Hepatic Cellular Models

A wide range of in vitro models are used in preclinical drug testing for the investigation of ADME-Tox (Absorption, Distribution, Metabolism, Excretion and oxicity) properties of New Chemical Entities (NCEs). The liver is the main organ with regards to ADME-Tox, it’s the place of more than 500 different functions, including: metabolism of lipids, carbohydrates, and vitamins, detoxification, production of bile, albumin, fibrinogen, globulin, etc (1). The liver lobule is composed of parenchymal cells (hepatocytes) and nonparenchymal cells (Kupffer cells, hepatic stellate cells, and sinusoidal endothelial cells).

Hepatocytes – the Gold Standard

Hepatocytes represent 80% of liver volume. Hepatocytes are commonly used in drug discovery and preclinical drug development to perform experiments requiring intact cellular systems. Intact hepatocytes contain the major hepatic drug-metabolizing enzymes required to study the four categories of xenobiotic biotransformation: hydrolysis, reduction, oxidation and conjugation.

Because of these enzymes, hepatocytes provide a viable and cost-effective alternative to in vivo testing. [Read more…]

HD-LCI video of HUVEC Transfection with Cytofect Kit

See human cells glow with green fluorescent protein in this time-lapse video. These images were captured over 20 hours via Lumascope 620. [Read more…]

Formation of Mesenchymal Tissues in Alvetex® Scaffold

The Alvetex®Scaffold is a novel substrate that enables a solution for simple and routine 3D culture. It is composed of a highly porous polystyrene scaffold that has been engineered into a 200 micron thick membrane to enable entry of cells and efficient exchange of gases and solutes. Cells enter the fabric of the scaffold, retain their natural 3D structure, and form close 3D interactions with adjacent cells. Unlike conventional 2D culture, cells in Alvetex®Scaffold do not grow as monolayers and do not undergo the flattened shape transition that can result in aberrant changes to gene and protein expression and consequently cellular function.

Mesenchymal stem cells (MSCs) are adherent multipotent cells derived from tissue such as bone marrow and which possess the ability to differentiate in vitro into a number of tissue types including bone, cartilage and muscle [1].

In this post, we demonstrate that MSCs extracted from the bone marrow of adult rats can be successfully cultured in 3D in Alvetex®Scaffold and induced to differentiate into osteogenic and adipogenic derivatives more efficiently than their 2D counterparts. We also report bone formation and the production of extracellular matrix by MG63 cells which represent an established cell line derived from a human osteosarcoma. The data generated here is supported by peer-reviewed literature [2,3] and clearly shows that Alvetex®Scaffold promotes enhanced in vitro differentiation. [Read more…]

Reprogramming-qualified B18R Recombinant Protein, Carrier-free

Reprogramming-qualified B18R Recombinant Protein, Carrier-free is of interest for cellular reprogramming and other applications requiring RNA-mediated gene delivery.  B18R protein is a Vaccinia virus-encoded receptor with specificity for mouse, human, rabbit, pig, rat and cow Type 1 interferons and has potent neutralizing capability acting as a decoy receptor for Type 1 interferons.

Biological Activity

  • Stemfactor B18R Recombinant Protein, Carrier-free bioactivity is demonstrated by the neutralization of IFN-alpha or IFN-beta effects in an assay of IL-12 production from CD40L-activated monocytes.
  • The biological function is validated by down-regulation of gene expression by qRT-PCR of TLR3, IFIT1, and OASI in response to B18R treatment on human fibroblasts to levels equivalent to non-transfected cells.
  • The efficiency of mRNA reprogramming is tested by performing mRNA reprogramming with OKSML on neonatal foreskin fibroblasts and demonstrating greater than 0.5% iPSC colony formation.
NuFF feeder cells plated at at 2.5 x 10E5 cells per well on Day -2. Images were captured 24 hours after plating and represent an appropriate NuFF feeder layer density for plating target cells on Day -1.

NuFF feeder cells plated at at 2.5 x 10E5 cells per well on Day -2.
Images were captured 24 hours after plating and represent an appropriate NuFF feeder layer density for plating target cells on Day -1.

Previously only available as part of the mRNA Reprogramming Kit or microRNA Booster KitReprogramming-qualified B18R Recombinant Protein, Carrier-free is now available individually.

PBMCs & drug development

Use of human samples including peripheral blood mononuclear cells (PBMCs) in drug discovery is critical for increasing the chances of success for a small molecule screen hit. A review based on recent published data!

PBMCs DD 1Several theories try to explain why many drug discovery and development projects have a moderate success rate. Even with the advances made in methods (HTS, HCA, library managment, etc), there are many hurdles for a small molecule on the long road to FDA or EMA approval.

