Contract Research Services, Training and Consultation

HemoGenix® - The Leader in In Vitro Stem Cell Hemotoxicity (Hematotoxicity)  Screening and Testing

HemoGenix® has been providing contract research services since the company started in 2000. With its own patented and proprietary assays, HemoGenix® is the only company in the world with high throughput screening capability for cells of the stem cell blood-forming (lympho-hematopoietic) system and the mesenchymal stem/stromal cell system. In vitro assays specifically developed and validated for contract research service predictive toxicity screening and testing have been further developed into assay kits for routine in-house implementation and licensing. To ensure that the implementation is rapid and efficient, HemoGenix® provides extensive training and technical support. We also provide scientific presentations and consultation. HemoGenix® has provided high quality and confidential contract research services to small, medium and many of the largest biopharmaceutical companies as well as government agencies.

Contract Research Services

HemoGenix® provides contract research services to the biopharmaceutical industry, environmental industry and agencies, stem cell transplantation centers and umbilical cord blood banks. In addition, we also work with basic research, veterinary science and medical facilities. These services include, but are not limited to:

• In vitro high throughput toxicity screening and testing using various primary stem cell and stem cell lines, fresh, primary explanted cells as well as transformed cell lines.
• In vitro testing of cells derived from animal studies.
• In vitro assay development and validation.
• Compound potency assays.
• Stem cell potency, quality and release assays for umbilical cord blood, mobilized peripheral blood, bone marrow and mesenchymal stem cells.

Contract Services Workflow

• The CSO of HemoGenix® will consult with our clients and advise regarding the best procedure and assays to perform to achieve the goal(s) of the study.
• A quote will be prepared and revised to suit the client's budget.
• GLP Study Phase Audits will be performed throughout the workflow.
• Prior to the start of any study, a detailed Study Plan will be prepared by the Study Director for the Study Monitor's approval.
• Sponsor ships test articles for study.
• The target cells/tissues for the study will be procured.
• The study will be initiated upon arrival of the target cells or tissues.
• Most in vitro studies are completed within 7-14 days.
• Phase I Report is usually provided within 4 - 7 business days after study completion.
• Phase II Final Report is generated and the study terminated.

Parameters Used to Define a Study

• Number of compounds or test articles.
• Dose range and number of doses (usually 6-12) to be tested (if applicable).
• Number and type of reference, positive and/or negative controls.
• Primary assay(s) to be used and add-on assays (if required) to be performed.
• Type and number of cell populations to be tested.
• Type and number of species to be tested. 
• Type of test compound addition (if applicable):
  • Timed pre-culture addition.
  • Direct addition for culture duration.
  • Timed post-culture addition.
• Special culture conditions, if required.
• Sample analysis / curve-fit analysis, type and number of EC/IC values (if relevant) and statistics (if applicable).
• GLP / non-GLP/QA audit.

Training

Depending on the application required, HemoGenix® provides a training session lasting 2-4 days for investigators to learn how to use HALO®, LUMENESC™ and LumiSTEM™ assays. These training sessions include the background biology used to interpret the results as well as hands-on practical work that will allow the immediate use of HemoGenix® products. Training can be performed upon request either at the HemoGenix® facility in Colorado Springs, Colorado or at the user’s facility. Please contact HemoGenix® for more information.

Consultation

With nearly 40 years of experience and expertise in the field of developmental, experimental and applied clinical stem cell hematology and stem cell biology, HemoGenix® can provide expert consultation in a number of important areas including hemotoxicity, stem cell toxicity, stem cell culture and expansion.

In addition, HemoGenix® is the only company that has developed specific assays and procedures to measure stem cell "quality" and potency of cellular therapeutic products. Please contact HemoGenix® for more information.

Species, Organs and Tissues, Cell Systems and Cell Types Available for Contract Research Studies

Species

Organs from which cells can be obtained

Cell Systems and Tissues available for study

 
Specialized Fresh or Cryopreserved Cells available for study

Procedures, Assays Multiplexing Capability and Mechanism of Action

Basic Cell Preparation Procedures for In Vitro Culture

In Vitro Cell Maintenance or Expansion Procedures

Mechanism of Action and Platform Multiplexing Assays

 

Culture Conditions

Virtually all cells have to be cultured in a fully humidified atmosphere containing CO₂, usually at 5%. It is not often that a company offers culture conditions under low oxygen tension. In fact, culturing cells under low oxygen tension helps reduce oxygen toxicity, keeps molecules in a reduced state and improves plating efficiency and therefore assay sensitivity. At HemoGenix® all cultures involving lympho-hematopoietic cells are performed under low oxygen tension approx. equivalent to venous oxygen tension (35mm Hg or 5% O₂). Mesenchymal stem cells are also best cultured under low oxygen tension. Some organs, however, exhibit wide ranges of oxygen tension. These include the liver and kidney. As a result, contract services using cells from these and other organs may be offered using atmospheric and low oxygen tension. 

