Preparation process of polylactic acid-glycolic acid copolymer/hydroxyapatite microcarrier by electrostatic spraying method and its effect evaluation / 吉林大学学报(医学版)

Objective: To investigate the preparation process of polylactic acid-glycolic acid copolymer/ hydroxyapatite (PLGA/HA) microcarriers by electrostatic spraying method, and to elucidate the superiority of PLGA/HA microcarriers. Methods: The PLGA/HA microcarriers were prepared by electrostatic spraying method using nano-HA (20 nm, 99.9%) and PLGA (LA/GA = 50/50, Mw30k), and the influence of different concentrations (1%, 3%, 5%) and different voltages (4. 0, 4. 5, > 5. 0 kV) of HA in the morphology of the microcarriers was investigated and the best ball making parameters were obtained. The PLGA microcarriers were used as PLGA group, PLGA+1%HA as 1% PLGA/HA group, PLGA + 3% HA as 3% PLGA/HA group, PLGA+ 5% HA as 5% PLGA/HA group, and the simple cells were used as blank control group. The characteristics of PLGA/HA microcarriers were detected by scanning electron microscope (SEM), cell proliferation test, cell fluorescence staining experiment, and Fourier transform infrared spectroscopy (FTIR). Results: The SEM results showed that the microcarrier particles were uniform, all of them were elliptical or circular, without abnormal shape spheres, with smooth surface, without sharp edges, adhesion between the spheres and aggregation, and there were no significant differences between the different concentrations of microcarriers. The cell proliferation test results showed that the order of adhesion cells was 5% PLGA/HA group > 3% PLGA/HA group > 1% PLGA/HA group > PLGA group > blank control group (P<0. 05); the number of cells was increased with the increasing of HA concentration; the microcarriers in 5% PLGA/HA group had the best cell affinity and the microcarriers had no cytotoxity. The cell fluorescence staining experiment showed that the MC3T3-E1 cells adhered well on the microcarriers. The FTIR analysis results showed that HA characteristic absorption peak was observed, indicating that the composite microcarrier contained PLGA and HA. Conclusion: The preparation process of PLGA/ HA microcarriers is successfully established by electrostatic spraying method. The method is simple and convenient to operate, and has excellent ball-making effect. It has broadly application prospects in bone tissue engineering.

Co-culture of Human Dental Pulp Stem Cells and Endothelial Cells Using Porous Biopolymer Microcarriers: A Feasibility Study for Bone Tissue Engineering

Delivery of stem cells with osteogenesis while enabling angiogenesis is important for vascularized bone tissue engineering. Here a three-dimensional (3D) co-culture system of dental pulp stem cells (DPSCs) and endothelial cells (ECs) was designed using porous microcarriers, and the feasibility of applying to bone tissue engineering was investigated in vitro. Highly porous spherical microcarriers made of degradable biopolymers were prepared with sizes of hundreds of micrometers. The microcarriers loaded with DPSCs were co-cultured with ECs embedded in a hydrogel of type I collagen. An optimal coculture medium that preserves the viability of ECs while stimulating the osteogenic differentiation of DPSCs was found to be a 10:1 of osteogenic medium:endothelial medium. The co-cultured constructs of DPSCs/ECs showed significantly higher level of alkaline phosphatase activity than the mono-cultured cells. Moreover, the expressions of genes related with osteogenesis and angiogenesis were significantly up-regulated by the co-cultures with respect to the mono-cultures. Results imply the interplay between ECs and DPSCs through the designed 3D co-culture models. The microcarrier-enabled co-cultured cell system is considered to be useful as an alternative tool for future vascularized bone tissue engineering.

Construction and in vitro functional evaluation on a new hybrid bioartificial liver / 中华肝胆外科杂志

Objective To design a new type of hybrid bioartificial liver (HBAL), evaluate its efficacy in vitro, and explore the feasibility in clinical application.Methods CL-1 human hepatocytes were cultured on microcarriers for 5 days, when cell count reached about 4.0 × 109 with cell density of about 4.0 × 107/ml.CL-1 cells cultured on microcarriers in home-made bioreactor constitute the biological part of the HBAL.The abiotic part included blood perfusion and bilirubin adsorption, and blood pump was employed as the circulation driver, which were parts of HBAL.The changes of the concentrations of indirect bilirubin (UBD), chenodeoxycholic acid (CDCD), cholic acid (CA), blood ammonia (AA), AST, ALT and LDH were observed under the condition of in vitro circulation.Meanwhile, the function, morphology and the cell activity of CL-1 cells were also observed.Results After in vitro circulation for 24 h, the concentrations of UBD, CDCD, CA and AA significantly decreased from (335.3 ± 6.0) μmol/L, (395.0 ± 5.6) μmol/L, (155.7 ± 4.5) μmol/L, (39.0 ± 2.6) μmol/L at 0 h to (106.0 ± 10.9) μmol/L, (131.8 ± 28.7) μmol/L, (42.2 ± 7.3) μmol/L, (3.5 ± 1.0) μmol/L, respectively.At 48 h, ALT, AST and LDH significantly increased from (25.9 ± 4.2) IU/L, (22.0 ± 3.6) IU/L, (0.28 ± 0.09) μmol/L to (31.0 ± 2.6) IU/L,(31.6 ± 8.0) IU/L, (0.41 ± 0.12) μmol/L, meanwhile the count and vitality of CL-1 cells were significant declined.Conclusions (1) In the new HBAL system, CL-1 cells can keep its viability and function in vitro;and (2) the HBAL appears to be effective in purifying the serum in liver failure simulation model by clearing out non-conjugated bilirubin, chenodeoxycholic acid, cholic acid and ammonium chloride, which seems to be a promising therapeutic option.

