Aerodynamically assisted jetting and threading for processing concentrated suspensions containing advanced structural,functional and biological materials

In recent years material sciences have been interpreted right across the physical and the life sciences. Essentially this discipline broadly addresses the materials, processing, and/or fabrication right up to the structure. The materials and structures areas can range from the micro- to the nanometre scale and, in a materials sense, span from the structural, functional to the most complex, namely biological (living cells). It is generally recognised that the processing or fabrication is fundamental in bridging the materials with their structures. In a global perspective, processing has not only contributed to the materials sciences but its very nature has bridged the physical with the life sciences. In this review we discuss one such swiftly emerging fabrication approach having a plethora of applications spanning the physical and life sciences.     

Hepatic stem cells: A viable approach for the treatment of liver cirrhosis

Liver cirrhosis is characterized by distortion of liver architecture, necrosis of hepatocytes and regenerative nodules formation leading to cirrhosis. Various types of cell sources have been used for the management and treatment of decompensated liver cirrhosis. Knowledge of stem cells has offered a new dimension for regenerative therapy and has been considered as one of the potential adjuvant treatment modality in patients with end stage liver diseases (ESLD). Human fetal hepatic progenitor cells are less immunogenic than adult ones. They are highly propagative and challenging to cryopreservation. In our earlier studies we have demonstrated that fetuses at 10-18 wk of gestation age contain a large number of actively dividing hepatic stem and progenitor cells which possess bi-potent nature having potential to differentiate into bile duct cells and mature hepatocytes. Hepatic stem cell therapy for the treatment of ESLD is in their early stage of the translation. The emerging technology of decellularization and recellularization might offer a significant platform for developing bioengineered personalized livers to come over the scarcity of desired number of donor organs for the treatment of ESLD. Despite these significant advancements long-term tracking of stem cells in human is the most important subject nowadays in order to answer several unsettles issues regarding the route of delivery, the choice of stem cell type(s), the cell number and the time-point of cell delivery for the treatment in a chronic setting. Answering to these questions will further contribute to the development of safer, noninvasive, and repeatable imaging modalities that could discover better cell therapeutic approaches from bench to bed-side. Combinatorial approach of decellularization and nanotechnology could pave a way towards the better understanding in determination of cell fate post-transplantation.     

Surface-based cryopreservation strategies for human embryonic stem cells: A comparative study

Human embryonic stem cells (hESC) hold tremendous potential in the emerging fields of gene and cell therapy as well as in basic scientific research. One of the major challenges regarding their application is the development of efficient cryopreservation protocols for hESC since current methods present poor recovery rates and/or technical difficulties which impair the development of effective processes that can handle bulk quantities of pluripotent cells. The main focus of this work was to compare different strategies for the cryopreservation of adherent hESC colonies. Slow‐rate freezing protocols using intact hESC colonies was evaluated and compared with a surface‐based vitrification approach. Entrapment within ultra‐high viscous alginate was investigated as the main strategy to avoid the commonly observed loss of viability and colony fragmentation during slow‐rate freezing. Our results indicate that entrapment beneath a layer of ultra‐high viscous alginate does not provide further protection to hESC cryopreserved through slow‐rate freezing, irrespectively of the cryomedium used. Vitrification of adherent hESC colonies on culture dishes yielded significantly higher recovery rates when compared to the slow‐rate freezing approaches investigated. The pluripotency of hESC was not changed after a vitrification/thawing cycle and during further propagation in culture. In conclusion, from the cryopreservation methods investigated in this study, surface‐based vitrification of hESC has proven to be the most efficient for the cryopreservation of intact hESC colonies, reducing the time required to amplify frozen stocks thus supporting the widespread use of these cells in research and clinical applications. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1079–1087, 2012     

Large-scale compatible methods for the preservation of human embryonic stem cells: current perspectives

Human embryonic stem cells (hESCs) and hESC-derived cells are of great interest, not only because of their therapeutic potential but also their prospective uses in in vitro drug and toxicity screening. The ability to preserve these cells is critical, allowing for the generation of quality-controlled stocks of cells, transport of cells between sites, and avoiding the need for expensive and time-consuming continuous culture. Current methodologies, namely conventional slow freezing and vitrification, can successfully preserve hESCs and their differentiated progeny, retaining the key characteristics of the cells. However, there is a significant gap between the number of cells potentially needed to either treat patients or run a high-throughput drug screen and how many cells can be preserved using these techniques.Therefore, this review focuses on the scalability of slow freezing and vitrification, identifying key barriers to success and whether they can be overcome. Given the precedent with other mammalian cells in using slow freezing to successfully preserve large quantities of cells and its compatibility with current and emerging culture methods for hESCs, it is likely to become the method of choice for cryopreserving these cells at scale. However, issues other than scale still exist; therefore, alternatives to cryopreservation should also be explored. Here,the potential to lyophilize hESCs for long-term storage is considered as one such alternative.     

Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single‐chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti‐PCNA scFvs were screened in vitro after being tagged with dimeric glutathione‐S‐transferase. Anti‐PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error‐prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C‐terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide‐conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody‐based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright © 2014 John Wiley & Sons, Ltd.     

Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues

Mesenchymal stem cell (MSC) research progressively moves towards clinical phases. Accordingly, a wide range of different procedures were presented in the literature for MSC isolation from human tissues; however, there is not yet any close focus on the details to offer precise information for best method selection. Choosing a proper isolation method is a critical step in obtaining cells with optimal quality and yield in companion with clinical and economical considerations. In this concern, current review widely discusses advantages of omitting proteolysis step in isolation process and presence of tissue pieces in primary culture of MSCs, including removal of lytic stress on cells, reduction of in vivo to in vitro transition stress for migrated/isolated cells, reduction of price, processing time and labour, removal of viral contamination risk, and addition of supporting functions of extracellular matrix and released growth factors from tissue explant. In next sections, it provides an overall report of technical highlights and molecular events of explant culture method for isolation of MSCs from human tissues including adipose tissue, bone marrow, dental pulp, hair follicle, cornea, umbilical cord and placenta. Focusing on informative collection of molecular and methodological data about explant methods can make it easy for researchers to choose an optimal method for their experiments/clinical studies and also stimulate them to investigate and optimize more efficient procedures according to clinical and economical benefits.     

Perfusion cultures of human tumor cells: a scalable production platform for oncolytic adenoviral vectors

This study demonstrates the novel use of the HeLaS3 human tumor cell line for propagating ONYX-411, a recombinant oncolytic adenoviral vector. HeLaS3 cells enabled high levels of vector production without the risk of generating vector recombinants, which is possible with HEK293 cells. The development of a high-cell-density perfusion process using ATF technology yielded production levels as high as 6 x 10(11) vp/mL, which was approximately sevenfold greater than the titers achieved in fed-batch bioreactors. Several experiments were performed at the bench (15 L) and pilot (70 L) scales to demonstrate the robust and scalable nature of this industrially relevant technology.     

Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity

To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO)/DG44 cell line by sequential homologous recombination. FUT8 encodes an alpha-1,6-fucosyltransferase that catalyzes the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNAc) in an alpha-1,6 linkage. FUT8(-/-) cell lines have morphology and growth kinetics similar to those of the parent, and produce completely defucosylated recombinant antibodies. FUT8(-/-)-produced chimeric anti-CD20 IgG1 shows the same level of antigen-binding activity and complement-dependent cytotoxicity (CDC) as the FUT8(+/+)-produced, comparable antibody, Rituxan. In contrast, FUT8(-/-)-produced anti-CD20 IgG1 strongly binds to human Fcgamma-receptor IIIa (FcgammaRIIIa) and dramatically enhances ADCC to approximately 100-fold that of Rituxan. Our results demonstrate that FUT8(-/-) cells are ideal host cell lines to stably produce completely defucosylated high-ADCC antibodies with fixed quality and efficacy for therapeutic use.     

Human CD8+ T cells transduced with an additional receptor bispecific for both Mycobacterium tuberculosis and HIV‐1 recognize both epitopes

Tuberculosis (TB) and human immunodeficiency virus type 1 (HIV‐1) infection are closely intertwined, with one‐quarter of TB/HIV coinfected deaths among people died of TB. Effector CD8⁺ T cells play a crucial role in the control of Mycobacterium tuberculosis (MTB) and HIV‐1 infection in coinfected patients. Adoptive transfer of a multitude of effector CD8⁺ T cells is an appealing strategy to impose improved anti‐MTB/HIV‐1 activity onto coinfected individuals. Due to extensive existence of heterologous immunity, that is, T cells cross‐reactive with peptides encoded by related or even very dissimilar pathogens, it is reasonable to find a single T cell receptor (TCR) recognizing both MTB and HIV‐1 antigenic peptides. In this study, a single TCR specific for both MTB Ag85B_199‐207 peptide and HIV‐1 Env_120‐128 peptide was screened out from peripheral blood mononuclear cells of a HLA‐A*0201⁺ healthy individual using complementarity determining region 3 spectratype analysis and transferred to primary CD8⁺ T cells using a recombinant retroviral vector. The bispecificity of the TCR gene‐modified CD8⁺ T cells was demonstrated by elevated secretion of interferon‐γ, tumour necrosis factor‐α, granzyme B and specific cytolytic activity after antigen presentation of either Ag85B_199‐207 or Env_120‐128 by autologous dendritic cells. To the best of our knowledge, this study is the first report proposing to produce responses against two dissimilar antigenic peptides of MTB and HIV‐1 simultaneously by transfecting CD8⁺ T cells with a single TCR. Taken together, T cells transduced with the additional bispecific TCR might be a useful strategy in immunotherapy for MTB/HIV‐1 coinfected individuals.