Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a p...Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.展开更多
BACKGROUND: The bioartificial liver (BAL) is considered a possible alternative method for treating liver failure. The core of the BAL system is culturing liver cells in vitro with high density and activity. Microcarri...BACKGROUND: The bioartificial liver (BAL) is considered a possible alternative method for treating liver failure. The core of the BAL system is culturing liver cells in vitro with high density and activity. Microcarrier culture is a mode of high-density culture. We set out to prepare a novel porous microcarrier to improve the activity of liver cells in vitro. METHODS: Chitosan was used to prepare a novel porous spherical microcarrier with interconnected structure. The chitosan porous microcarriers (CPMs) were modified with gelatin to improve their biocompatibility. CPMs were co-cultured with liver cells, HL-7702 (L-02), to evaluate their effect on cell culture. RESULTS: The average size of the CPMs was about 400 μm in diameter and their apertures were less than 30 μm. The pores of the microcarrier were interconnected. After fixation by sodium tripolyphosphate, the structure of the first freeze-dried CPMs was stable. To further improve the biocompatibility, the surface of CPMs was modified with gelatin through chemical crosslinking (GM-CPMs). Comparing the proliferation curves of L-02 cells cultured on simple CPMs, GM-CPMs and tissue culture polystyrene (TCPS, a mode of planar cell culture), the proliferation rates were similar in the first 5 days and the cells proliferated until day 8 in culture with microcarriers. The OD value of liver cells cultured on GM-CPMs was 1.97-fold higher than that on TCPS culture at day 8. Levels of urea and albumin in supernatants of cells cultured on GM-CPMs increased steadily for 8 days, and were clearly higher than those of cells cultured on TCPS (P<0.05).CONCLUSIONS: The novel CPMs were promising microcarriers for hepatocyte culture and the GM-CPM seemed better. Porous microcarrier culture was beneficial for hepatocyte function and activity.展开更多
AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion...AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion with collagenase.In culture onmicrocarriers,primary porcine hepatocyteswere inoculated at a concentration of 5×10~7/mLinto the static culture systems containing 2 g/LCytodex-3,then supplemented with 100 mL/Lfetal calf serum(FCS)or 100 mL/L porcineportal vein serum(PPVS)respectively.Inspheroidal aggregate culture hepatocytes wereinoculated into 100 mL siliconized flasks at aconcentration of 5.0×10~6/mL.RESULTS In culture on microcarriershepatocytes tended to aggregate on Cytodex-3obviously after being inoculated.Typical multi-cellular aggregated spheroids could be found inthe two systems 24 h-48 h after hepatocyteswere cultured.The morphological charact-eristics and synthetic functions were maintainedfor 5 wk in FCS culture system and 8 wk in PPVSculture system.In spheroidal aggregate cultureabout 80%-90% isolated hepatocytes becameaggregated spheroids 24h after cultured insuspension and mean diameter of the spheroidswas 100μm.The relationship among thehepatocytes resembled that in the liver in vivo.Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytescultured on monolayers.CONCLUSION As high-yields and high-activitymodes of culture on microcarriers or inspheroidal aggregate culture with portal veinserum are promising to provide biomaterials forbioartificial liver(BAL)efficiently.展开更多
objective: To cultivate human liver cell line (CL-1) on microcarriers and study the synthetic and transformational function of this culture system. Methods:CL-1 were cultivated on Cytodex-3 microcarriers. The cell gro...objective: To cultivate human liver cell line (CL-1) on microcarriers and study the synthetic and transformational function of this culture system. Methods:CL-1 were cultivated on Cytodex-3 microcarriers. The cell growth was kinetically inspected with light microscope and scanning electronic microscope on the lst, 3rd, 5th, 7th, 9th day, and the amount of diazepam transformation and albumin synthesis were deter mined at the same time. Results:On 7th day after inoculating, the CL-1 cell density could reach 2. 16 ×106/ ml ; the amount of diazepam trans formation was 619 μg and albumin synthesis 78. 23 μg. Conclusion:CL-1 can be cultivated to a high density on microcarriers and has hepatic specific biotransformation and biosynthesis functions. So the culture system may be further studied for being used as the biomaterial of bioartificial liver.展开更多
