Dynamic Culture System Research Articles

Behavior of Bone Marrow Mesenchymal Stem Cells on Collagen Scaffolds in a Dynamic Culture System

Aim: BMSCs cultivation in vitro under three-dimensional (3D) dynamic conditions. Methods: a novel bioreactor system without shear stress acting on cells was developed by the combined use of collagen gel, hollow fiber membranes (HFMs) and air-lift loop bioreactor (ALB). During the expansion period, the cell growth curves were measured and after 7 days, the cell viability and morphology were assayed by fluorescence staining microscopically. Moreover, the BMSC characters of expanded cells were identified by specific expressions of CD29, CD34, CD44 and CD45 and by multilineage differentiation into osteoblasts, chondrocytes and adipocytes. Results: the cultured BMSCs expanded about 17-fold under dynamic condition, 13-fold under static condition with the initial density of 5×105cells·mL-1; after 7 days, the cells maintained high viability of about 92% and spread with a spindle appearance and long cell protrusions in gel; the expanded cells were CD29 and CD44 positive, CD34 and CD45 negative, and they could differentiate into osteoblasts, chondrocytes and adipocytes; the cells expanded under dynamic condition spread more and showed stronger multi-differentiation potentials.

Novel dynamic culture system to support initiation of primordial follicle growth in prepubertal mouse ovaries

To evaluate the impact of dynamic in vitro culture on initiation of early follicular growth in prepubertal mouse ovaries. Ovaries from 8-day-old BALB/c mice were cultured either in a dynamic system (n=28) or in a static system (n=20) for 4 days. Uncultured 8-day-old (n=9) or 12-day-old (n=17) ovaries served as baseline or in vivo controls, respectively. Academic research center. Newborn female BALB/c mice (n=37). None. Histologic follicle classification and counting and assessment of follicular viability via immunofluorescent staining. The percentage of secondary follicles after dynamic culture was identical to the 12-day-old in vivo control. In contrast, after static culture ovaries showed a significantly higher percentage of secondary follicles. For immunofluorescent viability assessment 6.78 follicles per ovary could be isolated after dynamic culture, whereas only 3.8 follicles per ovary could be isolated after static culture. Dynamic in vitro culture supports physiologic follicular growth initiation, comparable to that observed in vivo. In contrast, accelerated follicular growth was observed after static culture. These findings add additional evidence to the idea that dynamic culture might be a beneficial first step to initiate follicle growth in vitro within the context of fertility preservation.

Effect of Purmorphamine on Osteogenic Differentiation of Human Mesenchymal Stem Cells in a Three-Dimensional Dynamic Culture System

We have previously demonstrated that purmorphamine exerts an osteoinductive effect on human bone marrow mesenchymal stem cells (hBM-MSCs) in a two-dimensional (2D) static culture system. In the present study, we have evaluated the effect of purmorphamine on osteogenic differentiation of human mesenchymal stem cells (hMSCs) in a three-dimensional (3D) dynamic culture system vs. a 2D static culture condition. In this study, hMSCs were seeded in either 3D collagen scaffolds using a perfusion bioreactor or on 2D culture plates. One day later, the medium was replaced with osteogenic medium supplemented with dexamethasone, l‐ascorbic acid and β-glycerophosphate in the presence or absence of purmorphamine. Histological staining, flow cytometry, and real-time PCR were conducted for evaluation of osteogenesis in the study groups. The expressions of RUNX-2, alkaline phosphatase (ALP), osteocalcin (OC), collagen I (Col I), and bone sialoprotein (BSP) were compared between the 3D and 2D culture systems at 14 and 21 days post-induction of differentiation. Based on our results, alizarin red staining showed deposition of the mineralized matrix in the 2D static and 3D dynamic cultures. Our flow cytometric analysis indicated that the number of ALP+/OC− cells increased in the presence of purmorphamine in the 2D culture rather than the 3D system at day14. One week later, the numbers of OC+/ALP− and OC+/Stro-1− cells increased significantly in the 3D dynamic model compared with the 2D cultures. Expression of RUNX-2 was upregulated in the presence of purmorphamine in both cultures at day 14. The purmorphamine response gene, Gli-1, was upregulated during both the early and late culture periods in the 3D dynamic system, while similar up-regulation was observed only during the early culture period in 2D culture. No difference was observed in expressions of collagen type I and BSP between groups. The present study indicates that purmorphamine as agonist of Shh signaling pathway affects on osteogenic differentiation program of hMSCs in static and dynamic culture systems.

