Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.     

Emergence,Development, and Maturity of the Gonad of Two Species of Chitons “Sea Cockroach” (Mollusca: Polyplacophora) through the Early Life Stages

This study describes and recognises, using histological and microscopical examinations on a morphometrical basis, several gonad traits through the early life stages of Chiton articulatus and C. albolineatus. Gonadal ontogenesis, gonad development stages, sexual differentiation, onset of the first sexual maturity, and growth sequences or “early life stages” were determined. In addition, allometry between lengths and body weight pooled for both sexes per each chiton were calculated using equation Y = aX^b. A total of 125 chitons (4≤TL≤40 mm, in total length “TL”) were used. All allometric relations showed a strong positive correlation (r), close to 1, with b-values above three, indicating an isometric growth. Gonadal ontogenesis and gonad development stages were categorised into three periods (“Pw” without gonad, “Pe” gonad emergence, and “Pf” gonadal sac formed) and four stages (“S0” gametocytogenesis, “S1” gametogenesis, “S2” mature, and “S3” spawning), respectively. Compound digital images were attained for each process. Periods and stages are overlapped among them and between species, with the following overall confidence intervals in TL: Pw 6.13–14.32 mm, Pe 10.32–16.93 mm, Pf 12.99–25.01 mm, S0 16.08–24.34 mm (females) and 19.51–26.60 mm (males), S1 27.15–35.63 mm (females) and 23.45–32.27 mm (males), S2 24.48–40.24 mm (females) and 25.45–32.87 mm (males). Sexual differentiation (in S0) of both chitons occurs first as a female then as a male; although, males reach the onset of the first sexual maturity earlier than females, thus for C. articulatus males at 17 mm and females at 32 mm, and for C. albolineatus males at 23.5 mm and females at 28 mm, all in TL. Four early life stages (i.e., subjuvenile, juvenile, subadult, and adult) are described and proposed to distinguish growth sequences. Our results may be useful to diverse disciplines, from developmental biology to fisheries management.     

MicroRNA and exosome: Key players in rheumatoid arthritis

Rheumatoid arthritis (RA) is known as one of important autoimmune disorders which can lead to joint pain and damage throughout body. Given that internal (ie, genetic and epigenetic alterations) and external factors (ie, lifestyle changes, age, hormones, smoking, stress, and obesity) involved in RA pathogenesis. Increasing evidence indicated that cellular and molecular alterations play critical roles in the initiation and progression of RA. Among various targets and molecular signaling pathways, microRNAs (miRNAs) and their regulatory networks have key roles in the RA pathogenesis. It has been showed that deregulation of many miRNAs involved in different stages of RA. Hence, identification of miRNAs and their signaling pathways in RA, could contribute to new knowledge which help to better treatment of patients with RA. Besides miRNAs, exosomes have been emerged as key messengers in RA pathogenesis. Exsosomes are nanocarriers which could be released from various cells and lead to changing of behaviors recipient cells via targeting their cargos (eg, proteins, messenger RNAs, miRNAs, long noncoding RNAs, DNAs). Here, we summarized several miRNAs involved in RA pathogenesis. Moreover, we highlighted the roles of exosomes in RA pathogenesis.     

CNTF overproduction hastens onset of symptoms in motor neuron degeneration (mnd) mice

Ciliary neurotrophic factor (CNTF) promotes the survival of motor neurons, in vitro and in vivo. Moreover, CNTF can block the degeneration of injured or diseased motor neurons in young rodents. Motor neuron degeneration (mnd) mutant mice display adult onset symptoms reflecting progressive motor debilitation and provide a model in which to test the hypothesis that CNTF can prevent the loss of these motor functions. We generated mnd mice that harbor a genomically integrated transgene, resulting in overexpression of the encoded CNTF protein in these mice. In contrast to the beneficial effects of CNTF in preventing motor neuron degeneration in other experimental paradigms, we report that overproduction of CNTF increased the rate of onset of motor disease symptoms in mnd mice and the presence of the transgene correlated with low adult body weight in mnd and wild-type genetic backgrounds. © 1996 John Wiley & Sons, Inc.     

Observation and establishment of gonad development stages in polyplacophorans (Mollusca): Chiton (Chiton) articulatus a case study

Gonad development stages (GDS) and, subsequently, the reproductive cycle are described by performing histology of some gonad portions. In polyplacophorans, gametogenesis is not enough to define GDS; further anatomical gonad features are relevant. In most adult polyplacophorans, the gonad is a simple anatomical structure that resembles and operates as one single gonadal acinus without glandular structure. These features have gone unnoticed causing inaccurate GDS assignment and, consequently, imprecise reproductive season in polyplacophorans. Here, dissection protocols that allow extracting a compact gonad are established. Emphasizing the anatomical structure of the whole gonad and the displacement of gametes, five GDS were assigned to both sexes of Chiton (Chiton) articulatus: I‐goniogenesis, II‐development, III‐ripe, IV‐spawning, V‐rest. Tissue platelets contribute importantly to GDS assignment and even help distinguishing between males and females. Neither a randomly selected portion of gonad nor a longitudinal section are recommended because it leads to misinterpretation higher than 50% in determining GDS and besides ignores displacement of gametes. A panoramic sweep across a complete transverse‐section of each gonad was validated as the best option for establishing GDS. This new methodology was tested on several polyplacophorans species, and seems generally applicable for histological assessment of reproductive cycle and reproductive season in polyplacophorans.     

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use,Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel? technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1600–1612, 2015     

Scalable Manufacturing of Human Mesenchymal Stromal Cells in the Vertical‐Wheel Bioreactor System: An Experimental and Economic Approach

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering applications and cell‐based therapies. Large cell doses (>1 × 10⁶ cells kg^?1) and Good Manufacturing Practices (GMP)‐compliant processes are however required for clinical purposes. Here, a serum‐ and xenogeneic‐free (S/XF) microcarrier‐based culture system is established for the expansion of human umbilical cord matrix (UCM)‐ and adipose tissue (AT)‐derived MSC using the Vertical‐Wheel system (PBS‐0.1 MAG; PBS Biotech). UCM and AT MSC are expanded to maximum cell densities of 5.3 ± 0.4 × 10⁵ cell mL^?1 (n = 3) and 3.6 ± 0.7 × 10⁵ cell mL^?1 (n = 3), respectively, after 7 days of culture, while maintaining their identity, according to standard criteria. An economic evaluation of the process transfer from T‐flasks to PBS‐0.1 MAG shows a reduction in the costs associated with the production of a dose for an average 70 kg adult patient (i.e., 70 million cells). Costs decrease from $17.0 K to $11.1 K for UCM MSC and from $21.5 K to $11.1 K for AT MSC, proving that the transition to Vertical‐Wheel reactors provides a cost‐effective alternative for MSC expansion. The present work reports the establishment of a scalable and cost‐effective culture platform for the manufacturing of UCM and AT MSC in a S/XF microcarrier‐based system.