•The hydrogel scaffold was produced using chitosan, β-glycerophosphate and collagen.•This novel hydrogel is in liquid phase at low temperature and is gelatinized at 37 °C.•The new hydrogel provides ADSCs a favorable 3D environment with highly maintenance of proliferation and cytoactive.•ADSCs seeded hydrogel differentiated into adipose tissue, indicating favorable ability of adipogenesis.•This attractive property of C/GP/CO hydrogel points to its value as an excellent scaffold for tissue engineering.In this study, the interaction of human adipose tissue-derived stem cells (ADSCs) with chitosan/β-glycerophosphate/collagen (C/GP/Co) hybrid hydrogel was test, followed by investigating the capability of engineered adipose tissue formation using this ADSCs seeded hydrogel. The ADSCs were harvested and mixed with a C/GP/Co hydrogel followed by a gelation at 37 °C and an in vitro culture. The results showed that the ADSCs within C/GP/Co hydrogels achieved a 30% of expansion over 7 days in culture medium and encapsulated cell in C/GP/Co hydrogel demonstrated a characteristic morphology with high viability over 5 days. C/GP/Co hydrogel were subcutaneous injected into SD-rats to assess the biocompatibility. The induced ADSCs-C/GP/Co hydrogel and non-induced ADSCs-C/GP/Co hydrogel were subcutaneously injected into nude mice for detecting potential of adipogenic differentiation. It has shown that C/GP/Co hydrogel were well tolerated in SD rats where they had persisted over 4 weeks post implantation. Histology analysis indicated that induced ADSCs-C/GP/Co hydrogel has a greater number of adipocytes and vascularized adipose tissues compared with non-induced ADSCs-C/GP/Co hydrogel.

Cartilage tissue engineering is believed to provide effective cartilage repair post-injuries or diseases. Biomedical materials play a key role in achieving successful culture and fabrication of cartilage. The physical properties of a chitosan/gelatin hybrid hydrogel scaffold make it an ideal cartilage biomimetic material. In this study, a chitosan/gelatin hybrid hydrogel was chosen to fabricate a tissue-engineered cartilage in vitro by inoculating human adipose-derived stem cells (ADSCs) at both dynamic and traditional static culture conditions. A bioreactor that provides a dynamic culture condition has received greater applications in tissue engineering due to its optimal mass transfer efficiency and its ability to simulate an equivalent physical environment compared to human body. In this study, prior to cell-scaffold fabrication experiment, mathematical simulations were confirmed with a mass transfer of glucose and TGF-β2 both in rotating wall vessel bioreactor (RWVB) and static culture conditions in early stage of culture via computational fluid dynamic (CFD) method. To further investigate the feasibility of the mass transfer efficiency of the bioreactor, this RWVB was adopted to fabricate three-dimensional cell-hydrogel cartilage constructs in a dynamic environment. The results showed that the mass transfer efficiency of RWVB was faster in achieving a final equilibrium compared to culture in static culture conditions. ADSCs culturing in RWVB expanded three times more compared to that in static condition over 10 days. Induced cell cultivation in a dynamic RWVB showed extensive expression of extracellular matrix, while the cell distribution was found much more uniformly distributing with full infiltration of extracellular matrix inside the porous scaffold. The increased mass transfer efficiency of glucose and TGF-β2 from RWVB promoted cellular proliferation and chondrogenic differentiation of ADSCs inside chitosan/gelatin hybrid hydrogel scaffolds. The improved mass transfer also accelerated a dynamic fabrication of cell-hydrogel constructs, providing an alternative method in tissue engineering cartilage.

Hematopoietic stem cells (HSCs) are capable to self-renew with multi-potency which generated much excitement in clinical therapy. However, the main obstacle of HSCs in clinical application was insufficient number of HSCs which were derived from either bone marrow, peripheral blood or umbilical cord blood. This review briefly discusses the indispensable utility of growth factors and cytokines, stromal cells, extracellular matrix, bionic scaffold and microenvironment aiming to control the hematopoiesis in all directions and provide a better and comprehensive understanding for in vitro expansion of hematopoietic stem cells.

