The majority of studies performed by HemoGenix® incorporate a Complete Service, Full Report that can be used for an IND application. More recently, Sponsors have requested a more streamlined study and studies involving high-throughput screening. For this reason, HemoGenix® now provides 3 types of study format. 

1. Complete Service, Full Report Study: Fully customized study that includes the Study Plan, Draft Text and Final Text Report with QA audit.
2. Rapid Toxicity Study: A customized study that includes the Study Plan and full protocol, raw results and graphical data in a single Excel Workbook. No formal text report and no QA audit is performed. This type of study is designed for early drug development. No interpretation or conclusions are provided.
3. High-Throughput Screening Module (presently only available for human studies): A screening study is designed for high-throughput screening of compounds during ADME/Tox screening in multiples of 5 on specific cell populations to provide the most important ranking information. The full protocol, raw results and graphical data are provided in an Excel Workbook. No interpretation or conclusions are provided. This type of Screening Study is part of the ComparaTOX™ HT Platform.

Mesenchymal cells is the general term that encompasses Mesenchymal Stem Cells (MSC), Mesenchymal Stromal Cells, (MSC) and Mesenchymal Progenitor Cells. That several different names apply to these cells, indicates the difficulty in specifying their properties and biological function. For the purposes of discussion, mesenchymal cells shall be designated as "MSC", since this is the term that has been used for decades. Whether they are actually stem cells, progenitor cells or stromal cells is still a matter of scientific debate. 

The term "stromal cell" has been applied to those cells in the bone marrow microenvironment that are, in part, an absolute requirement for a functioning bone marrow to produce lympho-hematopoietic cells. MSCs as "stromal cells" were first discovered by Owen and Frienstein as osteogenic precursors in 1988 (Ciba Foundation Symp. 1988: 136:42-60). Frienstein and co-workers published the first article on fibroblast colonies grown in monolayer cultures (Cell Tiss Kinet. 1970: 3:393-403). Based of the hematopoietic nomeclature of colony-forming cells in agar and later in methylcellulose, the fibroblast colonies were designated Colony-Forming Unit - Fibroblast (CFU-F). From this assays, MSCs were designated as an adherent, proliferating, fibroblastoid population of bone marrow cells, thereby assigning MSC those properties. 

Since that time, MSCs have been identified using other properties. In contrast to hematopoietic cells in the bone marrow, MSCs do not express CD45, CD14, CD34 or HLA membrane antigens. They do express CD29, CD44, CD73, CD90 and CD105. 

MSC are not only dereived from bone marrow, but can be obtained from umbilical cord blood, adipose tissue, amniotic fluid, endometrium, dental tissue and Wharton's jelly, to name but a few sources. 

All MSCs, regardless of the source, have to shown to differentiate in culture into osteocytes, chrondrocytes and adipocytes. They have also been shown to differentiate into hepatocytes, cardiomyocytes, pancreatic cells and neuronal cells. MSCs, therefore, can be designated as a multipotent cell population. 

MSCs have also been recently shown to exhibit a paracrine function in they can screte, under different situations, a multitude of cytokines and other factors. As such, MSCs have also been shown to demonstrate immunomodulatory functions and therefore being used for treatment of autoimmune inflammatory and degenerative diseases. In addition, MSCs are being studied in several neurodegenerative diseases, including Parkinson's disease and Alzheimer disease, rhumatoid arthritis, Type 1 diabetes and cardiovascular disease. 

Based on the properties and uses of MSCs, especially now in the era of the COVID-19 pandemic where many MSC studies and clinical trials are being performed, advanced and sophisticated assays that are both standardized and validated are required. This is the mainstay of all HemoGenix^® Contract Research Services.  

Despite the numerous sources of MSC available, the two most available sources of MSC for MSC toxicity testing and high-throughput screening are: 

• Bone marrow and,
• Umbilical cord blood.
• iPS-derived MSC are presently being tested as a source of MSC for toxicity applications.  

Native and naive primary MSC are never used since methods are unavailable to obtain sufficient unmanipulated cells.

For toxicity testing and high-throughput screening studies, fresh primary and non-cryopreserved bone marrow or cord blood is obtained and the mononuclear cell (MNC) fraction produced. These cells are then cultured and expanded to the 3-4th passage prior to use for any toxicity testing. The cells are phenotypically identified as MSCs using CD44⁺, CD73⁺, CD90⁺ and CD105⁺ and CD45^- by flow cytometry and population doubling time during expansion is performed using MSCGlo™ Real Time prior to use. MSC Real Time ensures that the cells exhibit the proliferation ability or "quality" required for toxicity testing.