The fact of microcarrier (MC) culture introduces new possibilities and makes possible the practical high-yield culture of anchorage-dependent cells has generated a considerable focus in this study. The objective of th...The fact of microcarrier (MC) culture introduces new possibilities and makes possible the practical high-yield culture of anchorage-dependent cells has generated a considerable focus in this study. The objective of this research was to study the comparison of Vero cell growth on different types of commercial microcarriers;Cytodex-1, Cytodex-3, Hil-lex? II and Plastic Plus in spinner vessel and two liters bioreactor cultured for 96 hours. Biological performance of the microcarrier in RPMI media showed the preference of Vero cell grew on Cytodex 3 microcarriers with highest maximum viable cell number (2.4 × 105 cells/ml) followed by Cytodex 1, Hillex and Plustic Plus. Vero cell on Cyto-dex-3 data in spinner flask was compared in bioreactor and result showed higher viable cell number in biorector. Thus, this dextran-crosslink gelatin microcarrier (Cytodex 3) provided the best surface for cell attachment and fast proliferation. At the end of this cell growth improvement will be used for virus transfection producing a vaccine in bioreactor.展开更多
The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate ...The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 10<sup>7</sup> FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.展开更多
Stem cell therapies have made great progress in the treatment of diabetic wounds during recent decades,while their short in vivo residence,alloimmune reactions,undesired behaviors,and dramatic losses of cell functions...Stem cell therapies have made great progress in the treatment of diabetic wounds during recent decades,while their short in vivo residence,alloimmune reactions,undesired behaviors,and dramatic losses of cell functions still hinder the translation of them into clinic.Here,inspired by the natural components of stem cell niches,we presented novel microfluidic hydrogel microcarriers with extracellular matrix(ECM)-like composition and adipose-derived stem cells(ADSCs)encapsulation for diabetic wound healing.As the hydrogel was synthesized by conjugating hyaluronic acid methacryloyl(HAMA)onto the Fibronectin(FN)molecule chain(FN-HAMA),the laden ADSCs in the microcarriers showed improved bioactivities and pro-regenerative capabilities.Based on these features,we have demonstrated that these ADSCs microcarriers exhibited significant promotion of neovascularization,follicular rejuvenation,and collagen deposition in a mouse diabetic wound model.These results indicated that the stem cell niche-inspired FN-HAMA microcarriers with ADSCs encapsulation have great clinical potential for diabetic wound treatment.展开更多
Bacterial infection causes wound inflammation and makes angiogenesis difficult.It is urgent to develop effectively antibacterial and pro-vascularizing dressings for wound healing.The hydrogel is developed with pH-resp...Bacterial infection causes wound inflammation and makes angiogenesis difficult.It is urgent to develop effectively antibacterial and pro-vascularizing dressings for wound healing.The hydrogel is developed with pH-responsive drug-releasing microcarriers which were loaded with vascular endothelial growth factor(VEGF)that promotes angiogenesis and actively respond to wound pH for control and prolong VEGF release.The surfaces of the microcarriers were coated with polydopamine which can reduce the silver nanoparticles(AgNPs)in situ,and dynamically crosslink with the polyacrylamide,which forms a stable slow-release system with different release behavior for the VEGF and AgNPs.The hydrogel inhib-ited bacterial formation and accelerated wound healing.With the hydrogel dressing,83.3%±4.29%of the wound heals at day 7,which is 40.9%±8.5%higher than the non-treatment group in defect infected model.The antibacterial properties of hydrogel down-regulate early inflammation-related cytokines,and the release of VEGF in the middle and late phases of wound healing in response to pH changes pro-motes angiogenesis and up-regulate the expression of angiogenesis-associated cytokine.The sequential release of antibacterial agents and pro-vascularizing agents in response to the change in wound microen-vironmental cues facilitate temporally controlled therapy that suites the need of different wound healing phases.Collectively,the hydrogel loaded with multifunctional microcarriers that enable controlled release of AgNPs and VEGF is an effective system for treating infected wounds.展开更多
Mechanical force is crucial in the whole process of embryonic development.However,the role of trophoblast mechanics during embryo implantation has rarely been studied.In this study,we constructed a model to explore th...Mechanical force is crucial in the whole process of embryonic development.However,the role of trophoblast mechanics during embryo implantation has rarely been studied.In this study,we constructed a model to explore the effect of stiffness changes in mouse trophoblast stem cells(mTSCs)on implantation:microcarrier was prepared by sodium alginate using a droplet microfluidics system,and mTSCs were attached to the microcarrier surface with laminin modifications,called T(micro).Compared with the spheroid,formed by the self-assembly of mTSCs(T(sph)),we could regulate the stiffness of the microcarrier,making the Young’s modulus of mTSCs(367.70±79.81 Pa)similar to that of the blastocyst trophoblast ectoderm(432.49±151.90 Pa).Moreover,T(micro)contributes to improve the adhesion rate,expansion area and invasion depth of mTSCs.Further,T(micro)was highly expressed in tissue migration-related genes due to the activation of the Rho-associated coiled-coil containing protein kinase(ROCK)pathway at relatively similar modulus of trophoblast.Overall,our study explores the embryo implantation process with a new perspective,and provides theoretical support for understanding the effect of mechanics on embryo implantation.展开更多
