In recent years,a number of studies have printed cell aggregates instead of 3D tissue scaffolds to create vascularized functional tissue,as scaffolding material inhibits cell-cell interactions and causes various complexities including inflammation,cytotoxicity,and tissue the cell sheet technique,tissue-specific cells are cultured on a temperature-responsive material to create a cell-ECM form vascularized tissue,a single monolayer sheet of ECs can be stacked with cell sheets of interest in a sandwich fashion or vascular cells can be co-cultured with tissue-specific cells to form vascularized multiple cell an illustration,Fig.9 shows an overall experimental scheme for fabricating an hMSC-based scaffold-free tissue engineered blood general,cell sheets can be attached and detached from a temperature-responsive culture dish incubating at either a higher(37 °C)or lower temperature(<25 °C).Nanometer-scale coating of poly(N-isopropylacrylamide)on polystyrene tissue culture surfaces facilitates attachment and detachment of endothelial cells and hepatocytes by shifting from hydrophobic to hydrophilic conditions at temperatures below 25°C[103].Vascularized cell sheets prepared in this way can be compiled to form thick instance,triple-layered cell sheets were produced from coculture of endothelial and cardiac cells overlaid on a resected tissue containing a vascular bed and perfused in a 3 days of perfusion culture,ECs formed luminal capillaries throughout the cardiac sheet upon connection with the vascular bed.The triple-layered vascularized cardiac tissues prepared in this way could beat and were increased tissue thickness,a six-layered cell sheet was prepared by compiling either two triple-layered or six single-layered cell sheets together and then the stack overlaid on the vascular bed.The combined two-triple layered sheets created thicker cardiac tissues with greater cell density compared to the compiled six-layer sheets due to improved cell viability and vascularization [104].Further,functional and vascularized tissue was obtained with 12 stacked cell sheets,indicating the efficacy of this approach to create vascularized thick tissue in transplantation of thin(～ 80 μm)cardiac cell sheets in vivo has also been found effective with respect to developing thick(～1 mm)vascularized one study,cell sheets grown from neonatal rat cardiomyocytes were repeatedly transplanted into rats at 1-,2-,or 3-day poly-surgical approach created a thick(～1mm),well-vascularized,and perfused myocardium tissue[105].Although vascularized tissue formation is possible with cell sheets,poor mechanical strength and tissue integrity of stacked cell sheets are major challenges that require further research.

pre-vascularization

A macro-scale tissue engineered construct containing a large cell population requires a sufficient supply of nutrients,oxygen gas,and biomolecules to maintain the metabolic activity,viability,and proliferation of embedded/seeded cells.In vitro,tissue scaffolds featuring a microfluidic network can be perfused with culture media to supply the necessary elements to the large cell ,the situation becomes complicated when scaffolds are implanted in vascular networks develop in the tissue construct by angiogenesis and vasculogenesis mechanisms that take time delay can cause ischemia in the large embedded cell population in a scaffold,and reduce cell viability by triggering apoptosis or address this issue,researchers have incorporated a microfluidic network into the tissue graft and then formed a monolayer of ECs by seeding(Fig.10).Such microfluidic network-embedded grafts were then sutured with host arteries/veins during in vivo thrombopoiesis-induced restenosis is often seen in tiny blood vessels,nutrient supply throughout the scaffold might be hindered due to the blockage of the microfluidic network in ,different studies have adapted several techniques to prevascularize the engineered construct,and then seed the graft with multiple cell types.In particular,scaffolds loaded with microvessel fragments,angiogenic factors,or vascular cells(ECs,SMCs)have been cultured in vitro or in vivo( loop)to form vascular networks prior to tissue-specific cell seeding[107,108].The prevascularized grafts take less time to inosculate with host vasculature,and thus support tissue growth,modeling,and example,when prevascularized collagen grafts seeded with fibroblasts,keratinocytes,and ECs were transplantated into mice,it took only 4 days to anastomose with host contrast,non-prevascularized scaffolds took 14 days to perfuse with native blood vessels[109].In a coculture system,prevascularization of tissue grafts largely depends on media composition,cell seeding technique,and ratio of multiple cell types.In a multiculture system,tri-culture of myoblasts, fibroblasts,and endothelial cells at certain ratios enhances capillaries compared to co-culture of myoblasts and endothelial cells after 4 weeks of culture[110].Furthermore,the tri-culture graft incubated with VEGF grew more blood vessels compared to those incubated with PDGF after 2 weeks of in vitro these cell-incorporated scaffolds achieved significant success in terms of prevascularization,formation of rapid,dense,mature,and functional capillary beds remains from in vitro prevascularization,scaffolds incorporating microvessel fragments have been used as prevascularized fact,these ECM matrices contain tissue-specific bio-chemical and topographic cues that regulate numerous cell-functions to form functional capillary collagen gel containing microvessel fragments in immunocompromised mice formed neovessels with lumen by day 11 and a mature functional microvascular bed by day 28[111].Although promising,allogeneic or xenogeneic microvessel fragments can cause immunological complexities in the host body.The success of in vitro prevascularization largely depends on the artificial physiological conditions applied during tissue number of studies maintained dynamic culture conditions including pulsation,variable flow rate,and dynamic pressure to form vascular example,cyclic mechanical strain and stress for 8 weeks promoted the proliferation,alignment,and collagen production of rabbit aortic SMCs seeded onto poly(L-lactide-co-caprolactone)(PLCL)scaffolds[112];dynamic sequential seeding of aortic SMCs and ECs onto poly(glycolic acid)(PGA)scaffolds and biomechanical stimulation for 25 days enhanced capillary formation and ECM deposition[113];and a controlled hypoxic environment influenced cells to secrete VEGF,which promoted vascularization in vitro[114].However,modulation of oxygen concentration in a co-culture system can alter the differentiation of stem cells into different lineages[115].Moreover,bidirectional flow in the biaxial bioreactor of a co-culture system(e.g.EPCs and MSCs)significantly reduces hypoxia-induced VEGF expression by eliminating the oxygen ,blood vessel formation is hindered in a bidirectional flow system compared to static culture and unidirectional flow systems[116].

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