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Applications

Assays for Cell Therapy

All assays described on this page are available as contract research services or as assay kits from our on-line catalog

 

HemoGenix® has used its bioluminomics™ technology to develop specific assays for cellular therapeutic applications involving hematopoietic stem cells and mesenchymal stem/stromal cells (MSC). Sources of hematopoietic stem cells include mobilized peripheral blood, bone marrow and umbilical cord blood used for transplantation purposes. The source of mesenchymal stem cells varies greatly from bone marrow, umbilical cord blood and Worton's jelly to MSCs derived from human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Primary stem cells derived from explanted tissues are also being used for experimental cellular therapies that have been approved by regulatory agencies. However, this new and rapidly advancing application area is only now coming under the scrutiny of regulatory agencies. In the United States, umbilical cord blood has been designated by the U.S. Food and Drug Administration (FDA) as a drug. As such, the use and processing of stem cells from cord blood should be compliant with the regulations governing the production and manufacture of classic drugs.

 

In 2007, HemoGenix® was the first company to recognize that specific assays were required for these application areas and introduced a quality control assay for human hematopoietic stem cells incorporating its bioluminomics™ technology. This technology includes the need for assay standardization and validation. Since that time, HemoGenix® has been a leader in providing specialized assays for the cellular therapeutic field, which although being highly advanced, are fast to learn, easy to use and incorporate all the attributes necessary to be compliant with regulatory requirements, including the ability to validate the assay.

 

NOTE: Although the community norm is to use the colony-forming unit (CFU) assay, such as CAMEO™-4, equivalent and alternative assays, such as STEMpredict™ and the HALO® assays described below, are acceptable by the FDA, providing they "satisfy the requirements of the applicable statutes and regulations". In addition, AABB Standards for Cellular Therapy Services also allows "equivalent functional assays for any product", and the National Marrow Donor Program (NMDP) allows testing "consistent with AABB Standards".

 

 

Assays for Hematopoietic Stem Cell Therapeutic Applications

Assays are divided into two primary groups, namely proliferation and differentiation assays. The proliferation assays are further divided into three groups depending on the readout or signal detection system used to measure proliferation, assay sensitivity and cost. The bioluminomics™ assays that measure stem cell product proliferation using ATP bioluminescence are the most advanced and sensitive and consequently are more expensive than the less sensitive absorbance assays.

 

 

Hematopoietic Stem and Progenitor Cell Product Differentiation Ability Assays

CAMEO™-4 for Stem Cell Processing Laboratories

CAMEO™-4 is a miniaturized, methylcellulose colony-forming unit (CFU) assay that saves time, reagents and cells compared to the "classic" MethoCult® reagents. In addition, all CAMEO™-4 assay kits include 50, 4-well plates for culturing the cells. There are four CAMEO™-4 assay kits available that contain exactly the same growth factor/cytokine cocktails as the MethoCult® reagents. These are listed in the table below. CAMEO-4 assays require an inverted microscope to view and count the colonies using a magnification of 40x - 100x.

 

 

Hematopoietic Stem and Progenitor Cell Product CFU Equivalent Proliferation Assays 

Bioluminomics™ Proliferation Assays

HALO®-96 PCAEQ is the bioluminomics™ version of CAMEO™-4 for stem cell processing laboratories. These four assays also utilize exactly the same growth factor/cytokine cocktails as the corresponding MethoCult® reagents. However, in contrast to both CAMEO™-4 and MethoCult®, HALO®-96 PCAEQ requires a plate luminometer or multiparameter plate reader with luminescence capability. The assays are quantitative and include an external ATP standard as well as high and low controls that allow the assay to be calibrated and standardized and, if required, validated according to regulatory requirements.

 

Fluorescence Proliferation Assays

HemoFLUOR-96 PCAEQ incorporates the same SEC™ Technology as HALO-96 PCAEQ, but requires a fluorescence plate reader or multiparameter plate reader with an excitation filter of 390-400nm and an emission filter at 505nm. HemoFLUOR™ assays do not include standards and controls.

 

Absorbance Proliferation Assays

HemoLIGHT™-96 PCAEQ is similar to HemoFLUOR™-96 PCAEQ and incorporate SEC™ Technology. These assays require an absorbance plate reader or multiparameter plate reader that can measure absorbance at 490nm.

 

Incorporating HALO®-96 PCAEQ, HemoFLUOR™-96 PCAEQ or HemoLIGHT™-96 PCAEQ can make the hematopoietic stem cell processing laboratory more cost-effective, efficient and compliant. The table below shows the assay kits available that are equivalent to and can replace MethoCult® reagents.