Microcarriers have attracted increasing interests in drug delivery. In order to develop this technique, it is prone to focus on the generation of functional particles through using simple approaches and novel but acce...Microcarriers have attracted increasing interests in drug delivery. In order to develop this technique, it is prone to focus on the generation of functional particles through using simple approaches and novel but accessible materials. Here, inspired by the formation mechanism of tofu that through the mixing of soymilk and brine for cross-linking soybean proteins, we present novel soybean protein microcarriers by using microfluidic generation approach for drug delivery. Since the soybean protein droplets are generated by microfluidic emulsification method, the tofu microparticles present highly monodisperse and homogeneous morphologies. Because of the excellent biocompatibility of the soybean protein and the interconnected porous structures throughout the whole microparticles after freeze-drying, various kinds of drugs and active molecules could be absorbed and loaded in the microcarriers, which makes them versatile for drug delivery. It can be anticipated that the microfluidic-generated tofu microcarriers will have great potential in the biomedical field.展开更多
Successful regeneration of cartilage tissue at a clinical scale has been a tremendous challenge in the past decades. Microcarriers (MCs), usually used for cell and drug delivery, have been studied broadly across a wid...Successful regeneration of cartilage tissue at a clinical scale has been a tremendous challenge in the past decades. Microcarriers (MCs), usually used for cell and drug delivery, have been studied broadly across a wide range of medical fields, especially the cartilage tissue engineering (TE). Notably, microcarrier systems provide an attractive method for regulating cell phenotype and microtissue maturations, they also serve as powerful injectable carriers and are combined with new technologies for cartilage regeneration. In this review, we introduced the typical methods to fabricate various types of microcarriers and discussed the appropriate ma-terials for microcarriers. Furthermore, we highlighted recent progress of applications and general design prin-ciple for microcarriers. Finally, we summarized the current challenges and promising prospects of microcarrier-based systems for medical applications. Overall, this review provides comprehensive and systematic guidelines for the rational design and applications of microcarriers in cartilage TE.展开更多
By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferati...By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesion and spreading stage.展开更多
Due to a worldwide focus on sustainable materials for human health and economy services, more and more natural renewable biomass are regarded as promising materials that could replace synthetic polymers and reduce glo...Due to a worldwide focus on sustainable materials for human health and economy services, more and more natural renewable biomass are regarded as promising materials that could replace synthetic polymers and reduce global dependence on petroleum resources. Cellulose is known as the most abundant renewable polymer in nature, varieties of cellulose-based products have been developed and have gained growing interest in recent years. In this review, a kind of water-soluble cellulose derivative, i.e., sodium cellulose sulfate (NaCS) is introduced. Details about NaCS's physicochemical properties like solubility, biocompatibility, biodegradability, degree of substitution, etc. are systematically elaborated. And promising applications of NaCS used as biomaterials for microcarriers' designing, such as microcell- carriers, micro-drug-carriers, etc., are presented.展开更多
Reusable microcarriers with appropriate surface topography,mechanical properties,as well as biological modification through decellularization facilitating repeated cell culture are crucial for tissue engineering appli...Reusable microcarriers with appropriate surface topography,mechanical properties,as well as biological modification through decellularization facilitating repeated cell culture are crucial for tissue engineering applications.Herein,we report the preparation of topological polyetheretherketone(PEEK)microcarriers via gas-driven and solvent exchange method followed by hydrothermal treatment at high temperature and pressure.After hydrothermal treated for 8 h,the resulting topological PEEK microcarriers exhibit walnut-like surface topography and good sphericity as well as uniform size distribution of 350.24619.44 mm.And the average width between ravine-patterned surface of PEEK microcarriers is 7806290 nm.After repeated steam sterilization by autoclaving for three times,topological PEEK microcarriers show nearly identical results compared with previous ones indicating strong tolerance to high temperature and pressure.This is a unique advantage for large-scale cell expansion and clinical applications.Moreover,PEEK microcarriers with special topography possess higher protein adsorption efficiency.In addition,the reutilization and biofunctionalization with repeated decellularization of topological PEEK microcarriers show highly beneficial for cell adhesion and proliferation.Therefore,our study is of great importance for new generation microcarriers with micro-and nano-scale surface feature for a broad application prospect in tissue engineering.展开更多
