Cell sheet(CS)-based approaches hold significant potential for tissue regeneration,relying on the extracellular matrix(ECM)for success.Like in native tissues,the ECM provides structural and biochemical support for cel...Cell sheet(CS)-based approaches hold significant potential for tissue regeneration,relying on the extracellular matrix(ECM)for success.Like in native tissues,the ECM provides structural and biochemical support for cellular homeostasis and function.Effective preservation strategies that maintain ECM integrity are critical to enhance the therapeutic potential of CS-based approaches.While cryogenic and hypothermic preservation methods offer potential solutions,their impact on CS ECM structure is not fully understood.Therefore,a comprehensive analysis of the ECM of hASCs CS following cryogenic and hypothermic preservation for 3 and 7 days,was conducted.Although proteomic analysis indicated that cryopreservation had no significant effect on the overall composition of the ECM,it induced significant ECM structural alterations,particularly disrupting collagen organization,which was not observed following hypothermic preservation.These structural changes were accompanied by alterations in mechanical properties,including a reduction in elastic modulus.In contrast,hypothermic preservation maintained ECM integrity and mechanical properties similar to the control.The notable ECM structural changes following cryogenic preservation can potentially impact cellular behavior,including adhesion,proliferation,and differentiation,thereby affecting the efficacy of CS therapies in vivo.This suggests that hypothermia may offer a promising alternative to cryopreservation for preserving CS integrity and functionality.展开更多
The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide.Tissue engineering offers a potential solution to address these shortages,but several challen...The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide.Tissue engineering offers a potential solution to address these shortages,but several challenges remain,with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products.Concurrently,a different challenge hindering the clinical implementation of such products,regards their efficient preservation from the fabrication site to the bedside.Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies.Its impact on prevascularization,however,has not been well studied.In this work,3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4◦C using Hypothermosol or basal culture media(α-MEM).Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation.In contrast,the use ofα-MEM led to a clear reduction in prevascular structures,with concurrent induction of high levels of apoptosis and autophagy at the cellular level.In vivo evaluation using a chorioallantoic membrane model demonstrated that,in opposition toα-MEM,Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue.These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization,in order to validate and streamline their clinical application.展开更多
基金funded by EU Horizon 2020 research and innovation program under the European Research Council(ERC)grant CapBed(805411)the Portuguese Science and Technology Foundation(FCT)UIDB/04539/2020,UIDP/04539/2020+1 种基金The National Mass Spectrometry Network(POCI-01–0145-FEDER-402–022125 Ref.ROTEIRO/0028/2013)SFR acknowledges doctoral fellowship PD/BD/135252/2017 and RAP acknowledges individual grant CEECIND/05623/2022.
文摘Cell sheet(CS)-based approaches hold significant potential for tissue regeneration,relying on the extracellular matrix(ECM)for success.Like in native tissues,the ECM provides structural and biochemical support for cellular homeostasis and function.Effective preservation strategies that maintain ECM integrity are critical to enhance the therapeutic potential of CS-based approaches.While cryogenic and hypothermic preservation methods offer potential solutions,their impact on CS ECM structure is not fully understood.Therefore,a comprehensive analysis of the ECM of hASCs CS following cryogenic and hypothermic preservation for 3 and 7 days,was conducted.Although proteomic analysis indicated that cryopreservation had no significant effect on the overall composition of the ECM,it induced significant ECM structural alterations,particularly disrupting collagen organization,which was not observed following hypothermic preservation.These structural changes were accompanied by alterations in mechanical properties,including a reduction in elastic modulus.In contrast,hypothermic preservation maintained ECM integrity and mechanical properties similar to the control.The notable ECM structural changes following cryogenic preservation can potentially impact cellular behavior,including adhesion,proliferation,and differentiation,thereby affecting the efficacy of CS therapies in vivo.This suggests that hypothermia may offer a promising alternative to cryopreservation for preserving CS integrity and functionality.
基金funded by EU Horizon 2020 research and innovation program under the ERC grant CapBed (805411)national funds+1 种基金trough the Portuguese Foundation for Science and Technology (FCT)project UIDB/50026/2020 and UIDP/50026/2020SFR acknowledges doctoral fellowship PD/BD/135252/2017,RPP individual grant IF/00347/2015,BSM individual grant DL 57/2016,LdS Scientific Employment Stimulus-Individual Call (CEEC Individual)2020.01541.CEECIND/CP1600/CT0024。
文摘The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide.Tissue engineering offers a potential solution to address these shortages,but several challenges remain,with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products.Concurrently,a different challenge hindering the clinical implementation of such products,regards their efficient preservation from the fabrication site to the bedside.Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies.Its impact on prevascularization,however,has not been well studied.In this work,3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4◦C using Hypothermosol or basal culture media(α-MEM).Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation.In contrast,the use ofα-MEM led to a clear reduction in prevascular structures,with concurrent induction of high levels of apoptosis and autophagy at the cellular level.In vivo evaluation using a chorioallantoic membrane model demonstrated that,in opposition toα-MEM,Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue.These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization,in order to validate and streamline their clinical application.
