Quality of Cell Products: Authenticity, Identity, Genomic Stability and Status of Differentiation

Abstract

Cellular therapies that either use modifications of a patient's own cells or allogeneic cell lines are becoming in vogue. Besides the technical issues of optimal isolation, cultivation and modification, quality control of the generated cellular products are increasingly being considered to be more important. This is not only relevant for the cell's therapeutic application but also for cell science in general. Recent changes in editorial policies of respected journals, which now require proof of authenticity when cell lines are used, demonstrate that the subject of the present paper is not a virtual problem at all. In this article we provide 2 examples of contaminated cell lines followed by a review of the recent developments used to verify cell lines, stem cells and modifications of autologous cells. With relative simple techniques one can now prove the authenticity and the quality of the cellular material of interest and therefore improve the scientific basis for the development of cells for therapeutic applications. The future of advanced cellular therapies will require production and characterization of cells under GMP and GLP conditions, which include proof of identity, safety and functionality and absence of contamination.

Fig. 1

Analysis of ‘human’ immature neuronal cells. A The short tandem repeat (STR) patterns of uncultivated male human donor cells isolated by positive magnetic cell sorting. STR loci tested were D5S818 (1), vWA (2), D13S317 (3), THO1 (4), D7S820 (5), TPOX (6), D16S539 (7), CSF1 (8) and amelogenin (X and Y) [37]. A unique human STR pattern without indication of cross-contamination was obtained. B The cultivated cells reacted with antibodies against neuron specific proteins, anti-human bassoon, a large multidomain protein in the presynaptic active zone (green fluorescence) and anti-human CaC, a voltage-gated Ca²⁺ channel protein (P/Q-type, [.alpha]-1A subunit) – red fluorescence). The antibodies were known to cross-react to other mammalian species such as rats. Insert: scanning electron microscopy of cultured cells. C Chromosome analysis of the cultured cells revealed that the cells are not of human origin. The large acrocentric chromosomes (arrows) and the small metacentric chromosomes (arrowheads) suggest a rat karyotype [67], which D was confirmed by PCR analysis of the cultured neurons.

Fig. 2

Bone marrow-derived Stem-1 cell line identified as SAOS-2 cells (osteosarcoma cell line). Comparison of STR analysis with a cell line database (DSMZ) indicated that the STR pattern of Stem1 is identical to the pattern of SAOS-2 (see table 1). The same STR loci as in figure 1 were used. Further analysis of different passages and samples confirmed the mix-up/contamination (data not shown).

Fig. 3

Example for characterization of dendritic cell products. STR analyses of freshly isolated CD14+ monocytes (A) and of mature dendritic cells after 7 days in cell culture (B) confirm authenticity. STR loci are numbered as in figure 1. Detection of X-amelogenin and Y-amelogenin indicates a male donor. C) Expression profiling of dendritic cells derived from CD14+ monocytes (day 0) of leukapheresis samples (LK53, LK61, LK68 and LK78) during cultivation and maturation (day 4 and day 7) using Affymetrix HG-U95a DNA arrays. Heat map of selected genes indicates changes in gene expression during maturation. Up-regulation of maturation specific genes is evident LK61, LK68 and LK78 D) Immunostimulatory function of the mature dendritic cell product was confirmed by interferon [γ] – ELISPOT assays: Tyrosinase-specific T cells were stimulated by superantigen staphylococcal enterotoxin (SEB) (unspecific positive control) and specifically by tyrosinase peptide-presenting mature dendritic cells (tyr) but not by > dendritic cells alone or dendritic cells loaded with an unrelated peptide. The experiments were performed in triplicate; mean and standard deviation are shown.