Micro-manufacturing : research, technology outcomes and development issues

Besides continuing effort in developing MEMS-based manufacturing techniques, latest effort in Micro-manufacturing is also in Non-MEMS-based manufacturing. Research and technological development (RTD) in this field is encouraged by the increased demand on micro-components as well as promised development in the scaling down of the traditional macro-manufacturing processes for micro-length-scale manufacturing. This paper highlights some EU funded research activities in micro/nano-manufacturing, and gives examples of the latest development in micro-manufacturing methods/techniques, process chains, hybrid-processes, manufacturing equipment and supporting technologies/device, etc., which is followed by a summary of the achievements of the EU MASMICRO project. Finally, concluding remarks are given, which raise several issues concerning further development in micro-manufacturing

Pharmacological Inhibition of Caspase and Calpain Proteases: A Novel Strategy to Enhance the Homing Responses of Cord Blood HSPCs during Expansion

Background: Expansion of hematopoietic stem/progenitor cells (HSPCs) is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB) derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion. Methodology/Principal Findings: CB derived CD34 + cells were expanded using a combination of growth factors with and without Caspase inhibitor-zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homingrelated molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors) caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice

Redox responsive Pluronic micelle mediated delivery of functional siRNA: a modular nano-assembly for targeted delivery

There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of similar to 291 nm in DLS and similar to 200-250 nm in SEM at 37 degrees C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in similar to 65% silencing of STAT3 mRNA in BT-13 gliospheres, while only similar to 20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.Peer reviewe

Improved expression of CD49d and CD49e integrins and higher adhesion of the expanded HSPCS.

<p>(A) Multicolour flow cytometry analysis showed a an increase of CD49d and Cd49e integrins in the primitive CD34⁺CD38⁻ fraction of inhibitor-treated HSPCs. Data are represented as mean percentage ± standard deviation of four experiments, *<i>p<</i>0.05. (B) Adhesion to extracellular matrix protein fibronectin was significantly increased (up to two fold) in the zVADfmk/zLLyfmk cultures compared to the control. Data are represented as mean ± standard deviation of four experiments, *<i>p<</i>0.05. (C) The adhered zVADfmk/zLLYfmk HSPCs retained a higher β1 integrin ligand binding status and the activation of focal adhesion kinase (D) compared to the control, green- HUTS 21, red – FAKpY397, blue – nuclei, scale 10 µm.</p

Actin dynamics of expanded HSPCs.

<p>(A) The expanded control and zVADfmk/zLLYfmk HSPCs were assessed for the polymerized actin (F actin) using fluorescein conjugated phalloidin. The presence of zVADfmk and zLLYfmk enhanced the of F actin fluorescence intensity compared to the control. Data are represented as mean ± standard deviation of four experiments, *<i>p</i>≤0.05. (B) Actin polymerization assay done by exposing the control and test HSPCs to SDF1α for different time intervals showed an early response and higher polymerization of actin at all time points analyzed in the zVADfmk/zLLYfmk sets. Data are represented as mean ± standard deviation of three experiments. Statistical analysis was always made between control vs. zVADfmk/zLLYfmk at all time points. *<i>p</i>≤0.05, **<i>p<</i>0.01. (C) The migrated population from the zVADfmk/zLLYfmk HSPCs showed a typical polarized morphology with a prominent localization of F actin towards the leading edge of the cells (white arrow heads & insets) where as in control cells a homogeneous distribution of F actin was seen without much visible cell polarization, green – F actin, bar = 10 µm. (D) The expression of RhoGTPase members RhoA, Rac1 and Cdc42 was found to be increased in the zVADfmk/zLLYfmk HSPCs, green –RhoA, Rac1 and Cdc42, bar = 10 µm (E) Quantitative real time PCR analysis showing a significant up regulation in the gene expression of RhoA upto (3.2 fold) and Cdc42 (up to 2.5 fold) in the zVADfmk/zLLYfmk expanded HSPCs compared to the control, whereas the Rac1 showed a modest increase in those sets. Data are represented as mean ± standard deviation of three experiments, *<i>p</i>≤0.05, **<i>p<</i>0.01.</p

Higher expression of adhesion molecules in the expanded HSPCs.

<p>(A) & (B & C) Flow cytometry analyses of HSPCs after staining for CD34 vs CD62L, CD34 vs CD54 and CD34 vs CD44 antibodies demonstrate higher positive population of these adhesion molecules on CD34⁺ progenitor cells. (D) Alternatively multi colour flow cytometry analyses showed that zVADfmk/zLLYfmk enhanced the presence of CD62L, CD44 and CD54 on CD34⁺CD38− subsets as well. Data are represented as mean percentage ± standard deviation of four experiments, *<i>p</i><0.05, **<i>p</i><0.01.</p

<i>In vitro</i> migration of expanded CB HSPCs.

<p>(A) The presence of zVADfmk/zLLYfmk during <i>ex vivo</i> expansion significantly increased the chemotaxis of HSPCs towards SDF1α compared to the cytokine-expanded (control) counterpart. Data are represented as mean ± standard deviation of four experiments, *<i>p</i>≤0.05, **<i>p</i>≤0.01. (B) The migration of expanded HSPCs towards SDF1α was reduced when a neutralizing antibody to CXCR4 was used, indicating the direct role of CXCR4 in maintaining the chemotactic responses. Data are represented as mean ± standard deviation of three experiments. (C) The colony forming assay of the migrated population showed a higher clonogenicity, especially the presence of primitive progenitors in terms of CFU mix/GEMM colonies in the zVADfmk/zLLYfmk cultures. Data obtained from three experiments are represented as mean ± standard deviation, *<i>p</i>≤0.05, **<i>p</i><0.01.</p

CXCR4 expression analysis of the cultured HSPCs.

<p>(A) The expanded cells were stained using anti CXCR4 antibody and the expression was assessed by flow cytometry. HSPCs cultured in the presence of zVADfmk/zLLYfmk showed a two-fold increase in the CXCR4⁺ population compared to the control counterpart. Data are represented as mean percentage ± standard deviation of five biological replicates, <i>*p≤0.05</i>, <i>**p≤0.01</i>. A representative flow-overlay is shown in the inset. (B) Confocal microscopy images further confirm higher expression of CXCR4 (green) on cell surface of HSPCs from zVADfmk/zLLYfmk cultures than that of controls. Cell nuclei (blue) were stained with DAPI (bar  = 10 µm, n = 3). (C) Flow cytometry analyses of HSPCs after multicolor staining (CD34 and CXCR4 or CD34, CD38 and CXCR4) demonstrate higher CXCR4 positive population in CD34⁺ and CD34⁺CD38⁻ compartments. Thus, zVADfmk/zLLYfmk facilitates higher production of CXCR4 expressing CD34⁺ and CD34⁺CD38⁻ hematopoietic progenitor cells. Data are represented as mean percentage ± standard deviation of four experiments, <i>*p≤0.5.</i></p