For all MSC expansion and culture studies, regardless of whether the MSCs are used for toxicity studies or other biological functional studies, the following conditions are available upon request:  

• Serum-free cell culture
• Low serum cell culture
• Humanized cell cutlure
• CRUXRUFA™, fibrinogen-depleted (FD), GMP produced and quarantined (Q) grade Human Platelet Lysate. 

Depending on the study, HemoGenix will suggest the best conditions for the study.

Competitors might suggest using the CFU-F assay or a fluorescence or absorbance assay for studying the response of MSCs to new drug candidates, environmental toxins or other perturbations. However, it is unlikely that these assays have:

(a) been designed for toxicity testing,

(b) incorporate controls and standards for calibration and standardization of the assay,

(c) have been validated according to FDA bioanalytical method validation guidelines or,

(d) provide measurement assurance parameters that ensure the assay is working correctly and provides trustworthy results. 

At HemoGenix™, these 4 assay features are incorporated into all of our proprietary assays and MSCGlo™-Tox HT is no exception.

MSCGlo™-Tox HT is a fully standardized and validated ATP bioluminescence (customized CellTiter-Glo^® from Promega) toxicity assay that measures functional viability of cells based on intracellular ATP (iATP) concentrations. In short, MSCGlo™-Tox HT is a metabolic viability assay designed specifically for MSCs. 

Since MSCs are a proliferating population, MSCGlo™-Tox HT is highly sensitive to quantitatively measure small changes in population kinetics as a result of insults subjected to the cells.

For toxicity studies, MSCs are grown to near confluence in an adherent state in either 96- or 384-well plates (used also for high-throughput screening). Test articles are prepared in a serial dose response and added using a liquid handler to the wells of the plate. The response to the test article(s) is determined 2-3 days after administration. In plate luminometer is calibrated using controls and an ATP standard curve is performed and the parameters compared to measurement assurance parameters for the readout. Compliance with these parameters allows the test plate samples to be processed and ATP measurements performed. Where appropriate, IC or EC values are directly calculated from the 4- or 5-parameter logistic curve fits using SoftMax pro software. 

MSCGlo™-Tox HT is available for toxicity testing in 96- or 384-well plates. HemoGenix™ will suggest the best format to use for a cost-effective study. 

HemoGenix® has implemented a High-Throughput Screening Module that allows compounds, in multiples of 5, to be screened for their effect on human bone marrow-derived MSCs. This type of screening is meant to be performed in conjunction with ADME/Tox screening. 

This type of high-throughput screening is also performed as part of the HemoGenix™ ComparaTOX™ Platform in which bone marrow MSC target cells are used together with other target cells from different biological systems (e.g. hematotoxicity, immunotoxicity, hepatotoxicity, cardiotoxicity etc) and, in some cases, even different species, (human, primate, dog, rat, mouse) to rank the toxicity of compounds.  

The ComparaTOX™ HT Platform is specifically for human cells derived from different tissues for high-throughput screening. It is used in conjunction with the High-Throughput Screening Module mentioned above. 

With the exponential increase of MSCs as a treatment for numerous diseases, the basic regulatory requirements of quality and potency are often forgotten. Rather than designing and developing a potency assay in parallel with increased scientific understanding of MSC use for a specific disease, this basic, but probably, most important assay of all, is often left to the end, when the new regenerative therapy is being prepared for review by the regulatory authorities.

In its most basic form, a potency assay is used to predict whether the biological action of the therapy will produce the intended response. For example, if MSCs are produced from a specific source and, without any further manipulation, administered to a patient, it will be necessary to know whether the MSCs exhibit sufficient quality and potency to elicit the intended response. The response may be as "simple" as proliferation, or producing a specific set of cytokines or as complex as interacting with immune cells. To know whether the MSCs have sufficient potency is to know how to quantitatively measure the intended biological response that is the endpoint.    

HemoGenix^® has designed and developed a quality and potency assay for MSCs that is initially based on the functional ability of MSCs to proliferate. The MSCGlo™ QC quality assay and MSCGlo™ SC-IPS strength or potency assay can form the basis of more advanced and sophisticated assays that can be applied to clinical grade MSCs used for a regenerative medicine therapy.