Porous microcarriers have aroused increasing attention recently by facilitating oxygen and nutrient transfer,supporting cell attachment and growth with sufficient cell seeding density.In this study,porous polyethereth...Porous microcarriers have aroused increasing attention recently by facilitating oxygen and nutrient transfer,supporting cell attachment and growth with sufficient cell seeding density.In this study,porous polyetheretherketone(PEEK)microcarriers coated with mineralized extracellular matrix(mECM),known for their chemical,mechanical and biological superiority,were developed for orthopedic applications.Porous PEEK microcarriers were derived from smooth microcarriers using a simple wet-chemistry strategy involving the reduction of carbonyl groups.This treatment simultaneously modified surface topology and chemical composition.Furthermore,the microstructure,protein absorption,cytotoxicity and bioactivity of the obtained porous microcarriers were investigated.The deposition of mECM through repeated recellularization and decellularization on the surface of porous MCs further promoted cell proliferation and osteogenic activity.Additionally,the mECM coated porous microcarriers exhibited excellent bone regeneration in a rat calvarial defect repair model in vivo,suggesting huge potential applications in bone tissue engineering.展开更多
Traditional monolayer culture is still the common method used in hepatocarcinoma cell culture in vitro. For lack of the structure similar to that in vivo, it is disadvantageous in the research of the relation between ...Traditional monolayer culture is still the common method used in hepatocarcinoma cell culture in vitro. For lack of the structure similar to that in vivo, it is disadvantageous in the research of the relation between structure and function. We established a three-dimensional (3-D) culture model of human hepatocarcinoma cell (BEL-7402) in vitro by using microcarrier cytodex-3 in static condition. The results of SEM, TEM, enzyme activity and flow cytometry indicated that the cells were polygonal-shaped and arranged in multilayers. Intercellular space was 0.5-2.0 (Am wide where lots of microvilli could be seen. Adjacent cells were connected with desmosomes and localized membrane projects. More than 90% of the cells were viable and maintained the consumption of glucose and the expression of EGF receptor. The intracellular ALT, AST and LDH-L activities were higher in 3-D culture than those of monolayer culture. Compared with monolayer culture, this 3-D culture with the structure similar to trabecular展开更多
Cell transplantation is an effective strategy to improve the repair effect of nerve guide conduits(NGCs).However,problems such as low loading efficiency and cell anoikis undermine the outcomes.Microcarriers are effici...Cell transplantation is an effective strategy to improve the repair effect of nerve guide conduits(NGCs).However,problems such as low loading efficiency and cell anoikis undermine the outcomes.Microcarriers are efficient 3D cell culture scaffolds,which can also prevent cell anoikis by providing substrate for adhesion during transplantation.Here,we demonstrate for the first time microcarrier-based cell transplantation in peripheral nerve repair.We first prepared macroporous chitosan microcarriers(CSMCs)by the emulsion-phase separation method,and then decorated the CSMCs with polylysine(pl-CSMCs)to improve cell affinity.We then loaded the pl-CSMCs with adipose-derived stem cells(ADSCs)and injected them into electrospun polycaprolactone/chitosan NGCs to repair rat sciatic nerve defects.The ADSCs-laden pl-CSMCs effectively improved nerve regeneration as demonstrated by evaluation of histology,motor function recovery,electrophysiology,and gastrocnemius recovery.With efficient cell transplantation,convenient operation,and the multiple merits of ADSCs,the ADSCs-laden pl-CSMCs hold good potential in peripheral nerve repair.展开更多
基金supported by the National Key Research and Development Program of China,Nos.2017YFE0122900(to BH),2019YFA0110800(to WL),2019YFA0903802(to YW),2021YFA1101604(to LW),2018YFA0108502(to LF),and 2020YFA0804003(to JW)the National Natural Science Foundation of China,Nos.31621004(to WL,BH)and 31970821(to YW)+1 种基金CAS Project for Young Scientists in Basic Research,No.YSBR-041(to YW)Joint Funds of the National Natural Science Foundation of China,No.U21A20396(to BH)。
文摘Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.
