Development of a Lyophilized Off-the-Shelf Mesenchymal Stem Cell-Derived Acellular Therapeutic.

The therapeutic activities elicited by mesenchymal stem cells (MSC) are in part mediated through paracrine action by the release of extracellular vesicles (EV) and secreted proteins. Collectively, these MSC-derived factors, referred to as the secretome product (SP), are intrinsically therapeutic and represent an attractive alternative to cell-based therapies. Herein, we developed a lyopreservation protocol to extend the shelf-life of the MSC-SP without compromising the structural or functional integrity of the vesicular components. The SP isolated from normoxia- and anoxia-exposed MSC elicited protective effects in an in vitro model of oxidative injury and the bioactivity was retained in the lyophilized samples. Three separate formulations of MSC-SP were isolated by tangential flow filtration using sucrose, trehalose, and mannitol as lyoprotectant agents. The MSC-SPs were lyophilized using a manifold protocol and the structural and functional integrity were assessed. The trehalose formulation of SP exhibited the highest EV and protein recovery after manifold-based lyophilization. To facilitate development as a therapeutic, a shelf lyophilization protocol was developed which markedly enhanced the recovery of EV and proteins. In conclusion, lyophilization represents an efficient method to preserve the structural and functional integrity of the MSC-SP and can be used to develop an off-the-shelf therapeutic.

Structural and Functional Equivalency Between Lyopreserved and Cryopreserved Chorions with Viable Cells.

Objective: Clinical studies have demonstrated that the use of cryopreserved amnion or trophoblast (TR)-free chorion, containing viable cells, in the treatment of chronic wounds results in high rate of wound closure. Recently, a new lyopreservation method has been developed for preservation of amnion that also retains the endogenous viable cells. The objective of this study was to use this method for lyopreservation of TR-free chorionic membrane (viable lyopreserved chorionic membrane [VLCM]) and compare it with the viable cryopreserved chorionic membrane (VCCM). A second objective was to investigate the immunogenicity of chorion, an important question that has not been fully addressed. Approach: Chorion immunogenicity was tested in vitro in a mixed lymphocyte reaction and lipopolysaccharide (LPS) challenge assay, and in vivo in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by LIVE/DEAD cell fluorescent staining. Inhibition of tumor necrosis factor α secretion by LPS-activated THP-1 cells and endothelial cell tubule formation assays were performed to evaluate the anti-inflammatory and proangiogenic properties, respectively. An in vivo rabbit abdominal adhesion model was used to evaluate the antifibrotic properties. Results: Chorionic membrane without trophoblast (CM) was shown to be nonimmunogenic. Tissue architecture, growth factors, and cell viability of fresh CM were maintained in VLCM and VCCM. In vitro studies showed that anti-inflammatory and angiogenic properties were retained in VLCM. Furthermore, VLCM prevents formation of postsurgical adhesions in a rabbit abdominal surgical adhesion model. Innovation: Characterization of structural and functional properties of VLCM is reported for the first time. Conclusion: Similar to VCCM, VLCM retains native components of fresh CM, including collagen-rich extracellular matrix, growth factors, and viable cells. In vitro and in vivo models demonstrate that VLCM is anti-inflammatory, proangiogenic and antifibrotic. Results of this study support the structural and functional equivalency between VLCM and VCCM.

A Viable Lyopreserved Amniotic Membrane Modulates Diabetic Wound Microenvironment and Accelerates Wound Closure.