The term "IPS" means Identity, Purity and Strength, the 3 basic requirements for defining a biological product, in this case a regenerative medicine product, that has the properties to indicate manufacturing consistency, stability and the correct biological function so as not to cause, on the one hand, lack of response or on the other, toxicity.

Please contact HemoGenix^® for more information on our MSC quality and potency services. For more information on cell quality and potency, please click here.   

In addition to MSC toxicity, studies may involve the response of MSC differentiation into chondrocytes, adipocytes or osteocytes.

To this end, it may be necessary to follow the differentiation, or lack of differentiation due to a drug, other compound or perturbation, as a function of time. Alternatively, the direct response on the MSC differentiated cell type might be needed.

HemoGenix^® has the capability to follow and determine MSC differentiation capacity using multiple assays. These include, but are not limited to:

• Culture systems that allow MSCs to differentiate into chondrocytes, adipocytes or osteocytes.
• MSCGlo™, MSCFluor™ or MSCLight™ to demonstrate decreased proliferation as MSCs differentiate into the different lineages.
• MSCGlo™ Real Time to measure the growth kinetics and simultaneous membrane markers for cells differentiating into any of the 3 lineages.
• Phenotyping of the differentiated cells by flow cytometry or other markers.
• Detection and measurement of cytokines and other factors using multiplexing technology.

For more information, please contact HemoGenix^® to discuss the study involving MSC differentiation.

HemoGenix® has an array of assays to study different aspects of the MSC system. Some of these have already been described above, but are listed here to provide the reader with an overview of capabilities.

• MSCGlo™-Tox HT for general toxicity testing and high-throughput screening, residual toxicity and drug sensitivity. In addition, MSCGlo™-Tox HT is also used for drug interaction assays.
• ComparaTOX™: Incorporating MSCGlo™-Tox HT with other proprietary ATP bioluminescence assays for other cells and tissues to compare and rank toxicity.
• MSCGlo™ Real Time: Measurement of population doubling time and time-related response coupled with flow cytometry.
• MSCGlo™ QC: MSC quality assay.
• MSCGlo™ SC-IPS: Identity, Purity and Strength (Potency) assay for MSCs.
• MSCGlo™: MSC proliferation assay using ATP bioluminescence as the readout.
• MSCFluor™: Non-lytic, Resazurin/Resorifin fluorescence assay for MSC proliferation.
• MSCLight™: Non-lytic, tetrozolium MTS absorbance assay for MSC proliferation.
• Flow cytometry for phenotypic analysis, cell cycle analysis, apoptosis, intracellular cytokines and more.
• Cytokine production and release assays.
• Caspase assays for apoptosis.
• LDH and membrane integrity assays.
• Mitochondrial dysfunction.
• Oxidative stress.
• Oxidative DNA damage (OxyFLOW™, see below).
• Kinase assays.
• Protease assays.

Toxicity to MSCs

To demonstrate how drugs, in particular anti-cancer drugs, can affect MSCs, consider the effect of doxorubicin (Adriamycin) on human bone marrow MSCs shown in the graph. The MSCs were tested at two different cell doses. In both cases, there was significant cytotoxicity. 

Cellular Drug-Drug Interaction

Drug-drug interaction can activate or inhibit CYP450 enzymes. Although occurring primarily in hepatocytes as the cells responsible for detoxification, drug interactions can occur in many other cell types. Although little, if anything, is known regarding the response of MSCs to drug interactions, drug-drug interaction assays can be performed in a similar manner to those for the lympho-hematopoietic stem cells using HALO®-DDI. For more information, please contact HemoGenix® directly.

You can perform MSC toxicity and other assays in-house using assay kits that are sold by Preferred Cell Systems™. Please click on the links below to take you to the assay kit page on the Preferred Cell Systems website.

MSCGlo™-Tox HT: MSC toxicity assays.

MSCGlo™ QC: MSC quality assays.

MSCGlo™ SC-IPS: MSC Identity, Purity and Strength (Potency) assays.

MSCGlo™: MSC proliferation assay using an ATP bioluminescence readout.

MSCFluor™: MSC proliferation assay using a Resazurin/Resorufin fluorescence readout.

MSCLight™ MSC proliferation assay using a tetrazolium/WST absorbance readout.