基金supported by grants from the National Natural Science Foundation of China (30672043, 30772105)the National 863 program of China (2008AA02Z417)
文摘BACKGROUND: The bioartificial liver (BAL) is considered a possible alternative method for treating liver failure. The core of the BAL system is culturing liver cells in vitro with high density and activity. Microcarrier culture is a mode of high-density culture. We set out to prepare a novel porous microcarrier to improve the activity of liver cells in vitro. METHODS: Chitosan was used to prepare a novel porous spherical microcarrier with interconnected structure. The chitosan porous microcarriers (CPMs) were modified with gelatin to improve their biocompatibility. CPMs were co-cultured with liver cells, HL-7702 (L-02), to evaluate their effect on cell culture. RESULTS: The average size of the CPMs was about 400 μm in diameter and their apertures were less than 30 μm. The pores of the microcarrier were interconnected. After fixation by sodium tripolyphosphate, the structure of the first freeze-dried CPMs was stable. To further improve the biocompatibility, the surface of CPMs was modified with gelatin through chemical crosslinking (GM-CPMs). Comparing the proliferation curves of L-02 cells cultured on simple CPMs, GM-CPMs and tissue culture polystyrene (TCPS, a mode of planar cell culture), the proliferation rates were similar in the first 5 days and the cells proliferated until day 8 in culture with microcarriers. The OD value of liver cells cultured on GM-CPMs was 1.97-fold higher than that on TCPS culture at day 8. Levels of urea and albumin in supernatants of cells cultured on GM-CPMs increased steadily for 8 days, and were clearly higher than those of cells cultured on TCPS (P<0.05).CONCLUSIONS: The novel CPMs were promising microcarriers for hepatocyte culture and the GM-CPM seemed better. Porous microcarrier culture was beneficial for hepatocyte function and activity.
基金the National Natural Science Foundation of China,No.39570212
文摘AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion with collagenase.In culture onmicrocarriers,primary porcine hepatocyteswere inoculated at a concentration of 5×10~7/mLinto the static culture systems containing 2 g/LCytodex-3,then supplemented with 100 mL/Lfetal calf serum(FCS)or 100 mL/L porcineportal vein serum(PPVS)respectively.Inspheroidal aggregate culture hepatocytes wereinoculated into 100 mL siliconized flasks at aconcentration of 5.0×10~6/mL.RESULTS In culture on microcarriershepatocytes tended to aggregate on Cytodex-3obviously after being inoculated.Typical multi-cellular aggregated spheroids could be found inthe two systems 24 h-48 h after hepatocyteswere cultured.The morphological charact-eristics and synthetic functions were maintainedfor 5 wk in FCS culture system and 8 wk in PPVSculture system.In spheroidal aggregate cultureabout 80%-90% isolated hepatocytes becameaggregated spheroids 24h after cultured insuspension and mean diameter of the spheroidswas 100μm.The relationship among thehepatocytes resembled that in the liver in vivo.Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytescultured on monolayers.CONCLUSION As high-yields and high-activitymodes of culture on microcarriers or inspheroidal aggregate culture with portal veinserum are promising to provide biomaterials forbioartificial liver(BAL)efficiently.
文摘objective: To cultivate human liver cell line (CL-1) on microcarriers and study the synthetic and transformational function of this culture system. Methods:CL-1 were cultivated on Cytodex-3 microcarriers. The cell growth was kinetically inspected with light microscope and scanning electronic microscope on the lst, 3rd, 5th, 7th, 9th day, and the amount of diazepam transformation and albumin synthesis were deter mined at the same time. Results:On 7th day after inoculating, the CL-1 cell density could reach 2. 16 ×106/ ml ; the amount of diazepam trans formation was 619 μg and albumin synthesis 78. 23 μg. Conclusion:CL-1 can be cultivated to a high density on microcarriers and has hepatic specific biotransformation and biosynthesis functions. So the culture system may be further studied for being used as the biomaterial of bioartificial liver.
