Xiangqin Li

Culture of neural stem cells in calcium alginate beads

Neural stem cells (NSCs) with the capacity of extensive self‐renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse‐derived NSCs were cultured in three‐dimensional calcium alginate beads (Ca‐Alg‐Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca‐Alg‐Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, β‐tubulin, and GFAP. The results show that the 2‐mm diameter Ca‐Alg‐Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 × 105 cells·mL−1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca‐Alg‐Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca‐Alg‐Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca‐Alg‐Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.

Fabrication and detection of tissue‐engineered bones with bio‐derived scaffolds in a rotating bioreactor

In order to explore the methods for commercialized bone tissue engineering, engineered bones should be cultivated in bioreactors to realize three‐dimensional culture under well‐defined culture conditions. In the present paper, osteoblasts isolated from the cranium of 1‐month‐old Zelanian rabbits were inoculated on to the BDBS (bio‐derived bone scaffolds) to investigate the three‐dimensional fabrication of engineered bone in an RWVB (rotating‐wall vessel bioreactor). The osteoblasts, after being transfected with green fluorescent protein, were respectively seeded at 2×106 and 1×106 cells·ml−1 on to the BDBS and then cultured in a T‐flask and an RWVB for 1 week. The morphologies and structure of the fabricated bone were investigated by using an inverted microscope, a scanning electron microscope and a laser confocal microscope using the stains haematoxylin/eosin and Toluidine Blue. After being digested from the scaffolds, the cells were assayed with ALP (alkaline phosphatase) stain, von‐Kossa staining on mineralized nodules, type I collagen and bone morphogenetic protein‐2 expression, and the cell expansion and growth curves using different culture methods were quantitatively determined with MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide). Furthermore, cell cycle and apoptosis were detected by using a flow cytometer, and total DNA was also assayed. For a comparative study, cell‐seeded constructs were also cultured under static conditions. The results show that the cell number cultured in the RWVB was five times that in the T‐flask. Bone tissues cultured in the RWVB with two different densities grew well, and the osteoblasts maintained their normal cycle and DNA content. The result demonstrates that, with the stress stimulation in the fluid in the RWVB, the active expression of ALP can be increased, rapid proliferation and differentiation of osteoblasts are possible and the three‐dimensional fabrication of engineered bone could be realized.

Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions.

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells by ectopic expression of defined transcription factors (TFs). However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs) without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs). The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.

Neural network analysis of boiling heat transfer enhancement using additives

Abstract A model was developed to evaluate and predict boiling heat transfer enhancement using additives. The model is based on the molecular structures of the additives and uses artificial neural network technology. The effects of 30 additives tested by the authors and other researchers on the augmentation of boiling heat transfer were analyzed with the model. The results show that the evaluation of all 30 additives is consistent with the experimental data, which means that the training accuracy of the model is 100%. In addition, the boiling heat transfer enhancement with sodium oleate and 11 other additives was also predicted, with a prediction accuracy of over 90% since the calculated results for 10 of the 11 additives were in agreement with the experimental results.

Analysis on forces and movement of cultivated particles in a rotating wall vessel bioreactor

The forces acting on a microcarrier or a small piece of tissue and its movement in the rotating wall vessel (RWV) bioreactor were analyzed. The tracks of a particle in RWV reactor were calculated under different inner and outer cylinder wall rotating speeds, different particle sizes and different density difference between culture medium and the particle. The results show that cells or particles experience partial microgravity only in the upper area of RWV, which changes with angle θ, while in the lower part of RWV, they experience overweight. The trajectory of a moving particle in RWV reactor is an eccentric helix under ground-based condition. And the eccentric degree increases with the decrease of outer wall rotating speed, and with the increase of density difference and particle size. The proper match of rotating speeds of inner and outer cylinder walls is the key to prevent the particle colliding with the walls. For a relatively large piece of cultivated tissue, it can move around the inner wall only when the rotating speed of the outer cylinder is high. And the drag force acting on a particle inside RWV increases with the particle size and the density difference.

Formation process of mixed fouling of microbe and CaCO3 in water systems

Abstract The mixed fouling process of Pseudomonas fluorescens and CaCO 3 on different solid surfaces in a simulated cooling water system has been investigated. The mixed fouling behavior on different solid surfaces under the condition of different CaCO 3 saturation levels and bulk velocities has been examined, and the growth curves of mixed fouling have been obtained. The results show that the mixed fouling behavior on various material surfaces depends mainly on the affinity of bacteria to a material. The mixed fouling mass developed on polymer materials is much more than that on metal surfaces. With the increase of saturation level of CaCO 3 , however, this difference becomes minimal. The results also indicate that the mixed fouling mass decreases with the increase of CaCO 3 saturation degree and velocity. The induction period of mixed fouling declines with the rising of velocity. Moreover, the induction period of mixed fouling is longer than that of pure biofouling when the saturation level of CaCO 3 is less than 1 and greater than 0. The induction period decreases with CaCO 3 saturation degree when the level is more than or equal to 1. In addition, the sequence of adhesion and deposit of the bacteria and CaCO 3 on a glass surface was measured with micro-video technology. The results show that CaCO 3 first deposits on the solid surface, while the bacteria need longer time to adhere on the surface.