•This study introduces a promising method for encapsulating ADSCs within hybrid gel-beads.•The gel-beads embedded with bone power provide a better biocompatible environment to mimic an in-vivo niche.•A spinner flask with a simple configuration was adopted to provide a dynamic condition for biomechanical stimuli.•Shear stress stimuli and bone powder were paramount to promote the cell differentiation and maturation of ADSCs.•This feasible approach was believed to fabricate engineered bone aggregates encapsulated by shape-adaptable gel-beads.Traditional treatment for bone diseases limits their clinical application due to undesirable host immune rejection, limited donator sources and severe pain and suffering for patients. Bone tissue engineering therefore is expected to be a more effective way in treating bone diseases. In the present study, hybrid calcium alginate/bone powder gel-beads with a uniform size distribution, good biocompatibility and osteoinductive capability, were prepared to be used as an in-vitro niche-like matrix. The beads were optimized using 2.5% (w/v) sodium alginate solution, 4.5% (w/v) CaCl2 solution and 5.0 mg/mL bone powder using an easy-to-use method. Human ADSCs were cultured and induced into chondrocytes and osteoblasts, respectively. The cells were characterized by histological staining showing the ADSCs were able to maintain their characteristic morphology with multipotent differentiation ability. ADSCs at density of 5 × 106 cells/mL were encapsulated into the gel-beads aiming to explore cell expansion under different conditions and the osteogenic induction of ADSCs was verified by specific staining. Results demonstrated that the encapsulated ADSCs expanded 5.6 folds in 10 days under dynamic condition via spinner flask, and were able to differentiate into osteoblasts (OBs) with extensive mineralized nodules forming the bone aggregates over 3 weeks postosteogenic induction. In summary, hybrid gel-beads encapsulating ADSCs are proved to be feasible as a new method to fabricate tissue engineered bone aggregation with potential to treat skeletal injury in the near future.

•P(NIPAAm-co-HPM) grafted onto microcarrier for producing a novel thermosensitive microcarrier•Grafted microcarriers are biocompatible to significantly enhance adhesion and growth of BMMSCs.•Well attached BMMSCs could be mostly removed from grafted microcarrier with high cell viability.•This study presents a feasible substitute for cell recovery and a 3D condition for cell growth.•This study makes it convenient and efficient to expand cells by combining a dynamic bioreactor.Traditional two-dimensional (2D) static culture environment for stem cells followed by enzymatic cell detachment or mechanical treatment is routinely used in research laboratories. However, this method is not ideal as stem cells expand slowly, with cell damage and partial loss of specific stemness. For this reason, a better culture condition is urgently needed to improve stem cell recovery. A novel thermosensitive P(NIPAAm-co-HPM)-g-TMSPM-g-microcarrier was prepared here as a three-dimensional (3D) culture substitute. This novel microcarrier was prepared by grafting NIPAAm and HPM to the surface of glass microcarrier using TMSPM through surface free radical copolymerization. The prepared material was tested in cell culture and via cooling harvest method. We found that NIPAAm was successfully grafted on to the surface of the microcarriers, providing an excellent biocompatible environment for BMMSC adhesion and growth. More importantly, BMMSCs could be fully removed from the thermosensitive glass microcarriers with remained cell viability.

•ADSCs/hybrid scaffold constructs are dynamically fabricated in a spinner flask with a special framework.•Inside convection in spinner flask made enough supplement of oxygen and nutrients far beyond the depth of passive diffusion.•3D culture environment accelerated mass transfer of nutrients and discharge of metabolites.•A special designed steel framework successfully avoided the collision between constructs and walls.Cartilage transplantation using in vitro tissue engineered cartilage is considered a promising treatment for articular cartilage defects. In this study, we assessed the advantages of adipose derived stem cells (ADSCs) combined with chitosan/gelatin hybrid hydrogel scaffolds, which acted as a cartilage biomimetic scaffold, to fabricate a tissue engineered cartilage dynamically in vitro and compared this with traditional static culture. Physical properties of the hydrogel scaffolds were evaluated and ADSCs were inoculated into the hydrogel at a density of 1 × 107 cells/mL and cultured in a spinner flask with a special designed steel framework and feed with chondrogenic inductive media for two weeks. The results showed that the average pore size, porosity, swelling rate and elasticity modulus of hybrid scaffolds with good biocompatibility were 118.25 ± 19.51 μm, 82.60 ± 2.34%, 361.28 ± 0.47% and 61.2 ± 0.16 kPa, respectively. ADSCs grew well in chitosan/gelatin hybrid scaffold and successfully differentiated into chondrocytes, showing that the scaffolds were suitable for tissue engineering applications in cartilage regeneration. Induced cells cultivated in a dynamic spinner flask with a special designed steel frame expressed more proteoglycans and the cell distribution was much more uniform with the scaffold being filled mostly with extracellular matrix produced by cells. A spinner flask with framework promoted proliferation and chondrogenic differentiation of ADSCs within chitosan/gelatin hybrid scaffolds and accelerated dynamic fabrication of cell–hydrogel constructs, which could be a selective and good method to construct tissue engineered cartilage in vitro.