Rabbit Mesenchymal Stem Cells Cultured in a Dynamic Culture System Displayed Superior Cell Proliferation and Osteogenetic Induction

Objective We evaluated the effect of three-dimensional static and dynamic culture on the proliferation, distribution, and differentiation of rabbit mesenchymal stem cells (MSCs) in a porous scaffold via autograft for bone regeneration. Methods Rabbit MSCs were seeded in a porous hydroxyapatite scaffold (MSCs/scaffold), and then cultured in petri dishes and a bidirectional flow reactor for 4 weeks for osteogenetic induction in vitro . Metabolic assay of the culture medium was carried out every 2 days; glucose, lactic acid, and calcium concentrations in the medium were also examined. Cell distribution in the scaffold was examined histologically. Cultured MSCs/scaffolds were implanted in rabbit condyles for the evaluation of bone regeneration in vivo . Results Histological sections showed that cells cultured in petri dishes grew only around the scaffolds and seldom in the inner part. However, the scaffolds cultured with MSCs in a bioreactor were almost fully occupied by cells. Metabolic results revealed that the average concentration of glucose in the medium was decreased as cells propagated. Glucose consumption was observed in both static and dynamic cultures, but higher lactic acid production was found in the static culture. Calcium ion concentration was reduced significantly in the dynamic culture after the addition of an osteogenetic induction medium, indicating progression of mineralization. The in vivo results showed that about 80% of the defect in the condyles with dynamically cultured MSCs/scaffold implants was filled with new bone tissue, a proportion much higher than that in the petri dish-cultured MSCs/scaffolds, in which only half of the bone regeneration occurred in the cavity. Conclusion This study provides evidence of the effectiveness of dynamic in vitro MSC culture for robust osteogenesis and indicates that it may impart superior potential for bone regeneration in vivo .

Three-Dimensional Dynamic Bioreactor Culture System Supports the Angiogenesis Directional of Human Umbilical Vein Endothelial Cells

Three-Dimensional Dynamic Bioreactor Culture System Supports the Angiogenesis Directional of Human Umbilical Vein Endothelial Cells

Effect of chitosan conduit under a dynamic culture on the proliferation and neural differentiation of human exfoliated deciduous teeth stem cells

Ex vivo engineering of artificial nerve conduit is a suitable alternative clinical treatment for nerve injuries. Stem cells from human exfoliated deciduous teeth (SHEDs) have been considered as alternative sources of adult stem cells because of their potential to differentiate into multiple cell lineages. These cells, when cultured in six-well plates, exhibited a spindle fibroblastic morphology, whereas those under a dynamic culture aggregated into neurosphere-like clusters in the chitosan conduit. In this study, we confirmed that SHEDs efficiently express the neural stem cell marker nestin, the early neural cell marker β-III-tubulin, the late neural marker neuron-specific enolase and the glial cell markers glial fibrillary acidic protein (GFAP) and 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). The three-dimensional chitosan conduit and dynamic culture system generated fluid shear stress and enhanced nutrient transfer, promoting the differentiation of SHEDs to neural cells. In particular, the gene expressions of GFAP and CNPase increased by 28- and 53-fold, respectively. This study provides evidence for the dynamic culture of SHEDs during ex vivo neural differentiation and demonstrates its potential for cell therapy in neurological diseases. Copyright © 2013 John Wiley & Sons, Ltd.

Tissue Engineering of a Human 3D <em>in vitro</em> Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system. Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns). Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow. In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D <em>in vitro</em> Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Heterotopic and orthotopic autologous chondrocyte implantation using a minipig chondral defect model