文摘The fact of microcarrier (MC) culture introduces new possibilities and makes possible the practical high-yield culture of anchorage-dependent cells has generated a considerable focus in this study. The objective of this research was to study the comparison of Vero cell growth on different types of commercial microcarriers;Cytodex-1, Cytodex-3, Hil-lex? II and Plastic Plus in spinner vessel and two liters bioreactor cultured for 96 hours. Biological performance of the microcarrier in RPMI media showed the preference of Vero cell grew on Cytodex 3 microcarriers with highest maximum viable cell number (2.4 × 105 cells/ml) followed by Cytodex 1, Hillex and Plustic Plus. Vero cell on Cyto-dex-3 data in spinner flask was compared in bioreactor and result showed higher viable cell number in biorector. Thus, this dextran-crosslink gelatin microcarrier (Cytodex 3) provided the best surface for cell attachment and fast proliferation. At the end of this cell growth improvement will be used for virus transfection producing a vaccine in bioreactor.
文摘The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 10<sup>7</sup> FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.
基金supported by the National Key Research and Development Program of China(2020YFA0908200)the National Natural Science Foundation of China(81974288,T2225003 and 52073060)+2 种基金the Strategic Priority Research Program of the Chinese Academy of Science(XDA16021103)the Guangdong Basic and Applied Basic Research Foundation(2021B1515120054)the Shenzhen Fundamental Research Program(JCYJ20190813152616459 and JCYJ20210324133214038).
文摘Stem cell therapies have made great progress in the treatment of diabetic wounds during recent decades,while their short in vivo residence,alloimmune reactions,undesired behaviors,and dramatic losses of cell functions still hinder the translation of them into clinic.Here,inspired by the natural components of stem cell niches,we presented novel microfluidic hydrogel microcarriers with extracellular matrix(ECM)-like composition and adipose-derived stem cells(ADSCs)encapsulation for diabetic wound healing.As the hydrogel was synthesized by conjugating hyaluronic acid methacryloyl(HAMA)onto the Fibronectin(FN)molecule chain(FN-HAMA),the laden ADSCs in the microcarriers showed improved bioactivities and pro-regenerative capabilities.Based on these features,we have demonstrated that these ADSCs microcarriers exhibited significant promotion of neovascularization,follicular rejuvenation,and collagen deposition in a mouse diabetic wound model.These results indicated that the stem cell niche-inspired FN-HAMA microcarriers with ADSCs encapsulation have great clinical potential for diabetic wound treatment.
文摘Bacterial infection causes wound inflammation and makes angiogenesis difficult.It is urgent to develop effectively antibacterial and pro-vascularizing dressings for wound healing.The hydrogel is developed with pH-responsive drug-releasing microcarriers which were loaded with vascular endothelial growth factor(VEGF)that promotes angiogenesis and actively respond to wound pH for control and prolong VEGF release.The surfaces of the microcarriers were coated with polydopamine which can reduce the silver nanoparticles(AgNPs)in situ,and dynamically crosslink with the polyacrylamide,which forms a stable slow-release system with different release behavior for the VEGF and AgNPs.The hydrogel inhib-ited bacterial formation and accelerated wound healing.With the hydrogel dressing,83.3%±4.29%of the wound heals at day 7,which is 40.9%±8.5%higher than the non-treatment group in defect infected model.The antibacterial properties of hydrogel down-regulate early inflammation-related cytokines,and the release of VEGF in the middle and late phases of wound healing in response to pH changes pro-motes angiogenesis and up-regulate the expression of angiogenesis-associated cytokine.The sequential release of antibacterial agents and pro-vascularizing agents in response to the change in wound microen-vironmental cues facilitate temporally controlled therapy that suites the need of different wound healing phases.Collectively,the hydrogel loaded with multifunctional microcarriers that enable controlled release of AgNPs and VEGF is an effective system for treating infected wounds.
基金supported by National Natural Science Foundation of China(T2222029 and U21A20396)Strategic Priority Research Program of Chinese Academy of Sciences(XDA16020802)+1 种基金CAS Project for Young Scientists in Basic Research(YSBR-012)CAS Engineering Laboratory for Intelligent Organ Manufacturing(KFJ-PTXM-039).
文摘Mechanical force is crucial in the whole process of embryonic development.However,the role of trophoblast mechanics during embryo implantation has rarely been studied.In this study,we constructed a model to explore the effect of stiffness changes in mouse trophoblast stem cells(mTSCs)on implantation:microcarrier was prepared by sodium alginate using a droplet microfluidics system,and mTSCs were attached to the microcarrier surface with laminin modifications,called T(micro).Compared with the spheroid,formed by the self-assembly of mTSCs(T(sph)),we could regulate the stiffness of the microcarrier,making the Young’s modulus of mTSCs(367.70±79.81 Pa)similar to that of the blastocyst trophoblast ectoderm(432.49±151.90 Pa).Moreover,T(micro)contributes to improve the adhesion rate,expansion area and invasion depth of mTSCs.Further,T(micro)was highly expressed in tissue migration-related genes due to the activation of the Rho-associated coiled-coil containing protein kinase(ROCK)pathway at relatively similar modulus of trophoblast.Overall,our study explores the embryo implantation process with a new perspective,and provides theoretical support for understanding the effect of mechanics on embryo implantation.