 

HemoGenix® Assays that are Equivalent to MethoCult® Reagents
CAMEO™-4 HALO®-96 PCAEQ HemoFLUOR™-96 PCAEQ HemoLIGHT™-96 PCAEQ MethoCult® Growth Factor/Cytokine Cocktail
KC-GEM2-50H K2-PCA1-1,2,4* K5-PCA1-1,2,4* K4-PCA1-1,2,4* H4434 EPO, GM-CSF, IL-3, SCF
KC-GEM3-50H K2-PCA2-1,2,4* K5-PCA2-1,2,4* K4-PCA2-1,2,4* H4034 EPO, GM-CSF, G-CSF, IL-3, SCF
KC-GM1-50H K2-PCA3-1,2,4* K5-PCA3-1,2,4* K4-PCA3-1,2,4* H4534 GM-CSF, IL-3, SCF
KC-GM2-50H K2-PCA4-1,2,4* K5-PCA4-1,2,4* K4-PCA4-1,2,4* H4035 GM-CSF, G-CSF, IL-3, SCF
*denotes that assay kits are available with 1, 2 or 4, 96-well plates
 
 

STEMpredict™ - The Fastest and Most Sensitive Hematopoietic Stem Cell Growth and Functionality Assay Available

We were asked to develop an assay that could detect whether stem cells in a processed product would exhibit growth and functionality. STEMpredict™ was the answer. STEMpredict™ is an ATP-based, stem cell assay that delivers results in 2-3 days. It is the fastest and most reliable stem cell growth, functionality and viability assay ever produced.
 
 

HALO®-96 SPC-QC - For Routine Quality Control Assessment of Hematopoietic Stem Cell Products

Whereas STEMpredict™ can assess the growth and functionality of a hematopoietic stem cell product very quickly,HALO®-96 SPC-QC is used for the routine assay of the multipotential hematopoietic stem cell population CFC-GEMM and can be combined with the more primitive lympho-hematopoietic stem cell population, HPP-SP (high proliferative potential - stem and progenitor cells). HALO®-96 SPC-QC measures stem cell "quality", which is defined as stem cell proliferation status or ability. It is used when large numbers of samples have to be processed, such as in cord blood banks, or when consistency and/or stability have to be measured prior to or after certain procedures, e.g. cryopreservation. For more information on HALO®-96 SPC-QC, please refer to the article entitled, "Bioluminescence Assays for Assessing Potency of Cellular Therapy Products", published in Cellular Therapy: Principles, Methods and Regulations in 2009 (Eds. Areman EM, Loper K).
 
 

HALO®-96 PQR - A Potency, Quality and Release Assay for Hematopoietic Stem Cell Products Used Just Prior to Transplantation

HALO®-96 PQR is the only reference standard-based potency assay commercially available. It allows stem cell potency and quality to be assessed simultaneously and helps define acceptance and rejection limits for release criteria, in a single assay. HALO®-96 PQR is compliant with FDA regulations for potency and potency assays. Like all bioluminomics™ assays, HALO®-96 PQR can be validated according to FDA regulations. For more information on measuring stem cell potency, please refer to the following articles:
  • "Bioluminescence Assays for Assessing Potency of Cellular Therapy Products", published in Cellular Therapy: Principles, Methods and Regulations in 2009 (Eds. Areman EM, Loper K).
  • Karen M. Hall, Holli Harper and Ivan N. Rich (2012). Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation, Advances in Hematopoietic Stem Cell Research, Rosana Pelayo (Ed.), ISBN: 978-953-307-930-1, InTech.
 
 

Assays to Determine Time to Engraftment and Patient Reconstitution Monitoring after Transplantation

Time to engraftment is determined by monitoring the time to reach a certain number of circulating neutrophils, platelets and/or red blood cells. These are differentiated, mature cells which are the result of growing CFU colonies of GM-CFC, Mk-CFC and/or BFU-E progenitor cells.
 
A faster, more reliable and cost-effective method is to measure whether these progenitor cell populations exhibit the proliferation ability to grow as a predictor for the production of circulating blood elements.
 
To measure "global" reconstitution of the hematopoietic system, a 5 population assay is available that detects two stem cell populations and all three hematopoietic cell lineage populations. In addition, HemoGenix also offers a "global" 7 population assay that includes the T- and B-progenitor cells. For more information on simultaneously assessing the presence and response of multiple lympho-hematopoietic cell populations, please refer to the article entitled, "Bioluminescence Assays for Assessing Potency of Cellular Therapy Products", published in Cellular Therapy: Principles, Methods and Regulations in 2009 (Eds. Areman EM, Loper K).
 