Currently, RWVB (Rotating wall vessel bioreactor) combined with a microcarrier used for in vitro expansion revealed that the suspended cells attached on the microcarrier will collide with outer and inner cylinders of RWVB inevitably, which leads to harmful results to the cells. Considering this, hollow fiber (HF) membrane module treated as a cell carrier is adopted to combine with RWVB to form a novel rotating wall hollow fiber membrane bioreactor (RWHMB) to avoid aforementioned harmful collision, since the cells cultured inside this bioreactor will mainly adhere to large specific surface of hollow fiber membrane module. Prior to cell experiment, mathematical simulations concerned with flow field inside RWHMB are performed by CFD, which includes the distributions of the total pressure, velocity, and shear stress with the variation of rotating speeds and directions, as well as the radial location and diameter of hollow fiber membrane. To further confirm the feasible parameters getting from the simulation, this RWHMB is adopted to expand osteoblasts isolated from SD rats within its dynamic conditions. Cell expansion in T-flask is carried out as a negative control. The results showed that with the same rotating direction and speed of 10 rpm, inner and outer cylinders of RWHMB generated cyclical stress stimulus, which was acceptable to cell expansion and facilitated the secretion of extracellular matrix. Besides, hollow fiber membrane carrier with a diameter of 0.2 mm has an excellent biocompatibility and their radial locations presented a tiny influence on flow field inside the culture chamber.

•PNIPAAm-grafted HFMs exhibited thermoresponsive characteristic.•The OB cells could adhere and spread well on the surface of PNIPAAm-grafted HFMs.•PNIPAAm-grafted HFMs do not significantly impact ALP activity and OCN protein expression level of OB cells.•Cell could be detached from PNIPAAm-grafted HFMs when temperature decreased from 37 °C to 20 °C.Hollow fiber membrane (HFM) culture system is one of the most important bioreactors for the large-scale culture and expansion of therapeutic cells. However, enzymatic and mechanical treatments are traditionally applied to harvest the expanded cells from HFMs, which inevitably causes harm to the cells. In this study, thermo-responsive cellulose acetate HFMs for cell culture and non-invasive harvest were prepared for the first time via free radical polymerization in the presence of cerium (IV). ATR-FTIR and elemental analysis results indicated that the poly(N-isopropylacrylamide) (PNIPAAm) was covalently grafted on HFMs successfully. Dynamic contact angle measurements at different temperatures revealed that the magnitude of volume phase transition was decreased with increasing grafted amount of PNIPAAm. And the amount of serum protein adsorbed on HFMs surface also displayed the same pattern. Meanwhile osteoblasts adhered and spread well on the surface of PNIPAAm-grafted HFMs at 37 °C. And Calcein-AM/PI staining, AB assay, ALP activity and OCN protein expression level all showed that PNIPAAm-grafted HFMs had good cell compatibility. After incubation at 20 °C for 120 min, the adhering cells on PNIPAAm-grafted HFMs turned to be round and detached after being gently pipetted. These results suggest that thermo-responsive HFMs are attractive cell culture substrates which enable cell culture, expansion and the recovery without proteolytic enzyme treatment for the application in tissue engineering and regenerative medicine.

The aim of this study is to analyze the growth and substance metabolism of neural stem cells (NSCs) cultured in biological collagen-based scaffolds. Mass transfer and metabolism model of glucose, lactic acid, and dissolved oxygen (DO) were established and solved on MATLAB platform to obtain the concentration distributions of DO, glucose, and lactic acid in culture system, respectively. Calculation results showed that the DO influenced their normal growth and metabolism of NSCs mostly in the in vitro culture within collagen-based scaffolds. This study also confirmed that 2-mm thickness of collagen scaffold was capable of in vitro cultivation and growth of NSCs with an inoculating density of 1 × 106 cells/mL.