基金supported by the National Natural Science Foundation of China(21473029,51522302)the National Scholastic Athletics Foundation of China(U1530260)the Scientific Research Foundation of Southeast University
文摘Microcarriers have attracted increasing interests in drug delivery. In order to develop this technique, it is prone to focus on the generation of functional particles through using simple approaches and novel but accessible materials. Here, inspired by the formation mechanism of tofu that through the mixing of soymilk and brine for cross-linking soybean proteins, we present novel soybean protein microcarriers by using microfluidic generation approach for drug delivery. Since the soybean protein droplets are generated by microfluidic emulsification method, the tofu microparticles present highly monodisperse and homogeneous morphologies. Because of the excellent biocompatibility of the soybean protein and the interconnected porous structures throughout the whole microparticles after freeze-drying, various kinds of drugs and active molecules could be absorbed and loaded in the microcarriers, which makes them versatile for drug delivery. It can be anticipated that the microfluidic-generated tofu microcarriers will have great potential in the biomedical field.
基金The authors thank Shuo Wang and Shen Ji for helpful discussion.This work was supported by the National Natural Science Foundation of China(Grant No.81773091)the Natural Science Foundation of Beijing Municipality(Grant No.7212020)+4 种基金Science and Technology Planning Project of Beijing Municipal Education Commission(Grant No.KM202110025013)the Beijing Municipal Excellent Talents Project(Grant No.2020A43)Strategic Priority Research Program of Chinese Academy of Sciences(Grant No.XDA16020802)CAS Engineering Laboratory for Intelligent Organ Manufacturing(Grant No.KFJ-PTXM-039)the National Natural Science Foundation of China(Grant No.82001848).
文摘Successful regeneration of cartilage tissue at a clinical scale has been a tremendous challenge in the past decades. Microcarriers (MCs), usually used for cell and drug delivery, have been studied broadly across a wide range of medical fields, especially the cartilage tissue engineering (TE). Notably, microcarrier systems provide an attractive method for regulating cell phenotype and microtissue maturations, they also serve as powerful injectable carriers and are combined with new technologies for cartilage regeneration. In this review, we introduced the typical methods to fabricate various types of microcarriers and discussed the appropriate ma-terials for microcarriers. Furthermore, we highlighted recent progress of applications and general design prin-ciple for microcarriers. Finally, we summarized the current challenges and promising prospects of microcarrier-based systems for medical applications. Overall, this review provides comprehensive and systematic guidelines for the rational design and applications of microcarriers in cartilage TE.
文摘By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesion and spreading stage.
基金China Postdoctoral Science Foundation (No. 2017M611998)the National Natural Science Foundation of China (Grant Nos. 21606002 and 21576233)+2 种基金the Natural Science Foundation of Anhui Province (CN)(No. 1708085QC64)the Doctoral Research Start-up Fund of Anhui University (J01001319)the Undergraduate Research Training Programs for Innovation (Nos. KYXL2017036, 201710357034 and 201710357268).
文摘Due to a worldwide focus on sustainable materials for human health and economy services, more and more natural renewable biomass are regarded as promising materials that could replace synthetic polymers and reduce global dependence on petroleum resources. Cellulose is known as the most abundant renewable polymer in nature, varieties of cellulose-based products have been developed and have gained growing interest in recent years. In this review, a kind of water-soluble cellulose derivative, i.e., sodium cellulose sulfate (NaCS) is introduced. Details about NaCS's physicochemical properties like solubility, biocompatibility, biodegradability, degree of substitution, etc. are systematically elaborated. And promising applications of NaCS used as biomaterials for microcarriers' designing, such as microcell- carriers, micro-drug-carriers, etc., are presented.
基金This research was financially supported by the National Natural Science Foundation of China(Projects.51473164 and 51673186)the joint funded program of Chinese Academy of Sciences and Japan Society for the Promotion of Science(GJHZ1519)the Special Fund for Industrialization of Science and Technology Cooperation between Jilin Province and Chinese Academy of Sciences(2017SYHZ0021).