Implantation of non-articular (heterotopic) chondrocyte-based implants might be an alternative approach to articular cartilage repair. This strategy could be helpful in cases in which there are no or too few articular chondrocytes available. Therefore, this study was undertaken to compare joint cartilage defect healing in the minipig model after implantation of heterotopic auricular and orthotopic articular chondrocytes. Poly-glycolic acid (PGA) associated three-dimensional (3D) constructs were prepared culturing autologous minipig-derived articular and auricular chondrocytes for 7 days in a dynamic culture system. Chondrocyte PGA constructs were implanted into 8mm diameter and ∼1.1mm deep chondral defects within the medial and lateral condyles of the minipig knee joints. Empty defects served as controls for assessment of the intrinsic healing response. Defect healing was monitored 6 months post implantation using a macroscopic and microscopic score system and biomechanical analysis. Neo-cartilage formation could be observed in the PGA constructs seeded with articular and auricular chondrocytes in vivo. The defect healing did not significantly differ at the macroscopic and histological level in response to implantation of either autologous articular or auricular chondrocytes seeded constructs compared with the empty defects. Although the differences were not significant, the auricular chondrocytes-based implants led to a slightly inferior repair quality at the macroscopic level, but a histologically superior healing response when compared with the empty defect group. However, biomechanical analysis revealed a higher stiffness in repair tissues produced by auricular chondrocyte implantation compared with the other groups. Deduced from these results, articular chondrocytes represent the preferable cell source for implantation.

The impact of culture conditions on early follicle recruitment and growth from human ovarian cortex biopsies in vitro

To investigate the effects of a dynamic fluidic culture system on early in vitro folliculogenesis in standardized ovarian cortex biopsies. Cortical small strips were cultured for 6 days in a conventional static or in a dynamic fluidic culture system. University-affiliated laboratory with an associated cryobank facility. Ovarian cortex from postpuberal female cancer patients (26.1 ± 1.3 y) who opted for cryopreservation of their tissue for fertility protection before gonadotoxic cancer therapy. With informed consent of the Institutional Ethics Committee, part of the tissue was available for patient-related research studies. None. The viability and proliferative capacity of the cortex biopsies were evaluated by chemiluminescent microparticle immunoassay for detection of in vitro produced E2 and P in the supernate, by viable follicle counting via calcein staining, by histologic analyses, and by total RNA preparation and reverse transcription for real-time polymerase chain reaction of selected early folliculogenesis genes. The data support the notion that early follicle development can be better achieved in vitro in a dynamic fluidic culture system. The findings are based on the presence of more viable follicles, higher expression levels of early folliculogenesis genes KIT-L, INHB, and GDF9, and the absence of premature luteinization of follicles. This study provides evidence that dynamic fluidic culture is a promising approach for investigating early follicular recruitment and growth in cortical biopsies. It may serve as a first step in a multistep culture system to design a complex in vitro system for complete folliculogenesis.

Organ Culture of Porcine Mitral Valves as a Novel Experimental Paradigm

Mitral valve diseases are prevalent disorders, but how these conditions develop is poorly understood. Organ culture systems offer the potential to investigate valve biology and pathology in vitro. The purpose of this research was to determine whether culture within an organ culture system preserves the normal valve phenotype. Porcine mitral valves cultured for 3 weeks in an organ culture system (n = 5) were characterized mechanically, biochemically and histologically and compared to freshly harvested (n = 7) and static culture valves (without mechanical stimulation, n = 6). The dynamic culture system provided mechanical stimulation through physiologically relevant pressures and flow rates. The organ culture system maintained function and was free of contamination, but experienced an increase in regurgitant flow, over the culture duration. The static culture valves had lower DNA content (cellularity) and radial tensile modulus, and greater thickness and hydration compared with the fresh valves, whereas the dynamic culture valves better preserved these characteristics. Both groups of cultured valves had lower tensile moduli of the chordae tendineae compared to chordae from freshly harvested valves, and both groups demonstrated a reduction of glycosaminoglycans and proteoglycans, particularly hyaluronan and versican. The investigated organ culture system was able to maintain several aspects, but not all aspects, of normal mitral valve mechanics and microstructure. Challenges in maintaining normal forward flow likely altered the valve hemodynamics, and will require further refinement of the organ culture system. Nonetheless, organ culture systems offer considerable promise as a new experimental paradigm for studies of valve biology and pathology.