The Ca-alginate/gelatin (CAG) microbeads were prepared and evaluated through assays for their mechanical strength, permeability, and the feasibility as a cell carrier for in vitro culture of neural stem cells. The effects of different concentrations of sodium alginate, gelatin, and calcium chloride on the mechanical strength of CAG microbeads were determined using a self-made puncture force tester. Following this, the microbeads were immersed in DMEM media for a specified period to test its decay resistance. A diffusion model including a calculation formula of diffusion coefficient was built to investigate the diffusion of glucose and bovine serum albumin (BSA) through the wall of the microbeads. Furthermore, the feasibility of the microbeads for in vitro culture was identified using neural stem cells from Kunming mouse. Through a systematic approach and comprehensive analysis, the optimal gelatin conditions for microbead preparation were determined; the final combination of parameters of 1.5 % (wt%) sodium alginate (SA), 0.5 % (wt%) gelatin, and 4 % (wt%) CaCl2 were the best conditions for NSC cultures. This experiment demonstrated that CAG microbeads had good cytocompatibility that made it suitable for the culture and successfully maintained stemness of neural stem cells.

The in vitro dynamic fabrications of tissue-engineered bones were performed to assess the advantages of human adipose-derived stem cells (hADSCs) combined with acellular cancellous bone scaffold coming from fresh pig femur in a spinner flask compared with traditional static culture. In this study, the bio-derived cancellous bone was regarded as a biomimetic scaffold, and its surface appearance was observed under scanning electron microscopy (SEM). Moreover, its modulus of elasticity and chemical composition were measured with universal testing machine (UTM) and infrared detector, respectively. hADSCs were inoculated into cancellous bone scaffold at a density of 1 × 106 cells/mL and cultured in spinner flask and T-flask with osteogenic medium (OM) for 2 weeks, respectively. Following to this, the osteogenic differentiation was qualitatively and quantitatively detected with alkaline phosphatase (ALP) kits, and the cell growth and viability were assayed using Live/Dead staining; cell adhesion and extracellular matrix secretion were observed under a SEM. The average pore size of cancellous bone scaffold was 284.5 ± 83.62 μm, the elasticity modulus was 41.27 ± 15.63 MPa, and it also showed excellent biocompatibility. The hADSCs with multidifferentiation potentials were well proliferated, could grow to 90 % fusion within 5 days, and were therefore suitable to use as seed cells in the construction of tissue-engineered bones. After 2 weeks of fabrication, cells were well-distributed on scaffolds, and these scaffolds still remained intact. Compared to static environment, the ALP expression, cell distribution, and extracellular matrix secretion on cancellous bones in spinner flask were much better. It confirmed that three-dimensional dynamic culture in spinner flask promoted ADSC osteogenic differentiation, proliferation, and matrix secretion significantly to make for the fabrication of engineered bone substitutes.

In this paper, two-dimensional flow field simulation was conducted to determine shear stresses and velocity profiles for bone tissue engineering in a rotating wall vessel bioreactor (RWVB). In addition, in vitro three-dimensional fabrication of tissue-engineered bones was carried out in optimized bioreactor conditions, and in vivo implantation using fabricated bones was performed for segmental bone defects of Zelanian rabbits. The distribution of dynamic pressure, total pressure, shear stress, and velocity within the culture chamber was calculated for different scaffold locations. According to the simulation results, the dynamic pressure, velocity, and shear stress around the surface of cell-scaffold construction periodically changed at different locations of the RWVB, which could result in periodical stress stimulation for fabricated tissue constructs. However, overall shear stresses were relatively low, and the fluid velocities were uniform in the bioreactor. Our in vitro experiments showed that the number of cells cultured in the RWVB was five times higher than those cultured in a T-flask. The tissue-engineered bones grew very well in the RWVB. This study demonstrates that stress stimulation in an RWVB can be beneficial for cell/bio-derived bone constructs fabricated in an RWVB, with an application for repairing segmental bone defects.