文摘Reusable microcarriers with appropriate surface topography,mechanical properties,as well as biological modification through decellularization facilitating repeated cell culture are crucial for tissue engineering applications.Herein,we report the preparation of topological polyetheretherketone(PEEK)microcarriers via gas-driven and solvent exchange method followed by hydrothermal treatment at high temperature and pressure.After hydrothermal treated for 8 h,the resulting topological PEEK microcarriers exhibit walnut-like surface topography and good sphericity as well as uniform size distribution of 350.24619.44 mm.And the average width between ravine-patterned surface of PEEK microcarriers is 7806290 nm.After repeated steam sterilization by autoclaving for three times,topological PEEK microcarriers show nearly identical results compared with previous ones indicating strong tolerance to high temperature and pressure.This is a unique advantage for large-scale cell expansion and clinical applications.Moreover,PEEK microcarriers with special topography possess higher protein adsorption efficiency.In addition,the reutilization and biofunctionalization with repeated decellularization of topological PEEK microcarriers show highly beneficial for cell adhesion and proliferation.Therefore,our study is of great importance for new generation microcarriers with micro-and nano-scale surface feature for a broad application prospect in tissue engineering.
文摘Porous microcarriers have aroused increasing attention recently by facilitating oxygen and nutrient transfer,supporting cell attachment and growth with sufficient cell seeding density.In this study,porous polyetheretherketone(PEEK)microcarriers coated with mineralized extracellular matrix(mECM),known for their chemical,mechanical and biological superiority,were developed for orthopedic applications.Porous PEEK microcarriers were derived from smooth microcarriers using a simple wet-chemistry strategy involving the reduction of carbonyl groups.This treatment simultaneously modified surface topology and chemical composition.Furthermore,the microstructure,protein absorption,cytotoxicity and bioactivity of the obtained porous microcarriers were investigated.The deposition of mECM through repeated recellularization and decellularization on the surface of porous MCs further promoted cell proliferation and osteogenic activity.Additionally,the mECM coated porous microcarriers exhibited excellent bone regeneration in a rat calvarial defect repair model in vivo,suggesting huge potential applications in bone tissue engineering.
基金This work was supported by the Space Cell Culture.
文摘Traditional monolayer culture is still the common method used in hepatocarcinoma cell culture in vitro. For lack of the structure similar to that in vivo, it is disadvantageous in the research of the relation between structure and function. We established a three-dimensional (3-D) culture model of human hepatocarcinoma cell (BEL-7402) in vitro by using microcarrier cytodex-3 in static condition. The results of SEM, TEM, enzyme activity and flow cytometry indicated that the cells were polygonal-shaped and arranged in multilayers. Intercellular space was 0.5-2.0 (Am wide where lots of microvilli could be seen. Adjacent cells were connected with desmosomes and localized membrane projects. More than 90% of the cells were viable and maintained the consumption of glucose and the expression of EGF receptor. The intracellular ALT, AST and LDH-L activities were higher in 3-D culture than those of monolayer culture. Compared with monolayer culture, this 3-D culture with the structure similar to trabecular
基金National Natural Science Foundation of China(Grant No.51773018,51973018)Key Research and Development Projects of People’s Liberation Army(BWS17J036).
文摘Cell transplantation is an effective strategy to improve the repair effect of nerve guide conduits(NGCs).However,problems such as low loading efficiency and cell anoikis undermine the outcomes.Microcarriers are efficient 3D cell culture scaffolds,which can also prevent cell anoikis by providing substrate for adhesion during transplantation.Here,we demonstrate for the first time microcarrier-based cell transplantation in peripheral nerve repair.We first prepared macroporous chitosan microcarriers(CSMCs)by the emulsion-phase separation method,and then decorated the CSMCs with polylysine(pl-CSMCs)to improve cell affinity.We then loaded the pl-CSMCs with adipose-derived stem cells(ADSCs)and injected them into electrospun polycaprolactone/chitosan NGCs to repair rat sciatic nerve defects.The ADSCs-laden pl-CSMCs effectively improved nerve regeneration as demonstrated by evaluation of histology,motor function recovery,electrophysiology,and gastrocnemius recovery.With efficient cell transplantation,convenient operation,and the multiple merits of ADSCs,the ADSCs-laden pl-CSMCs hold good potential in peripheral nerve repair.