The Leader in Hemotoxicity Testing and Studies on the Blood-Forming (Lympho-Hematopoietic) System

The blood-forming or lympho-hematopoietic system is the most intensely studied biological system. More is known about the biology, physiology, organization and regulation of this system than any other in the body. The knowledge that has accumulated over decades of work allows results obtained from this system to be some of the most predictive. Indeed, investigators at Hoffmann-La Roche have shown using the HemoGenix® HALO® Hemotoxicity Platform (described below) that there is greater than 80% concordance between in vitro and in vivo data (Toxicol Let. 2009; 188/2:98-103).

The HALO® - The "Gold Standard" in Predictve Hemotoxicity Testing

In 2002, HemoGenix® introduced the HALO® Hemotoxicity Platform to the biopharmaceutical community. Since that time, HemoGenix® has been the leader in hemo-, hematotoxicity testing with small, medium and the largest biopharmaceutical companies either using its contract research services or implementing HALO® in-house for routine screening and testing. Originally developed from the "classic" methylcellulose, colony-forming cell (CFC) assay, which HemoGenix® provides as its proprietary CAMEO™-4 Platform, the HALO® Predictive Hemotoxicity Platform is now a highly refined, quantitative and validated assay system that is methylcellulose-free and can be performed using 96- or 384-well plate formats.
In addition to high throughput HALO® assays for proliferation and cytotoxicity studies, HemoGenix® offers its CAMEO™-4 CFC assay to detect effects on differentiation pathways and CAMEO™-96 that can detect both proliferation and differentiation. All this is available to detect multiple cell populations from multiple species. In addition, HALO® and CAMEO™-96 have been designed for multiplexing, thereby allowing signficant information to be obtained from the same sample.

Types of Studies

• Myelotoxicity studies. This term is often used to denote toxicity that results in neutropenia. It is also considered a form of immunotoxicity. In actual fact, myelo or muelos (Greek) merely refers to the marrow. Therefore, myeloid refers to the bone marrow and hence myelotoxicity refers to toxicity of the bone marrow, regardless of what cells are affected. (See also In Vitro Immunotoxicity). 
• Predictive in vitro hemotoxicity screening and testing for all stages of drug development and patient monitoring during clinical trials.
• In vivo to in vitro testing during pre-clinical animal studies. HemoGenix® works with other CROs performing animal studies from which cells derived from organs and tissues can be more precisely analyzed for treatment effects on primitive cells that cannot be otherwise detected using traditional toxicology procedures. 
• Drug-drug interaction studies.
• Residual stem cell drug sensitivity studies.
• Xenobiotic agent screening of chemicals and other agents.
• Cell stimulation/potentiation studies.
• Growth factor / cytokine studies, including potency testing.
• Stem cell quality control and potency studies for hematopoietic stem cell transplantation and cord blood banks.

Assays available

HemoGenix® has developed three assay platforms by which the lympho-hematopoietic stem, progenitor and precursor cells can be detected and measured. These are:

• HALO®-96 and HALO®-384 for all studies in which cell proliferation ability and potential have to be measured.
• CAMEO™-96 for all studies in which both cell proliferation and differentiation have to be detected and measured.
• CAMEO™-4 for all studies in which differentiation ability and potential have to be detected.

HALO® and CAMEO™-96 can both be used (and were designed) for multiplexing with other assays described on the previous page.

All three assay platforms are available for use with the following species:

• Human
• Non-human primate (Macaca fascicularis or cynomologus (Cyno) and Macaca mulatta (Rhesus)
• Horse
• Pig
• Sheep
• Dog
• Rat (various strains)
• Mouse (various strains)

Lympho-Hematopoietic Cells Populations Detected and Measured using HALO®, CAMEO™-96, CAMEO™-4 for Contract Service Applications 

Cells Detected	HALO®	CAMEO™-96	CAMEO™-4
	LTC-IC*	LTC-IC*	LTC-IC*
Quiescent primitive L-H stem cell	HPP-SP 1	HPP-SP 1	HPP-SP 1
Induced primitive L-H stem cell	HPP-SP 2	HPP-SP 2	HPP-SP 2
Primitive hematopoietic stem cell	CFC-GEMM 1	CFC-GEMM 1	CFC-GEMM 1
Primitive hematopoietic stem cell	CFC-GEMM 2	CFC-GEMM 2	CFC-GEMM 2
Mature hematopoietic stem cell	CFC-GEM 1	CFC-GEM 1	CFC-GEM 1
Mature hematopoietic stem cell	CFC-GEM 2	CFC-GEM 2	CFC-GEM 2
Mature hematopoietic stem cell	CFC-GEM 3	CFC-GEM 3	CFC-GEM 3
Primitive erythropoietic progenitor cell	BFU-E 1	BFU-E 1	BFU-E 1
Primitive erythropoietic progenitor cell	BFU-E 2	BFU-E 2	BFU-E 2
Erythropoietic precursor cell	Not available	CFU-E	CFU-E
Primitive granulocyte-macrophage progenitor cell	GM-CFC 1	GM-CFC 1	GM-CFC 1
Primitive granulocyte-macrophage progenitor cell	GM-CFC 2	GM-CFC 2	GM-CFC 2
Mature granulocyte-macrophage progenitor cell	GM-CFC 3	GM-CFC 3	GM-CFC 3
Granulocyte precursor cell	Not available	G-CFC	G-CFC
Macrophage precursor cell	Not available	M-CFC	M-CFC
Primitive megakaryocytic progenitor cell	Mk-CFC 1	Mk-CFC 1	Mk-CFC 1
Primitive megakaryocytic progenitor cell	Mk-CFC 2	Mk-CFC 2	Mk-CFC 2
T-lymphocyte progenitor cell	T-CFC	T-CFC	T-CFC
B-lymphocyte progenitor cell	B-CFC	B-CFC	B-CFC