Objective: Wound healing is a complex process involving the dynamic interplay of various types of cells and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of the process, resulting in non-healing chronic wounds. Human amniotic membrane (AM) has been shown to be effective in the management of chronic wounds. Recently, a viable lyopreserved AM (VLAM) has been developed. The VLAM retains the structural, molecular, and functional properties of fresh AM with the advantage of a long shelf life for living tissue at ambient temperatures. The objective of this study was to evaluate the effects of VLAM on the impaired wound microenvironment and wound closure in db/db mice. Approach: VLAM or saline gel (control) was applied weekly to 7-mm excisional wounds in diabetic (db/db) mice. Wound appearance and size were assessed weekly. Inflammation and redox state in wounds were tested by cytokine gene and protein expression, and by catalase and glutathione peroxidase activities, respectively. Wound tissue granulation and neovascularization were assessed histologically. Results: Diabetic wounds treated with VLAM closed faster than control wounds. On an average, VLAM-treated wounds closed 4 days faster than the control wounds, with a significantly faster rate of closure at days 7 and 14 as compared with control wounds. The faster closure correlated with a decrease in the expression of proinflammatory factors and oxidative stress, and an increase in angiogenesis and dermal thickness. Innovation: Effects of VLAM on a chronic wound microenvironment and underlying molecular mechanisms were investigated for the first time. Conclusion: VLAM accelerates wound closure in db/db mice by decreasing inflammation and oxidative stress and supporting wound tissue granulation, neovascularization, and re-epithelialization.

Role of Intrinsic Disorder in Animal Desiccation Tolerance.

This review compares the molecular strategies employed by anhydrobiotic invertebrates to survive extreme water stress. Intrinsically disordered proteins (IDPs) play a central role in desiccation tolerance in all species investigated. Various hypotheses about the functions of anhydrobiosis-related intrinsically disordered (ARID) proteins, including late embryogenesis abundant (LEA) and tardigrade-specific intrinsically disordered proteins, are evaluated by broad sequence characterization. A surprisingly wide range in sequence characteristics, including hydropathy and the frequency and distribution of charges, is discovered. Interestingly, two clusters of similar proteins are found that potentially correlate with distinct functions. This may indicate two broad groups of ARID proteins, composed of one group that folds into functional conformations during desiccation and a second group that potentially displays functions in the hydrated state. A broad range of physiochemical properties suggest that folding may be induced by factors such as hydration level, molecular crowding, and interactions with binding partners. This plasticity may be required to fine-tune the ARID-proteome response at different hydration levels during desiccation. Furthermore, the sequence properties of some LEA proteins share qualities with IDPs known to undergo liquid-liquid phase separations during environmental challenges.

The effect of glass-forming sugars on vesicle morphology and water distribution during drying.

Cryopreservation requires that stored materials be kept at extremely low temperatures and uses cryoprotectants that are toxic to cells at high concentrations. Lyopreservation is a potential alternative where stored materials can remain at room temperatures. That storage process involves desiccating cells filled with special glass-forming sugars. However, current desiccation techniques fail to produce viable cells, and researchers suspect that incomplete vitrification of the cells is to blame. To explore this hypothesis, a cell is modelled as a lipid vesicle to monitor the water content and membrane deformation during desiccation. The vesicle is represented as a moving, bending-resistant, inextensible interface and is tracked by a level set method. The vesicle is placed in a fluid containing a spatially varying sugar concentration field. The glass-forming nature is modelled through a concentration-dependent diffusivity and viscosity. It is found that there are optimal regimes for the values of the osmotic flow parameter and of the concentration dependence of the diffusivity to limit water trapping in the vesicle. Furthermore, it is found that the concentration dependencies of the diffusivity and viscosity can have profound effects on membrane deformations, which may have significant implications for vesicle damage during the desiccation process.

Thermostability of biological systems: fundamentals, challenges, and quantification.

This review examines the fundamentals and challenges in engineering/understanding the thermostability of biological systems over a wide temperature range (from the cryogenic to hyperthermic regimen). Applications of the bio-thermostability engineering to either destroy unwanted or stabilize useful biologicals for the treatment of diseases in modern medicine are first introduced. Studies on the biological responses to cryogenic and hyperthermic temperatures for the various applications are reviewed to understand the mechanism of thermal (both cryo and hyperthermic) injury and its quantification at the molecular, cellular and tissue/organ levels. Methods for quantifying the thermophysical processes of the various applications are then summarized accounting for the effect of blood perfusion, metabolism, water transport across cell plasma membrane, and phase transition (both equilibrium and non-equilibrium such as ice formation and glass transition) of water. The review concludes with a summary of the status quo and future perspectives in engineering the thermostability of biological systems.