Applications of microspheres in tissue engineering / 国际药学研究杂志

Tissue engineering has always been a hot spot in the life science area over the past few years. However, there are some problems, such as the multiplication of seeding cells, the preparation of tissue engineering scaffolds, and the controlled release of drugs from scaffolds and so on. Recently microspheres have been widely used to solve these problems. For example, microspheres are used as microcarriers to culture seeding cells. In addition, microsphers can be either used to prepare engineering scaffolds or directly used as scaffolds. Microspheres can be used to load drugs and control the drug release as well. In this paper the application of microspheres in tissue engineering is reviewed and the up-to-date methods are introduced, including ways of improving performances of microcarriers, preparing scaffolds with microspheres and adding drug-loaded microspheres into scaffolds.

Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system

Future clinical applications of human embryonic stem (hES) cells will require high-yield culture protocols. Currently, hES cells are mainly cultured in static tissue plates, which offer a limited surface and require repeated sub-culturing. Here we describe a stirred system with commercial dextran-based microcarriers coated with denatured collagen to scale-up hES cell production. Maintenance of pluripotency in the microcarrier-based stirred system was shown by immunocytochemical and flow cytometry analyses for pluripotency-associated markers. The formation of cavitated embryoid bodies expressing markers of endoderm, ectoderm and mesoderm was further evidence of maintenance of differentiation capability. Cell yield per volume of medium spent was more than 2-fold higher than in static plates, resulting in a significant decrease in cultivation costs. A total of 10(8) karyotypically stable hES cells were obtained from a unitary small vessel that needed virtually no manipulation during cell proliferation, decreasing risks of contamination. Spinner flasks are available up to working volumes in the range of several liters. If desired, samples from the homogenous suspension can be withdrawn to allow process validation needed in the last expansion steps prior to transplantation. Especially when thinking about clinical trials involving from dozens to hundreds of patients, the use of a small number of larger spinners instead of hundreds of plates or flasks will be beneficial. To our knowledge, this is the first description of successful scale-up of feeder- and Matrigel™-free production of undifferentiated hES cells under continuous agitation, which makes this system a promising alternative for both therapy and research needs.

Research on multiplication of the H9N2 subtype avian influenza virus in large-scale microcarrier-based MDCK cell culture system / 中国人兽共患病学报

To explore the regularity for the multiplication of avian influenza virus subtype H9N2 in large-scale microcarrier-based MDCK cell culture system, and to determine the optimal proliferation conditions. H9N2 subtype of avian influenza virus was inoculated into the MDCK cell growing on 24 well plate, and the HA titers of virus at different time were detected in the conditions of different infectious doses,different concentrations of TPCK- trypsin and different pH. The optimal conditions were determined. Then the H9N2 subtype avian influenza virus was grown in microcarrier-based MDCK cell in 250mL and 5L roller bottles. It was demonstrated that high viruse yield with a hemagglutination unit of 9 log2(1:512) could be obtained under the optimal conditions of multiplication . The result indicated the H9N2 subtype avian influenza virus could be produced in microcarrier-based MDCK cell in a large-scale culture system with a high virus yield and demonstrates the feasibility of the development of mammalian cell-based in influenza vaccine in microcarrier culture systems.

Transplantation of mesenchymal stem cells and gelatin porous microcarriers into normal rat heart tissue / 第三军医大学学报

Objective To investigate the feasibility of transplantation of human fetal liver-delivered mesenchymal stem cells(HFMSCs) and porous microcarries into normal heart tissue and whether it can improve heart function and regeneration of heart tissue.Methods SD rats were divided into HFMSCs injection group(n=9),microcarrier injection group(n=9) and control group(n=4),in which 80-100 ?l Perfadex with HFMSCs or gelatin porous microcarriers or pure Perfadex was injected into the wall of left ventricle.Heart function was evaluated by UCG before and 7 d after transplantation.On day 7,14,the survival of HFMSCs was tested by fluorescent in situ hybridization(FISH),regeneration or cardiac differentiation by immunohistological staining against desmin,tropomyosin and lectin,cellular immune response by the infiltration of macrophages,and lymphocyte reaction to HFMSCs by mixed lymphocyte culture(MLC) in vitro.Results Seven days after injection,the HFMSCs survived and improved the heart function,though no sign of differentiation into cardiomyocytes was seen.On day 14,a large amount of macrophages infiltrated into injection sites,and MLC showed prominent enhancement of proliferation of lymphocytes,when no transplanted cells were detected in the myocardium.On day 7,14,the microcarriers retained their round shape at the injection sites and were attatched by a large quantity of cells which were proven not cardiomyocytes or capilaries by immunohistological staining.Conclusion Transplantation of HFMSCs into normal heart improves heart function by short-period survival without differentiation,but the transplanted cells disappeared because of immune reaction.Transplantation of porous microcarriers into normal heart could not improve heart function either by regeneration of heart tissue or capilaries.