Contract Services

Why use primary cells to test your substance of interest?

In vitro assays using primary cells are:

• quantitative and qualitative
• robust
• flexible and customizable
• clinically relevant, giving more predictive data
• cost effective (reducing the number of animals required)  

Contract Assay Services can evaluate the effects of your compound on the following cell types and sources:

*Clinical samples from diseased or normal patients can also be evaluated – inquire for more information.


FIGURE 1: Illustration of hematopoietic and neural stem and progenitor cells and the mature cell types that they produce.

Hematopoietic in vitro assays

Our most popular service involves the hematopoeitic colony-forming cell (CFC) assay, an assay that has been validated for the determination of maximum tolerated doses (MTD) by ECVAM.^1,2  We use STEMCELL’s MethoCult^® product line, recognized as the global gold standard for the detection and quantification of hematopoietic progenitors using CFC assays. 

Hematopoietic CFC assays can be used to examine simultaneously the effects of substances (lead compounds/clinical candidates, mimetics, cytokines or chemokines) on the processes of proliferation and differentiation. 

Hematopoietic progenitors from different species can be assessed:

Evaluation of Stimulation

Substances thought to stimulate and/or enhance proliferation of hematopoietic progenitors can be tested for functionality in the CFC assay.

FIGURE 1: Stimulation by Erythropoietin (EPO)




Stimulation of erythroid progenitor proliferation by erythropoietin (EPO) is both quantifiable (Figure A) and qualitative (Figure B, colony size increases with increasing [EPO]). In vivo models can be used to confirm stimulatory activity of molecules.

Evaluation of Inhibition or Toxicity

Substances thought to inhibit the proliferation and/or differentiation of hematopoietic progenitors can be screened to assess their effects on the different lineages of progenitors: myeloid, erythroid, and/or megakaryocytes using the CFC assay.  Both IC₅₀ and IC₉₀ values can be generated to rank test compounds.

TABLE: Comparison of myelotoxicity (CFU-GM) in different species




FIGURE 2: Determination of IC50 values for 5-Fluorouracil

Dose response curves and IC₅₀ values for both human BM derived erythroid and myeloid progenitors incubated with 5-Fluorouracil

FIGURE 3: Human BM Colonies in the presence of a toxic compound


The size of CFCs will change in the presence of an inhibitory compound, serving as a sensitive toxicity assay readout.

References:

1. Pessina A, Albella B, Bueren J, Brantom P, Casati S, Gribaldo L, Croera C, Gagliardi G, Foti P, Parchment R, Parent-Massin D, Sibiril Y, Van Den Heuvel R. Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay. Toxicol In Vitro 15:729-740, 2001
   
2. Pessina A, Parent-Massin D, Albella B, Van Den Heuvel R, Casati S, Croera C,Malerba I, Sibiril Y, Gomez S, de Smedt A, Gribaldo L. Application of human CFU-Mk assay to predict potential thrombocytotoxicity of drugs. Toxicol In Vitro 23:194-200, 2009
   
   

Neural in vitro assays

STEMCELL scientists have worked with leaders in the neural stem cell field to develop standardized reagents and assays for the culture of neural stem and progenitor cells. 

Neurospheres¹ are highly dynamic, three-dimensional floating spheres containing cells from different stages of neural development – neural stem cells, progenitors and mature cells.  These neurospheres are the basis of both our proliferation (NCFC) assay and differentiation assay.

We use the NCFC assay to assess the effects of your substance of interest on primary neural cells.


FIGURE 1: Assays for neural stem and progenitor cells

 

References:

1. Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 10:1-13, 1996
   
   
   

Mesenchymal in vitro assays

Drug candidates destined for the tissue engineering market can be assessed for stimulatory or inhibitory effects on the bone marrow microenvironment (mesenchymal cell matrix). These cells, under the appropriate conditions, can differentiate into structural cell types that make up adipose, cartilage, bone and muscle. 

Functional assays using mesenchymal stem and progenitor cells from human bone marrow or mouse compact bone can be used to quantify and qualitatively assess the effects of compounds:

• Measure the effects of test compound on progenitor frequency
• Assess progenitor proliferative potential or overall expansion potential
• Quantitation of mesenchymal progenitor content in bone marrow
• Test the differentiation potential of mesenchymal stem cells into adipose, osteogenic and chrondrocyte lineages

The Contract Assay Services group has developed new assays for the quantification of mouse CFU-F from compact bone, a source known to be richer than the bone marrow in mesenchymal stem and progenitor cells. Mouse CFU-F frequency increases when culturing compact bone-derived MSCs under low oxygen conditions.

FIGURE 1: Effects of Doxorubicin on Human CFU-F

Dose response curve and IC₅₀ value for human BM derived CFU-F incubated with Doxorubicin. Inhibitory compounds can affect the size and density of the CFU-F colony.