* The LTC-IC is an assay that takes 5-7 weeks to perform. Although the LTC-IC stem cell population is probably the most primitive in vitro stem cell population that can be detected, HemoGenix® does not recommend performing this assay for screening purposes. The HPP-SP primitive stem cell population can provide almost similar conclusions in a significantly shorter time frame and at a fraction of the cost. 

L-H = Lympho-hematopoietic; a stem cell population that can produce both lymphoietic and hematopoietic stem and progenitor cells.

What is the Relationship Between CAMEO™-4, CAMEO™-96 and HALO^®?

An important question often asked is, "what is the relationship between the in vitro assays for lympho-hematopoietic cells"? Since both CAMEO™-96 and HALO® were both derived from the "classic" colony-forming cell assay, it would be expected that a relationship exists between all three. This is precisely the case. The graphs in the diagram shown on the left (click to enlarge) demonstrate that there is a direct correlation between CAMEO™-4, CAMEO™-96, HALO®-96 and HALO®-384 HT. This correlation means that the results from one assay will predict results from one of the other assays. In addition, the correlations demonstrates that one assay can replace the other. However, for virtually all toxicology applications where a sensitive and validated assay is required, HALO®, either in the 96-well or 384-well format should be used. Only in cases where a compound affects the downstream differentiation and/or maturation process, should CAMEO™-96 or CAMEO™-4 be used.

Examples of Stem Cell Hemotoxicity

The diagram to the left shows the dose response relationship between a number of familiar drugs and the effect on in vitro hematopoietic multipotential mature stem cells, CFC-GEMM, derived from human bone marrow mononuclear cells. These results were obtained using HALO®-384 HT. The upper graph shows that Omeprezole, Cyclosporine, Cimetidine and Warfarin have little if any effect on hematopoietic stem cells over the dose range studied. In contrast, Tamoxifin shows partial cytotoxicity at high doses, while Verapamil is cytotoxic at high doses. The lower graph demonstrates that while AZT and Cisplatin produce an inhibition at the highest doses, both 5-Fluorouracil and Mitomycin-C are highly toxic.

Can Results from the HALO® Predictive Hemotoxicity Platform Help Estimate in vivo Starting Doses?

The answer to this question is, yes. Not only is HALO® one of the most predictive in vitro toxicity assays, available, but can also be used to estimate drug dosing both in animals and humans. This is because HALO® has been validated against the Registry of Cytotoxicity Prediction Model. The Registry of Cytotoxicity contains a list of compounds for which the LD50 values in rats or mice have been documented. If the LD50 for a group of reference compounds is plotted against the IC50 values obtained from an in vitro assay, in this case HALO®, the points should allow a linear regression to be drawn with specific parameters. This not only allows the assay to be validated as a cytotoxicity assay, but allows an even more interesting aspect to be investigated. This is shown in the table. Using the Registry of Cytotoxicity Prediction Model, it is possible to convert in vitro IC50 values into clinically relevant doses, either in mg/kg or as mg/m². The results of this conversion are shown in the table on the left. The doses on the far right of the table, are those used to treat various forms of human cancer. It can be seen that the estimated doses for the anti-cancer drugs shown and obtained using the IC50 values derived from HALO® are in the same order of magnitude as those used for treatment. Only one drug, namely vinblastine, is the exception. 

The "Global" Predictive Hemotoxicity Platform

Knowing the response of the stem cells to a drug or agent will provide an excellent prediction of the effects observed at later times in the periphery or circulation. The addition of the early progenitor cells provides an added "global" view of the whole system indicating whether one lineage is affected more than another and ranking the cell types according to the compound response. This is why HemoGenix® developed the HALO® "Global" Predictive Hemotoxicity Platform, an example of which is shown for Daunorubicin on the stem and progenitor cells of the lympho-hematopoietic system. Although normally 7 cell populations are assessed using this unique and powerful in vitro predictive toxicity assay, the response of the quiescent primitive stem cell population (HPP-SP 1) is also shown. The results from this population indicate that, even in a mostly quiescent state, very primitive stem cells can be affected by drugs and other compounds. Action of a compound at this very primitive stage of lympho-hematopoiesis will have an affect on the whole system and could be associated with drastic repercussions.