Growth modes of condensates on nano-textured surfaces and mechanism of partially wetted droplet formation

Condensed droplets on different nano-textured surfaces may appear in three distinct wetting states, the Cassie–Baxter state with composite wetting, Wenzel state with complete wetting, and the partially wetted (PW) state. To maintain the super-hydrophobicity of a textured surface, condensed drops on it are usually expected to be in a Cassie–Baxter or PW state. Therefore, it is of importance to clarify the relation between condensed droplet wetting states and the nano-pillar geometries of surfaces. In view of the fact that all condensed droplets in diverse wetting states originate from the nuclei and/or condensate spots growing along different pathways, we think that the distinct growth modes of a condensate correspond to different energy increasing rates (EIRs), and a condensed drop should grow along the route with the minimum EIR. In this paper, accordingly, the EIRs of a droplet on different textured surfaces were analyzed during its growth along three pathways. The results show that the smallest initial EIR of a condensate spot occurs in the increasing contact angel (CA) mode, so that it will grow with the CA enlarging and the base area initially remaining unchanged. Then the EIR of the increasing CA mode becomes much higher than that of the other two modes. The base area of the drop begins to enlarge while the CA remains unchanged. During this period, the increasing base area can be either in a wetted or composite state, resulting in a Wenzel or PW droplet forming, respectively. The growth mode and the wetting state of a condensed droplet are strongly related to the nano-structure of the surface. Additionally, the calculation results of this model are consistent with experimental observations in the literature for the wetting states of condensed drops on nano-textured surfaces, with an accuracy of 91.9%, which is higher than the accuracy of results calculated with previously reported formulas.

Thermo-responsive poly(N-isopropylacrylamide)-grafted hollow fiber membranes for osteoblasts culture and non-invasive harvest

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.

Theoretical analysis of droplet transition from Cassie to Wenzel state

Whether droplets transit from the Cassie to the Wenzel state (C–W) on a textured surface is the touchstone that the superhydrophobicity of the surface is still maintained. However, the C–W transition mechanism, especially the spontaneous transition of small droplets, is still not very clear to date. The interface free energy gradient of a small droplet is firstly proposed and derived as the driving force for its C–W evolution in this study based on the energy and gradient analysis. Then the physical and mathematical model of the C–W transition is found after the C–W driving force or transition pressure, the resistance, and the parameters of the meniscus beneath the droplet are formulated. The results show that the micro/nano structural parameters significantly affect the C–W driving force and resistance. The smaller the pillar diameter and pitch, the minor the C–W transition pressure, and the larger the resistance. Consequently, the C–W transition is difficult to be completed for the droplets on nano-textured surfaces. Meanwhile if the posts are too short, the front of the curved liquid–air interface below the droplet will touch the structural substrate easily even though the three phase contact line (TPCL) has not depinned. When the posts are high enough, the TPCL beneath the drop must move firstly before the meniscus can reach the substrate. As a result, the droplet on a textured surface with short pillars is easy to complete its C–W evolution. On the other hand, the smaller the droplet, the easier the C–W shift, since the transition pressure becomes larger, which well explains why an evaporating drop will collapse spontaneously from composite to Wenzel state. Besides, both intrinsic and advancing contact angles affect the C–W transition as well. The greater the two angles, the harder the C–W transition. In the end, the C–W transition parameters and the critical conditions measured in literatures are calculated and compared, and the calculations accord well with the experimental results.

Differentiation of reprogrammed human adipose mesenchymal stem cells toward neural cells with defined transcription factors.

Somatic cell reprogramming may become a powerful approach to generate specific human cell types for cell-fate determination studies and potential transplantation therapies of neurological diseases. Here we report a reprogramming methodology with which human adipose stem cells (hADSCs) can be differentiated into neural cells. After being reprogrammed with polycistronic plasmid carrying defined factor OCT3/4, SOX2, KLF4 and c-MYC, and further treated with neural induce medium, the hADSCs switched to differentiate toward neural cell lineages. The generated cells had normal karyotypes and exogenous vector sequences were not inserted in the genomes. Therefore, this cell lineage conversion methodology bypasses the risk of mutation and gene instability, and provides a novel strategy to obtain patient-specific neural cells for basic research and therapeutic application.