Copolymer cell/scaffold constructs for bone tissue engineering: Co‐culture of low ratios of human endothelial and osteoblast‐like cells in a dynamic culture system

The aim of this study was to compare the effect of different ratios of human umbilical vein endothelial cells (HUVECs) on osteogenic activity of human osteoblast-like cells (HOB) and capillary-like structure (CLS), seeded into copolymer scaffolds in a dynamic culture system. HOB and HUVEC were co-cultured into poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)] scaffolds at ratios of 5:1 (5:1 group) and 2:1 (2:1 group). Samples were collected after 5, 15, and 25 days. Cross-sections were processed and the CLS from HUVEC was disclosed in both groups. Cell viability was determined by dsDNA assay. Cells seeded at the ratio of 5:1 had good viability. Total RNA was isolated and the reverse transcription reaction was performed. The influences on the expression of several osteogenic genes were various with regarding to different ratios of HUVEC demonstrated by the PCR array. The RT-PCR results was in consistent with the PCR array results that several osteogenesis related genes had higher expression in the 5:1 group than in the 2:1 group, especially at day 25, such as alkaline phosphatase, insulin-like growth factor 1 (IGF1), and so forth. ELISA showed that the production of IGF1 after 25 days of incubation were higher in cells co-cultured at the 5:1 ratio than at the 2:1 ratio. The results show that under dynamic culture conditions, co-culture of HOB with a low ratio of HUVEC in copolymer scaffolds results in CLS formation and significantly influenced the expression of osteogenic markers.

Fabrication of porous PLGA microspheres with BMP-2 releasing polyphosphate-functionalized nano-hydroxyapatite for enhanced bone regeneration

This paper introduces a novel bone-regenerative scaffold that is based on the systematic combination of porous polymer microspheres, nano-hydroxyapatite, and bone morphogenetic protein-2 (BMP-2), where each component was rationally incorporated to express its intrinsic activity in bone tissue formation. Poly(lactide-co-glycolide) (PLGA) microspheres, with interconnected pore structures, were fabricated by a gas-forming method in a water-in-oil-in-water double emulsion and solvent evaporation process. Polyphosphate-functionalized nano-hydroxyapatite (PP-n-HAp) was employed as a main component and was immobilized on the pore surface of the PLGA microspheres to controllably incorporate and release BMP-2. The surface polyphosphate functionalities of PP-n-HAp enabled the stable chemical immobilization of nano-hydroxyapatite (n-HAp) on the amine-treated pore surface of the PLGA microspheres. Field-emission scanning electron microscopy (FE-SEM) and X-ray photoelectron spectroscopy (XPS) confirmed the nano-level exposure of n-HAp on the pore surface of the PLGA microspheres. BMP-2 with a positive charge was bound at a high efficiency onto the anionic phosphates of surface-immobilized PP-n-HAp and was controllably released for approximately 1 month. The release rate was manipulated by adjusting the amount of loaded BMP-2. The osteogenic differentiation and proliferation of human adipose-derived stem cells (hADSCs) within the n-HAp/BMP-2-incorporated microspheres were monitored in a dynamic 3D cell culture system. Histological, immunohistochemical, and quantitative real-time polymerase chain reaction (PCR) analyses showed that the PP-n-HAp-immobilized surface promoted cell adhesion/proliferation and osteoconduction. With its osteoinductive property, the sustained release of BMP-2 further enhanced the bone tissue regenerative activity of the porous microspheres. Open image in new window

In vitro construction of tissue-engineered cardiac tissue sheet by small intestinal submucosa with bone mesenchymal stem cells

Objective To explore the feasibility of in vitro construction of tissue-engineered cardiac sheet by small intestinal submucosa(SIS) with bone mesenchymal stem cells(BMSCs). Methods BMSCs separated from rat bone marrow were induced by 5-azacytidine(5-Aza) for 3 weeks,and were seeded on the serosal side of acellular SIS scaffold.The tissue-engineered cardiac tissue sheet was established in vitro after co-culture in dynamic culture system for 2 weeks,and its properties were evaluated by histological,ultrastructural and immunohistochemical examinations. Results After co-culture for 2 weeks,HE staining revealed that the seeded cells were not restricted in the upper area of SIS,forming multiple uniform layers on SIS,and some cells penetrated and migrated into the porous matrix.Scanning electronic microscopy revealed that BMSCs adhered,proliferated and migrated well on SIS,and secreted a large amount of extracellular matrix.Immunohistochemical staining demonstrated that the cells on SIS were cardiomyocyte-like cells with the expression of α-actin,cTnⅠ and connexin-43. Conclusion The engineered cardiac tissue sheet has been successfully constructed by SIS with 5-Aza-induced BMSCs in vitro,and SIS is an ideal bio-scaffold material for cardiac tissue engineering.

