Wednesday, November 21, 2012

Oncolytic viruses=Cancer Killers

Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, researchers enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors.

Protein expression  levels were determined using Neuromics' Goat Polyclonal MMP-9 antibody: Simon Schäfer, Stephanie Weibel, Ulrike Donat, Qian Zhang, Richard J Aguilar, Nanhai G Chen and Aladar A Szalay. Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors. BMC Cancer 2012, 12:366 doi:10.1186/1471-2407-12-366.

Images and Figures: MMP-9 expression in PC-3 tumor sections and collagen IV quantification. Intratumoral expression of MMP-9 (red) was visualized using MMP-9 labeled PC-3 tumor sections. Nuclei (white) were stained with Hoechst 33258 dye. GFP (green) is a VACV reporter gene. Tumors were obtained at 7 days p.i. from PC-3 tumor-bearing mice injected with PBS, GLV-1h68 or GLV-1h255. All images are representative examples. (B) Quantification of MMP-9 expression was done by microscopic analysis. Mean fluorescence intensities were measured with ImageJ. (C) Quantification of collagen IV 7 days p.i. in GLV-1h68 or GLV-1h255 infected areas of PC-3 tumor sections. Images were taken at a 100× magnification (Leica MZ16 FA) and converted from RGB to grayscale using Photoshop. For image analysis ImageJ was used, the threshold value was 8/255.

Figure and Images: Expression of functional MMP-9 by GLV-1h255-infected tumor cells. (A) Expression cassettes of GLV-1h68 and GLV-1h255. In GLV-1h255 the insert in the Tk locus was replaced by the human mmp-9 gene under control of the PSE promoter. PSEL, synthetic early/late promoter; PSE, synthetic early promoter; P7.5, VACV p7.5 K early/late promoter; P11, VACV p11 late promoter; Tk, thymidine kinase locus, Ha, hemagglutinin locus. (B) Expression of virus-encoded MMP-9 (92 kDa) in GLV-1h255 infected PC-3 cells and supernatants in vitro, β-actin (42 kDa) was used as a loading control. (C) Activity of the MMP-9 protein was tested by gelatin zymography. Lysates and supernatants of infected A549 cells were isolated and separated by non-reducing SDS-PAGE. In zymography, cleavage of the substrate by MMP-9 resulted in a clear band.

This study revealed that the degradation of collagen IV (ECM) by VACV-encoded MMP-9 may represent a new option to significantly enhance the oncolytic effect of rVACV in PC-3 xenografts. We confirmed that the degradation of collagen IV facilitated viral infection of the tumor tissue, represented by significantly higher viral tumor titers and an accelerated tumor regression. Furthermore, both oncolytic viruses, parental GLV-1h68 and mmp-9-encoding GLV-1h255, significantly reduced the size of lumbar and renal lymph node metastases, indicating that MMP-9 enhances both virotherapy of the primary tumor and sustains the rVACV-metastasis reducing effect.

I will keep you posted on further studies.

Saturday, November 10, 2012

More On Petaka Mini Bioreactors

Getting Culture Conditions Right Every Time

I would like to provide yet more information on the capabilties of our new and innovative Petaka Mini Bioreactors. This posting focuses how oxygen concentrations are tightly controlled in a way that is consistent with the cultured cells natural environment.

The length and cross-section of the respiratory duct is purposely engineered to partially restrict the diffusion of oxygen from the high levels of ambient air to create lower, physiologic levels of dissolved oxygen in the reaction chamber. In accordance to Fick’s Law, as oxygen is consumed inside the culture chamber, decreasing the partial pressure of oxygen in the media, oxygen diffuses in from the outside atmospheric (higher) partial pressure, through the respiratory duct, to the lower partial pressure inside. Diffusion is proportional to the concentration gradient, as regulated by the engineered design of the respiratory duct, and occurs entirely spontaneously and without any manual intervention whatsoever.

Image: Petaka®G3 Ducted Respiratory Chamber (DRC). (1) cell culture chamber;(2) injection port; (3) respiratory duct; (4) 0.2μ filter; (5) water vapor condensers and capillary breakers; (6) unique barcode. The DRC is shown upright in a silicone stand (7).

At the same time, the respiratory duct partially retains carbon dioxide from cellular metabolism to maintain a physiologically normal mild acidosis to balance pH. All gas exchange with the outside environment occurs via a 0.2 micron filtered vent, preserving the internal sterility of the device but allowing exchange of gas diffusion and flow to prevent pressurization issues when filling and emptying the bioreactors.
Therefore, cell culture in DRC’s/Petaka G3 does not require supplemental oxygen-nitrogen balancing, CO2 and humidity sources, eliminating the entire panoply of gas tanks, regulators, sensors, microprocessors and water pans. This creates a double benefit: not only are cells cultured in more normal physiologic conditions, but the mechanics, logistics, risks and costs of cell culture are greatly simplified and reduced.

The Results are Stunning!

Here are result using Petaka® G3 LOT for culturing our UCB Derived hMSCs and Mouse MSCs.
Images: (A) Immuno-fluorescence microscopy of a mouse MSC in differentiation progression. Culture under 20 mmHg of O2 partial pressure. Red fluorescence positive staining of GFAP. hMSC 3 hours after seeding in Petaka G3 with low serum media and without matrix (B) and 96 h later (C). Photos: Jim Musick. Vitro-Biopharma. September, 2012.

Next up using Petaka DRCs for GMP. Stay tuned.

Sunday, November 04, 2012

True Physiologic Conditions in Cell Culturing

The Petaka Advantage!

A cornerstone of Neuromics' strategy is to enable better science by finding ways to help our customers and collaborators to both improve and lower the overall costs of their cell based assays.

We embrace new technologies if they prove capable of providing cell culture environment that more closely mimic in vivo environments. This enables basic and drug discovery researchers learn more about the true potential of targets. More informed decisions early in the process reduce downstream costs.

This is why the use of engineered mini bioreactors with Ducted Respiratory Chambers (DRCs) like the Petaka® G3 LOT needs to be considered. (detailed information @ Ducted Respiratory Chamber Bioreactors.© 2012 Genetic Engineering & Biotechnology News All Rights Reserved). Instead of attempting to impose “normal” gas conditions on the cells through active incubator controls, this system passively allows cells to maintain their own oxygen, carbon dioxide, and humidity levels.
Image: Petaka's DRC design. The cell culture chamber is isolated on the injection side from the atmosphere by a self-sealing silicone injection port that allows the closed introduction of media and cells, including most eukaryotic cells types, small early-stage embryos, tissue fragments, and even needle biopsies.

Because the cells control their own gas environment, there is little effect from outside gas conditions, and the DRC is effective in atmospheres from 500 meters below sea level up to elevations of 4,000 meters, and in relative humidity levels between 10% and 100%.
Figure: Self-regulating gas management in the DRC. Cells consume O2, with restricted O2 ingress, causing a first proliferative cell phase to evolve into a second differentiating cell phase for protein production and gene expression.

The DRC changes the paradigm of cell culture, replacing nearly a century of active attempts to humanly intervene to manipulate gas exchange with a self-regulating design driven by natural laws of diffusion to create dependable, and truly physiologic, gas environments.