The in vitro basic biological characteristics and directed differentiation potential towards cardiomyocytes of adult adipose-derived stem cells (ADSCs) induced by angiotensin II were both investigated. ADSCs were isolated from adult adipose tissue and cultured in vitro, and were subsequently induced into adipocytes, chondrocytes, and osteoblasts for assays of multipotential differentiation. The morphological characteristics of ADSCs were observed under an inverted microscope in bright field and phase-contrast ways and a confocal laser scanning microscopy. Moreover, the directional differentiation potential was observed by Oil Red, alkaline phosphatase, von Kossa, and toluidine blue stainings, respectively. The expressions of CD34, CD44, CD45, CD105, and HLA-DR were also detected via flow cytometry. Following to this, ADSCs were induced by angiotensin II and basic fibroblast growth factor for the purpose of directional differentiation towards cardiomyocyte-like cells, and the cells treated with 5-azacytidine were regarded as the control. The results showed that the isolated and cultured ADSCs presented a typical morphology of fusiform shape and also expressed CD44, CD105, but not CD34, CD45, and HLA-DR with assays of flow cytometry. The multi-differentiations to adipocytes, chondrocytes, and osteoblasts confirmed that the isolated cells maintained the stem characteristics generating from adipose tissues. After 4 weeks of induction by angiotensin II, the cells expressed myosin heavy chain, troponin I, and connexin43 by immunocytochemistry staining, but without beating of the cells. This current study indicated that ADSCs possessed the characteristics of mesenchymal stem cells and angiotensin II could induce ADSCs into cardiomyocyte-like cells.

Human adipose-derived adult stem cells (hADSCs) can express human telomerase reverse transcriptase phenotypes under an appropriate culture condition. Because adipose tissue is abundant and easily accessible, hADSCs offer a promising source of stem cells for tissue engineering application and other cell-based therapies. However, the shortage of cells number and the difficulty to proliferate, known as the “Hayflick limit” in vitro, limit their further clinical application. Here, hADSCs were transfected with human telomerase reverse transcriptase (hTERT) gene by the lentiviral vector to prolong the lifespan of stem cells and even immortalize them. Following to this, the cellular properties and functionalities of the transfected cell lines were assayed. The results demonstrated that hADSCs had been successfully transfected with hTERT gene (hTERT-ADSCs). Then, hTERT-ADSCs were initially selected by G418 and subsequently expanded over 20 passages in vitro. Moreover, the qualitative and quantitative differentiation criteria for 20 passages of hTERT-ADSCs also demonstrated that hTERT-ADSCs could differentiate into osteogenesis, chondrogenesis, and adipogenesis phenotypes in lineage-specific differentiation media. These findings confirmed that this transfection could prolong the lifespan of hADSCs.

Nutrient depletion within three-dimensional (3D) scaffolds is one of the major hurdles in the use of this technology to grow cells for applications in tissue engineering. In order to help in addressing it, we herein propose to use the controlled release of encapsulated nutrients within polymer microspheres into chitosan-based 3D scaffolds, wherein the microspheres are embedded. This method has allowed maintaining a stable concentration of nutrients within the scaffolds over the long term. The polymer microspheres were prepared using multiple emulsions (w/o/w), in which bovine serum albumin (BSA) and poly (lactic-co-glycolic) acid (PLGA) were regarded as the protein pattern and the exoperidium material, respectively. These were then mixed with a chitosan solution in order to form the scaffolds by cryo-desiccation. The release of BSA, entrapped within the embedded microspheres, was monitored with time using a BCA kit. The morphology and structure of the PLGA microspheres containing BSA before and after embedding within the scaffold were observed under a scanning electron microscope (SEM). These had a round shape with diameters in the range of 27–55 μm, whereas the chitosan-based scaffolds had a uniform porous structure with the microspheres uniformly dispersed within their 3D structure and without any morphological change. In addition, the porosity, water absorption and degradation rate at 37 °C in an aqueous environment of 1% chitosan-based scaffolds were (92.99 ± 2.51) %, (89.66 ± 0.66) % and (73.77 ± 3.21) %, respectively. The studies of BSA release from the embedded microspheres have shown a sustained and cumulative tendency with little initial burst, with (20.24 ± 0.83) % of the initial amount released after 168 h (an average rate of 0.12%/h). The protein concentration within the chitosan-based scaffolds after 168 h was found to be (11.44 ± 1.81) × 10− 2 mg/mL. This novel chitosan-based scaffold embedded with PLGA microspheres has proven to be a promising technique for the development of new and improved tissue engineering scaffolds.Highlights► A novel method was shown to improve the diffusivity limitations of scaffolds. ► The incorporation of PLGA microspheres containing nutrients was prepared. ► These hybrid scaffolds were shown to have an improved release of nutrients.