Bioluminomics™ Assays to Measure Time to Engraftment and Patient Reconstitution after Transplantation
HALO®-96 PMT is available for individual GM-CFC, Mk-CFC and BFU-E progenitor cell populations to detect time to engraftment. In addition 5- and 7-population assays to monitor reconstitution are also available. These assays incorporate SEC Technology and include reagents to calibrate and standardize the assays allowing results to be compare over time. These assays require a plate luminometer or multiparameter plate reader with luminescence measuring capability.
 
Fluorescence Assays to Measure Time to Engraftment and Patient Monitoring after Transplantation
HemoFLUOR™-96 PMT is also available for individual GM-CFC, Mk-CFC and BFU-E progenitor cell populations. In addition, a 5-population assay can also be obtained to monitor reconstitution. These assays require a fluorescence plate reader or multiparameter plate reader with an excitation filter at 390-400nm and an emission filter at 505nm.
 
Absorbance Assays to Measure Time to Engraftment and Patient Monitoring after Transplantation
HemoLIGHT™-96 PMT is also available for individual GM-CFC, Mk-CFC and BFU-E progenitor cell populations. In addition, a 5-population assay can also be obtained to monitor reconstitution. These assays require an absorbance plate reader or multiparameter plate reader that can measure absorbance at 490nm. 
 

Assays for Immune Cells

The function and response of immune and associated cells play a signficant role on cellular therapeutic applications. For this reason, HemoGenix has introduced two important assay platforms, namely lymphocyte proliferation assays and mixed lymphocyte culture (MLC) or mixed lymphocyte reaction (MLR) assays. Each of these assays is available with a bioluminomics™, fluorescence or absorbance readout. Use of one or other of the assays will depend on the instrumentation available, sensitivity required and cost.
 
Bioluminomics™ Assays for Immune Cells
These are the most sensitive all assays and require a plate luminometer or multiparameter plate reader capable of measuring luminescence. All bioluminomics assays allow for assay calibration and standardization allowing results to be compared over time. In addition, these assays can be validated according to regulatory requirements.
ImmunoGlo™-96 is a bioluminomics lymphocyte proliferation assay.
ImmunoGlo™-MLC can be used for a 1- or 2-way mixed lymphocyte culture or reaction.
 
Fluorescence Assays for Immune Cells
Both ImmunoFLUOR™-96 and ImmunoFLUOR™-MLC are the fluorescence equivalent assays to ImmunoGlo™-96 and ImmunoGlo™-MLC. The fluorescence assays require a fluorescence plate reader or multiparameter plate reader with an excitation filter at 390-400nm and an emission filter at 505nm.
 
Absorbance Assays for Immune Cells
ImmunoLIGHT™-96 and ImmunoLIGHT™-MLC are the least sensitive, but also the most cost-effective. These assays require an absorbance plate reader or multiparameter plate reader, available in most laboratories, with a 490nm absorbance filter. 
 
 
 

Assays for Mesenchymal Stem / Stromal Cell Therapeutic Applications

 

Regenerative medicine is a rapidly growing therapeutic area involving many different types of cells. Mesenchymal stem/stromal cells (MSC) are fibroblast-like cells that can differentiate into several different cell types, including chondrocytes, adipocytes, osteoblasts, skeletal cells and even neural cells. In collaboration with Vitro BioPharma™ that produces and manufactures high performance MSC media that shows superior growth properties and a longer shelf life than any other media, HemoGenix® has developed two assays to measure MSC quality and potency.

 

LUMENESC™-96 HuQC - A MSC Quality Control Assay

Similar in operation to that of its hematopoietic counterpart assay, HALO®-96 SPC-QC, LUMENESC™-96 HuQC measures the "quality" of mesenchymal stem/stromal cells and can be used to routinely compare different procedures during manufacture. The assay is available with low or serum-free medium or even humanized medium.

 

LUMENESC™-96 PQR - A Potency, Quality and Release Assay for MSCs

LUMENESC™-96 PQR is the counterpart to HALO®-96 PQR. Like the latter, this assay is the only commercially available potency assay that includes a reference standard for MSCs. The procedure for measuring MSC potency and quality is similar to that for hematopoietic stem cells.

 

MethoCult® is a registered trademark of StemCell Technologies

Hemogenix Inc.