One way that may improve predicted efficacy and toxicity of drug leads is to use human samples, such as blood, early on in drug discovery programs. Peripheral blood mononuclear cells  (PBMCs) can be easily used in a variety of ways during the drug discovery process to gain a better understanding of the effects of a small molecule. [Read more…]

Case study: 3D Cancer Cell Cytotoxicity in vitro Assessment

Nowadays in Cancer research, there is a strong need for three-dimensional (3D) in vitro experimental approaches that mimick as much as possible the in vivo tumour ecosystem. In addition to solving issues related to animal models, these in vitro 3D models allow precise tuning of experimental design when investigating cancer cell biology but also, compound screening and disease modelling. In this post, we’ll take a look at the growth of the popular breast cancer cell line, MCF-7, using Alvetex®Scaffold technology (Reinnervate – a ReproCell company) to create a 3D structure that closely resembles the structure of tumour tissues grown in vivo.

[Read more…]

Cancer immunotherapy and PBMCs

Patients who receive cancer immunotherapy treatments in clinical trials must have their peripheral blood mononuclear cell (PBMC) samples collected at baseline and at later time points. Immune monitoring facilities provide laboratory testing of these PBMC samples in order to gauge the response of the patient’s immune system to the test treatment.

A primary focus of immune monitoring facilities is therefore to develop cutting-edge technologies whilst also standardizing and validating immune assays with rigorous quality-control standards to ensure data reliability. After all, the weight of clinical trial results depends on the accuracy, precision, and reliability of data generated from these PBMC samples.

The fragile nature of biological samples makes standardization of laboratory procedures an especially important focus. Parameters that can affect data include the anticoagulant used for preserving PBMC samples, the time frame between sampling and processing, storage/shipping temperature en route to the central processing lab, the cryopreservation and thawing process, as well as the cell culture media.[1]

[Read more…]

Cell counting made easy!

After you’ve read this post, I hope that you’ll agree that what the title of the post announces is the right assumption. How? Read on!

I invite you to take a look at Cyto-X, which is a ready-for-use cell viability reagent for proliferation and cytotoxicity assays. When added to cell culture, a highly water-soluble tetrazolium salt is reduced by dehydrogenases in live cells to form an orange colored formazan product that is soluble in culture medium. The amount of formazan is directly proportional to the enzymatic activity of dehydrogenases and the number of living cells.

With its high sensitivity and low cytotoxicity, Cyto-X outperforms traditional assays using other tetrazolium salts such as MTT, XTT, MTS or WST-1.

Here are 3 good reasons to try Cyto-X Colorimetric cell counting reagent…

1. It’s more sensitive

 

Cyto-X 1

 

Left: Human Dermal Fibroblasts (HDF) were seeded at different concentrations. Cells were counted the next day using the Cyto-X (blue) and MTT (orange) assays.
Right: Bovine Aortic Endothelial Cells (BAOEC) were treated with staurosporine for 16 hours. Cell number was determined by Cyto-X and MTT assays.

 

2. It’s less toxic

Cyto-X 2

Left: Cyto-X was added to Human Umbilical Vein Endothelial Cells (HUVEC) for 24 hours. Following the Cyto-X cell counting assay, HUVEC are still proliferating and capable of uptaking DiL-Ac-LDL, demonstrating HUVEC are metabolically active, and that downstream applications are possible.
Right: By contrast, toxic formazan is formed after the MTT assay and HUVEC are dead.

 

3. It’s user friendly: fewer steps & less waiting time

Cyto-X 3

So, doesn’t this make cell counting easy? I could also claim “Cell counting made easy and inexpensive!”. Not only is this protocol quicker, it also uses less reagents vs. MTT (no phenol-red medium, no extraction solution, no wash solution, etc) and is as cheap as 0.36 € per test point.

Convinced? Leave your comments or get in touch below!

A polystyrene scaffold for 3D endocervix model

In this published work, Novel Three Dimensional Human Endocervix Cultures respond to 28-day hormone treatment (1) , the authors’ goal was to develop a robust three-dimensional (3D) endocervix model that was a reliable representation of the in vivo tissues and to identify the physiological responses to changing levels of steroid hormones during a 28-day time period. Human endocervical cells were grown on polystyrene scaffolds and the morphologic and hormonal responses of cultured cells were assessed in response to fluctuating levels of estradiol (E2) or progesterone (P4). [Read more…]

5 most popular posts in ADME-Tox in 2014!

Take a look at the 5 posts on our blog in the field of ADME-Tox that saw the most visits in 2014!

[Read more…]