CAMEO™-96 STD
Stem and progenitor Cell Potency Assays
by
Combining the Power of Instrument-Based ATP Bioluminescence Proliferation Assays to Standardize the Colony-Forming Cell Differentiation Assay
INTRODUCTION
The colony-forming cell (CFC) assay was never developed or designed as a stem cell potency assay, but rather as an investigative tool. After 1971, when human stem cell transplantation began to take hold as a potential therapy to treat malignancies of the lympho-hematopoietic system, it was necessary to have an assay that could detect the growth potential of the cells. The CFC assay was the only test system that could detect the growth potential of granulocyte-macrophage progenitor cells. As a result, processing laboratories started to incorporate the assay for the granulocyte-macrophage colony-forming progenitor cells into the quality control procedure. However, in the early 1980’s, flow cytometry made an appearance and the CD34 antigen was discovered. Together with nucleated cell counts, the introduction of flow cytometric gating protocols to detect the presence of viable CD34+ cells, a rapid and validated assay gradually became the procedure of choice for many stem cell processing laboratories. But, cell counts, CD34 enumeration and viability do not measure, and cannot replace, a measurement for growth potential and therefore the potency of the product.
Why are their Disadvantages of the Colony-Forming Cell Method as a Potency Assay?
As describe on other pages of this website, the CFC assay suffers from many drawbacks. As a quality control assay to measure potency, the CFC assay is a subjective assay because the assay requires manual enumeration of colonies. And because of this manual readout, the CFC assay cannot be calibrated and therefore cannot standardized. In addition, and as a direct result, it can only be properly validated and then only with considerable effort. There are also other aspects that have to be taken into account.
What are the Regulatory Issues involved?
To see a discussion on regulatory issues pertaining to stem cell potency assays for stem cell transplantation, click here.
What is CAMEO™-96 STD?
CAMEO™-96 STD is the first and only standardized colony-forming cell assay
The HALO® Platform and, in particular, the HALO® Stem and Progenitor Cell - Quality Control (SPC-QC) Platform, was the first standardized stem cell potency assay to be specifically designed for stem cell processing laboratories.
The HALO® SPC-QC Platform exists in two formats, one with methylcellulose (HALO®-96 MeC SPC-QC) and the other without methylcellulose (HALO®-96 SEC SPC-QC). Both take all the considerations into account necessary for a validated potency assay. In addition, both are performed in half the time or less than the traditional CFC assay.
However, realizing that people have difficulty in changing to a new assay after over 40 years, HemoGenix® has developed an innovative way of standardizing the CFC assay so that it can conform to the majority of characteristics for a potency assay. Like the traditional colony-forming cells assay, it still takes 14 days to obtain results, but the results that are obtained are standardized. For a 5 or 7 day stem and progenitor cell potency assay without having to manually count colony, click here.
The protocol for the CAMEO™-96 STD Platform is shown below and involves a two stage process.
When the CFC assay, HALO®-96 MeC and HALO®-96 SEC are all performed under exactly the same conditions, the results from all three assays can be correlated. This means not only are the two HALO® assays interchangeable, but both are alternatives, and in most cases, can replace, the traditional colony-forming cell assay. However, because the readouts of three assays correlate with each other, this relationship allows total colony counts from the CFC assay at 14 days of culture to be expressed in ATP concentration equivalents of the HALO®-96 MeC performed after 7 days of culture or HALO®-96 SEC perform only after 5 days of culture. (Follow this link for more information).
These correlative results led to the development of a combined colony-forming cell and HALO® assay called CAMEO™™-96 STD, a bi-functional assay that measures both proliferation and differentiation in the same culture at 14 days and, simultaneously allows the CFC assay to be standardized from an instrument-based, calibrated ATP bioluminescence measurement.
CAMEO™-96 STD is a miniaturized, methylcellulose colony-forming cell assay performed in a 96-well plate in two primary stages.
Stage 1. Add cells to the pre-dispensed Master Mix provided with the kit. Dispense 6 x 0.1ml replicate cultures from a single sample into the 96-well plate provided and incubate for 14 days. Count and/or identify the colonies in each well. At this point, you have now measured the differentiation potential of the stem and/or progenitor cells in the sample. (Steps 1-5 in the diagram above).
Stage 2. Perform a simple bioluminescnce ATP standard dose response in another 96-well plate (provided with the kit). This allows conversion of the plate luminometer output to standardized ATP concentrations. Then process the sample plate, measure the luminescence. (You do not have to use the whole 96-well plate to perform this assay). Since intracellular ATP concentration is directly proportional to proliferation, you have just performed a proliferation assay on your sample(s). Your plate luminometer software can be programmed to calculate sample ATP concentrations. By plotting total colony counts against the ATP concentrations from the same culture, the linear regression obtained allows colony counts to be expressed as ATP concentration equivalents, thereby standardizing the CFC assay against an external ATP standard.
By culturing the cells under exactly the same conditions, you have just performed a fully standardized duel proliferation and differentiation assay on the same sample. You are now in a position to standardized other procedures that depend on a potency assay as well as validating your results internally and with other laboratories.
The graph below shows the correlation between the number of large colonies manually counted and the total number of colonies counts (subtraction of the two provides the number of small colonies counted) and ATP production as a function of cell concentration / well. The linear regressions obtained allow the colony number obtained to be expressed in ATP concentration (µM) equivalents. Since the readouts from both assay were obtained from the same sample cultured under exactly the same conditions in a 96-well plate, the standardized ATP proliferation assay can be used to standardized the colony-forming cell assay.
What cell populations can I detect using CAMEO™™-96 STD?
The 14 day CAMEO™™-96 STD Potency Assay Kits are similar to the HemoGenix® 7 day HALO®-96 MeC SPC-QC and the 5 day HALO®-96 SEC SPC-QC, in that the same configurations all provided for all three platforms depending on the processing laboratory applications.
Single Stem Cell Potency Assay: Detects and measures proliferation and differentiation potential of the multipotential stem cell population (CFC-GEMM). No. of samples/96-well plate: 16 at 6 replicates/sample.
Duel Stem Cell Potency Assay: Detects and measures proliferation and differentiation potential of the primitive High Proliferative Potential Stem and Progenitor (HPP-SP) Stem Cell, which produces both lymphopoietic and hematopoietic lineages, and the mature CFC-GEMM population. No. of samples/96-well plate: 8 at 6 replicates/sample.
4-Population Potency Assay: Detects and measures proliferation and differentiation potenial of CFC-GEMM, BFU-E, GM-CFC and Mk-CFC. No. of samples/96-well plates: 4 at 6 replicates/sample.
7-Population Potency Assay: Detects and measures proliferation and differentiation potential of HPP-SP, CFC-GEMM, BFU-E, GM-CFC, Mk-CFC, T-CFC, B-CFC and a background control. No. of samples/96-well plate: 2 at 6 replicates/sample.
What's in the CAMEO™-96 STD Box?
All CAMEO™-96 STD Kits come complete to perform the assay. Just add cells.
Each kit contains the following contents:
CAMEO™™-96 STD Kits are available in 1, 2, 3 and 4 plate kit sizes.
Downloads and Links
Download the CAMEO™-96 STD Flyer
Download the CAMEO™-96 STD Protocol
Download the CAMEO™-96 STD Manual (available soon)
