. But the mechanism of action involved in both the cases is different. Here, we propose histamine-3 antagonist with acetyl cholinesterase (AchE) inhibitor activity as a novel class of drugs which can be used to treat Alzheimer‘s disease with less adverse peripheral effects caused by excessive AchE inhibitor. Our present study can be divided into two parts. In the first part, homology modeled structure of H3 active site and available crystal structure of AchE was used to collect the information for pharmacophore identification. The important descriptors were identified based on comparative 2D-QSAR and 3D-QSAR study of 28 druggable compounds for H3 receptor collected from the literature. In the second part, five hybrid molecules were generated based on the pharmacophore of H3 receptor and known pharmacophore of AchE inhibitors. All five hybrid molecules were screened through ADME/tox filters. The hybrid molecule was validated through GOLD docking score in both AchE and H3 receptor. The best hybrid compound (hybrid-3) was then evaluated by molecular dynamics (MD) simulation in water solvent model using 3D model of human H3 receptor (build based on bovine rhodopsin structure)

Preparation and Evaluation of Gelatin-Chitosan-Nanobioglass 3D Porous Scaffold for Bone Tissue Engineering

The aim of the present study was to prepare and characterize bioglass-natural biopolymer based composite scaffold and evaluate its bone regeneration ability. Bioactive glass nanoparticles (58S) in the size range of 20–30 nm were synthesized using sol-gel method. Porous scaffolds with varying bioglass composition from 10 to 30 wt% in chitosan, gelatin matrix were fabricated using the method of freeze drying of its slurry at 40 wt% solids loading. Samples were cross-linked with glutaraldehyde to obtain interconnected porous 3D microstructure with improved mechanical strength. The prepared scaffolds exhibited >80% porosity with a mean pore size range between 100 and 300 microns. Scaffold containing 30 wt% bioglass (GCB 30) showed a maximum compressive strength of 2.2±0.1 MPa. Swelling and degradation studies showed that the scaffold had excellent properties of hydrophilicity and biodegradability. GCB 30 scaffold was shown to be noncytotoxic and supported mesenchymal stem cell attachment, proliferation, and differentiation as indicated by MTT assay and RUNX-2 expression. Higher cellular activity was observed in GCB 30 scaffold as compared to GCB 0 scaffold suggesting the fact that 58S bioglass nanoparticles addition into the scaffold promoted better cell adhesion, proliferation, and differentiation. Thus, the study showed that the developed composite scaffolds are potential candidates for regenerating damaged bone tissue

Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas.

Bissoyi A, Reicher N, Chasnitsky M, et al. Ice Nucleation Properties of Ice-binding Proteins from Snow Fleas. Biomolecules. 2019;9(10): 532.Ice-binding proteins (IBPs) are found in many organisms, such as fish and hexapods, plants, and bacteria that need to cope with low temperatures. Ice nucleation and thermal hysteresis are two attributes of IBPs. While ice nucleation is promoted by large proteins, known as ice nucleating proteins, the smaller IBPs, referred to as antifreeze proteins (AFPs), inhibit the growth of ice crystals by up to several degrees below the melting point, resulting in a thermal hysteresis (TH) gap between melting and ice growth. Recently, we showed that the nucleation capacity of two types of IBPs corresponds to their size, in agreement with classical nucleation theory. Here, we expand this finding to additional IBPs that we isolated from snow fleas (the arthropod Collembola), collected in northern Israel. Chemical analyses using circular dichroism and Fourier-transform infrared spectroscopy data suggest that these IBPs have a similar structure to a previously reported snow flea antifreeze protein. Further experiments reveal that the ice-shell purified proteins have hyperactive antifreeze properties, as determined by nanoliter osmometry, and also exhibit low ice-nucleation activity in accordance with their size