88 COMPARISON BETWEEN STATIC AND DYNAMIC CULTURE RESULTS USING A NOVEL AIR ACTUATION SYSTEM

Mammalian embryos experience not only hormonal stimuli, but also mechanical stimuli (MS), such as shear stress (SS), compression and friction force, in the fallopian tube before nidation. Embryo development performed using previously described in vitro dynamic culture systems is significantly better than that performed using conventional static culture systems. Previously, we found that thawed human embryos showed developmental improvement in the blastocyst stage following a tilting embryo culture system (TECS) culture compared with static culture. However, a disadvantage of the system is the need to use electric devices inside the incubator under humidified conditions. To solve the problem, we developed a dynamic embryo culture system using air actuation and evaluated the applied MS and embryo culture results. We developed an air actuation system with microfluidic channels to apply MS by deforming a 0.1-mm-thick poly(dimethylsiloxane) (PDMS) membrane. The PDMS microfluidic device was placed in a humidified incubator and the mechanical actuator was placed outside the incubator. The embryos' motion in the microfluidic channel was recorded using an inverted microscope and a colour CCD camera with a frame rate of 30 frames s–1. Syringe velocity (VS) was controlled using a software model of the actuation system. The observed maximum velocity of the embryos (VE) and fluid velocity (VF) were calculated by tracking the images of the embryos and the particles in the medium, respectively. The experiments were repeated 3 times. Frozen 2-cell-stage embryos of imprinting control region (ICR) mouse were thawed. 10 to 13 embryos were applied into the microfluidic channel and cultured in ∼200 μL of potassium simplex optimized embryo culture medium covered with mineral oil for 3 days in a humidified environment of 5% CO2 in air at 37°C. The experiments were repeated 5 times. Chi-squared test and Student's t-test were used to determine differences in the blastocyst development rate and in the number of cells in the blastocysts between the groups, respectively. A P-value <0.05 was considered significant. Results: When syringe velocity (VS) was 0.5 mm over a period of seconds, the embryos rotated and did not slide. When VS and fluid velocity (VF) increased, the embryos slipped, did not come in contact with the floor. We conclude that different types and amounts of MS can be applied to the embryos by changing VS. We compared embryo development from the 2-cell stage to the blastocyst stage between static and dynamic cultures in the medium channel. Dynamic culture significantly improved the rate of development to the blastocyst stage (dynamic, 74% (n = 126); static, 62% (n = 118); P < 0.05). The average number of cells (mean ± standard error of the mean) in blastocysts obtained in dynamic and static cultures was 83 ± 3 (n = 54) and 76 ± 3 (n = 51) (P < 0.05), respectively. When the mouse embryos moved at VE of 0.2 mm s–1, there were significant differences in both blastocyst development rate and the average cell number of blastocysts between the 2 groups.

Design and characterization of a dynamic vibrational culture system

To engineer a functional vocal fold tissue, the mechanical environment of the native tissue needs to be emulated in vitro. We have created a dynamic culture system capable of generating vibratory stimulations at human phonation frequencies. The novel device is composed of a function generator, a power amplifier, an enclosed loudspeaker and a circumferentially-anchored silicone membrane. The vibration signals are translated to the membrane aerodynamically by the oscillating air pressure underneath. The vibration profiles detected on the membrane were symmetrical relative to the centre of the membrane as well as the resting position over the range of frequencies (60-300 Hz) and amplitudes tested (1-30 µm). The oscillatory motion of the membrane gave rise to two orthogonal, in-plane strain components that are similar in magnitude (0.47%) and are strong functions of membrane thickness. Neonatal foreskin fibroblasts (NFFs) attached to the membrane were subjected to a 1 h vibration at 60, 110 and 300 Hz, with the displacement at the centre of the membrane varying in the range 1-30 µm, followed by a 6 h rest. These regimens did not cause morphological changes to the cells. An increase in cell proliferation was detected when NFFs were driven into oscillation at 110 Hz with a normal displacement of 30 µm. qPCR results showed that the expression of genes encoding some extracellular matrix proteins was altered in response to changes in vibratory frequency and amplitude. The dynamic culture device provides a potentially useful in vitro platform for evaluating cellular responses to vibration.

Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds

Cord blood expansion ex vivo can be achieved in liquid suspension through the addition of cytokines at the expense of often undesirable cell differentiation. In order to derive a cytokine-free dynamic culture system, we hypothesised that a three-dimensional (3D) environment in the form of highly porous scaffolds made of poly ( d, l-lactide-co-glycolide) (PLGA) or polyurethane (PU) for the biomimetic growth of cord blood mononuclear cells (CBMNCs), would facilitate expansion of hematopoietic cells without exogenous cytokines. Both scaffolds supported cellular expansion ex vivo. Cytokine-free, long-term culture was best in PU coated with collagen type I (54-fold expansion). In contrast, traditional 2D well-plate cultures collapsed within 4 days in the absence of cytokines. CBMNCs cultured in the scaffolds were visualised by scanning electron microscopy and immunophenotypic/immunostaining analysis and the studies validated the presence of a dynamic culture containing erythroid precursors (CD45 −/CD71 +/CD235a +), hematopoietic stem/progenitor cells (CD38 −CD34 +, CD117 +), maturing myeloid cells (CD38 +, MPO +), CD4 + and CD8 + T-lymphocytes and megakaryocytes (FVIII +). Colony forming unit (CFU) assays indicated that BFU-E and CFU-GM increased ( p < 0.05) whereas CFU-GEMM were maintained at week 4. In conclusion, this 3D culture system is capable of long-term, cytokine-free expansion of CBMNCs, enabling the study of hematopoiesis and providing a potential platform for drug discovery and therapeutic applications ex vivo.

Microfluidic dynamic embryo culture increases the production of top quality human embryos through reduction in embryo fragmentation

Microfluidics has facilitated engineering of a dynamic environment for embryo culture that more closely recapitulates the female reproductive tract. This microfluidic dynamic culture system significantly improved mouse embryo development and pregnancy rates. We aimed to compare human embryo development in traditional static culture (SCS) and dynamic culture (DCS) systems.

Multiwalled carbon nanotube-coating of 3D collagen scaffolds for bone tissue engineering

Surface coating treatment with multiwalled carbon nanotubes (MWCNTs) was applied to 3D collagen scaffold for bone tissue engineering. Rat primary osteoblasts (ROBs) were cultured on an MWCNT-coated collagen sponge (MWCNT-coated sponge) in a 3D dynamic flow cell culture system and differentiation markers were measured. Alkaline phosphatase activity at 1 day, and calcium and osteopontin contents of the MWCNT-coated sponges at 7 days were significantly higher than those of uncoated sponges. ROBs on the MWCNT-coated sponge differentiated earlier than on the uncoated sponge. In addition, the tissue response to the MWCNT-coated sponge was evaluated. Slight inflammation was observed around MWCNTs at 7 and 28 days after implantation in subcutaneous tissue. Significantly more bone formation was observed around the MWCNT-coated sponges than around the uncoated sponges and new bone attached to MWCNTs directly at 28 and 56 days after implantation in the femur. Moreover, at 28 days after implantation of the MWCNT-coated sponge with osteoblasts cultured for 1 day, bone tissues were successfully formed in the pores according to its honeycomb structure. Therefore, MWCNT coating appears to be effective for bone tissue engineering.

Human urine-derived stem cells seeded in a modified 3D porous small intestinal submucosa scaffold for urethral tissue engineering

The goal of this study was to determine whether urothelial cells (UC) and smooth muscle cells (SMC) derived from the differentiation of urine-derived stem cells (USC) could be used to form engineered urethral tissue when seeded on a modified 3-D porous small intestinal submucosa (SIS) scaffold. Cells were obtained from 12 voided urine samples from 4 healthy individuals. USC were isolated, characterized and induced to differentiate into UC and SMC. Fresh SIS derived from pigs was decellularized with 5% peracetic acid (PAA). Differentiated UC and SMC derived from USC were seeded onto SIS scaffolds with highly porous microstructure in a layered co-culture fashion and cultured under dynamic conditions for one week. The seeded cells formed multiple uniform layers on the SIS and penetrated deeper into the porous matrix during dynamic culture. USC that were induced to differentiate also expressed UC markers (Uroplakin-III and AE1/AE3) or SMC markers (α-SM actin, desmin, and myosin) after implantation into athymic mice for one month, and the resulting tissues were similar to those formed when UC and SMC derived from native ureter were used. In conclusion, UC and SMC derived from USC could be maintained on 3-D porous SIS scaffold. The dynamic culture system promoted 3-D cell–matrix ingrowth and development of a multilayer mucosal structure similar to that of native urinary tract tissue. USC may serve as an alternative cell source in cell-based tissue engineering for urethral reconstruction or other urological tissue repair.