A consensus introduction to serum replacements and serum-free media for cellular therapies.

The cell therapy industry is a fast-growing industry targeted toward a myriad of clinical indications. As the cell therapy industry matures and clinical trials hit their pivotal Phase 3 studies, there will be a significant need for scale-up, process validation, and critical raw material quality assurance. Part of the well discussed challenges of upscaling manufacturing processes there is a less discussed issue relating to the availability of raw materials in the needed quality and quantities. The FDA recently noted that over 80% of the 66 investigational new drug (IND) applications for mesenchymal stem cell (MSC) products analyzed described the use of FBS during manufacturing. Accumulated data from the past years show an acceleration in serum consumption by at least 10%-15% annually, which suggests that the global demand for serum may soon exceed the supply. Ongoing concerns of safety issues due to risks of various pathogen contaminations, as well as issues related to the aforementioned serum variability that can affect final product reproducibility, are strong motivators to search for serum substitutes or serum-free media. it is important to note that there are no accepted definitions for most of these terms which leads to misleading's and misunderstandings, where the same term might be defined differently by different vendors, manufacturer, and users. It is the drug developer's responsibility to clarify what the supplied labels mean and to identify the correct questions and audits to ensure quality. The paper reviews the available serum replacements, main components, basic strategies for replacement of serum and suggests definitions.

Detection and enrichment of hematopoietic stem cells by side population phenotype.

A flow cytometric procedure has recently been described to isolate hematopoietic stem cells from mouse bone marrow based on the efflux properties of the vital dye Hoechst 33342. The assay defines a subset of cells-termed the "side population" (SP)-by simultaneously measuring fluorescence of the dye at two wavelengths (~450 nm and >670 nm). In this chapter, SP protocols are provided to detect candidate hematopoietic stem cells in mouse bone marrow and human cord blood. In the standard method, SP profiles are readily observed on a stream-in-air cell sorter using 30 mW of 351-356 nm ultraviolet excitation from a krypton-ion laser. Alternatively, SP profiles can be resolved on an analytical flow cytometer with cuvette flow cell using 8 mW of 325-nm ultraviolet excitation from a helium-cadmium laser. The ability to perform the SP assay on an analytical instrument facilitates optimization of staining conditions to identify hematopoietic and other stem cells in a variety of tissues. It is also demonstrated that SP profiles of slightly lower resolution can be obtained on a stream-in-air cell sorter using 100 mW of 407-nm violet excitation from a krypton-ion laser, raising the possibility that with appropriate validation the SP assay could be performed on flow cytometers that are not equipped with ultraviolet lasers.

Multiparameter flow cytometry of fluorescent protein reporters.

Reporters based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and GFP-like proteins from other marine organisms provide valuable tools to monitor gene transfer and expression noninvasively in living cells. Stable cell lines were generated from the Sp2/0-Ag14 hybridoma that express up to three spectral enhanced versions of GFP, the enhanced cyan fluorescent protein (ECFP), the enhanced green fluorescent protein (EGFP), and the enhanced yellow fluorescent protein (EYFP), and/or a variant of the Discosoma coral red fluorescent protein (DsRed). The panel of lines was used to demonstrate a flow cytometric procedure for simultaneous analysis of all four fluorescent proteins that utilizes dual-laser excitation at 488 nm and 407 nm. Additional schemes for simultaneous detection of two, three or four of these fluorescent proteins are also presented.