Cryopreservation of liver-cell spheroids with macromolecular cryoprotectants

Spheroids are a powerful tool for basic research and to reduce or replace in vivo (animal) studies but are not routinely banked nor shared. Here, we report the successful cryopreservation of hepatocyte spheroids using macromolecular (polyampholyte) cryoprotectants supplemented into dimethyl sulfoxide (DMSO) solutions. We demonstrate that a polyampholyte significantly increases post-thaw recovery, minimizes membrane damage related to cryo-injury, and remains in the extracellular space making it simple to remove post-thaw. In a model toxicology challenge, the thawed spheroids matched the performance of fresh spheroids. F-actin staining showed that DMSO-only cryopreserved samples had reduced actin polymerization, which the polyampholyte rescued, potentially linked to intracellular ice formation. This work may facilitate access to off-the-shelf and ready-to-use frozen spheroids, without the need for in-house culturing. Readily accessible 3-D cell models may also reduce the number of in vivo experiments

High molecular weight polyproline as a potential biosourced ice growth inhibitor : synthesis, ice recrystallization inhibition, and specific ice face binding

Ice-binding proteins (IBPs) from extremophile organisms can modulate ice formation and growth. There are many (bio)technological applications of IBPs, from cryopreservation to mitigating freeze–thaw damage in concrete to frozen food texture modifiers. Extraction or expression of IBPs can be challenging to scale up, and hence polymeric biomimetics have emerged. It is, however, desirable to use biosourced monomers and heteroatom-containing backbones in polymers for in vivo or environmental applications to allow degradation. Here we investigate high molecular weight polyproline as an ice recrystallization inhibitor (IRI). Low molecular weight polyproline is known to be a weak IRI. Its activity is hypothesized to be due to the unique PPI helix it adopts, but it has not been thoroughly investigated. Here an open-to-air aqueous N-carboxyanhydride polymerization is employed to obtain polyproline with molecular weights of up to 50000 g mol–1. These polymers were found to have IRI activity down to 5 mg mL–1, unlike a control peptide of polysarcosine, which did not inhibit all ice growth at up to 40 mg mL–1. The polyprolines exhibited lower critical solution temperature behavior and assembly/aggregation observed at room temperature, which may contribute to its activity. Single ice crystal assays with polyproline led to faceting, consistent with specific ice-face binding. This work shows that non-vinyl-based polymers can be designed to inhibit ice recrystallization and may offer a more sustainable or environmentally acceptable, while synthetically scalable, route to large-scale applications

Selaginella bryopteris

The effective long-term cryopreservation of human mesenchymal stem cells (MSCs) is an essential prerequisite step and represents a critical approach for their sustained supply in basic research, regenerative medicine, and tissue engineering applications. Therefore, attempts have been made in the present investigation to formulate a freezing solution consisting of a combination of Selaginella bryopteris water-soluble extract with and without dimethyl sulfoxide (Me2SO) for the efficient long-term storage of human umbilical cord blood- (hUCB-) derived MSCs. The cryopreservation experiment using the formulated freezing solution was further performed with hUCB MSCs in a controlled rate freezer. A significant increase in postthaw cell viability and cell attachment of MSCs was achieved with freezing medium containing Selaginella bryopteris water extract along with 10% Me2SO as compared to the freezing medium containing Me2SO (10% v/v) alone. Furthermore, the decreasing apoptotic events and reactive oxygen species production along with increasing expression of heat shock proteins also confirmed the beneficial effect of Selaginella bryopteris water extract. The beneficial effect of Selaginella bryopteris water extract was validated by its ability to render postpreservation high cell viability. In conclusion, the formulated freezing solution has been demonstrated to be effective for the standardization of cryopreservation protocol for hMSCs

Review Article Recent Advances and Future Direction in Lyophilisation and Desiccation of Mesenchymal Stem Cells

Mesenchymal Stem Cells (MSCs) are a promising mammalian cell type as they can be used for the reconstruction of human tissues and organs. MSCs are shown to form bone, cartilage, fat, and muscle-like cells under specific cultivation conditions. Current technology of MSCs cryopreservation has significant disadvantages. Alternative technologies of mammalian cells preservation through lyophilisation or desiccation (air-drying) are among the upcoming domains of investigation in the field of cryobiology. Different protectants and their combinations were studied in this context. Loading of the protectant in the live cell can be a challenging issue but recent studies have shown encouraging results. This paper deals with a review of the protectants, methods of their delivery, and physical boundary conditions adopted for the desiccation and lyophilisation of mammalian cells, including MSCs. A hybrid technique combining both methods is also proposed as a promising way of MSCs dry preservation

Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants