Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

BACKGROUND: Automated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell-based products, according to Ph. Eur. 2.6.27. Additionally, a study to select the best vial to detect fungus contamination was performed. METHODS: Microorganisms representing Gram-negative, Gram-positive, aerobic, anaerobic, spore-forming, slow-growing bacteria, yeast and mold were prepared in either Dulbecco's PBS or seven biological matrices containing approximately 5, 10, and 15 colony-forming units (CFU) per sample. These preparations were inoculated to the specific media required for each test method: (i) BACTEC aerobic and anaerobic vials; (ii) aerobic and anaerobic media for direct inoculation; and (iii) Trypcase soy 3P or Brucella blood agar plates. Colonies from cultures were identified using MALDI-TOF mass spectrometry. RESULTS: The BACTEC FX40 system, in both Dulbecco's PBS and the biological matrices with a 5-CFU inoculum, detected most of the microorganisms significantly faster than the conventional method, despite the presence of a matrix containing gentamicin and several matrices containing 10% DMSO. Conversely, it showed an extremely delayed detection of Candida albicans compared with the conventional method. The addition of a Mycosis IC/F (MYC) vial decreased radically the time to detection (TTD) of C. albicans (28.2 ± 1.8 h) compared with the conventional method (36 h). CONCLUSIONS: When a MYC vial was added to the standard aerobic and anaerobic vials to test each sample, BACTEC FX40 was shown to be a superior alternative sterility method for cell-therapy products contaminated with low inocula, with a faster TTD for microbial growth compared with the conventional method (5 versus 14 days). The studies were carried out in different cell-based matrices with sensitivities and specificities of 100% for all the tested strains at 15-, 10- and 5-CFU inoculum, with the exception of Kocuria rhizophila at 5 CFU (90.48% sensitivity and 100% specificity).

Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products.

BACKGROUND AIMS: Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). METHODS: In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. RESULTS: Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. CONCLUSIONS: Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Mesenchymal properties of SJL mice-stem cells and their efficacy as autologous therapy in a relapsing-remitting multiple sclerosis model.

INTRODUCTION: Mesenchymal stem cells (MSCs) are a multipotent population of adult stem cells, which may represent a promising therapeutic approach for neurological autoimmune diseases such as multiple sclerosis. The mouse is the most used species for obtaining and studying the characteristics of MSC and their potential as autologous transplants in pre-clinical models. However, conflicting data have been published disclosing intraspecies variations. The choice of the mouse strain and the tissue source appear, among others, as important factors in the experimental application of MSCs. METHODS: Adipose tissue-derived MSCs obtained from the SJL/JCrl mouse strain (SJL-AdMSC) have been cultured for a long time (from passage 0 up to 15) under controlled experimental conditions, and their growth rate, morphology, stromal and haematopoietic marker expression profiles and differentiation capacity towards adipocytes, osteocytes and chondrocytes have been determined. Moreover, their preclinical efficacy has been assessed by autologous transplant in relapsing-remitting experimental autoimmune encephalomielitis (RR-EAE)-induced SJL mice (a well established mice model for the study of RR-multiple sclerosis). RESULTS: We demonstrate that SJL-AdMSCs show the same fibroblastic shape, growth rate, profile of markers expression and multipotency described for MSCs in every passage evaluated (up to passage 15). Additionally, SJL-AdMSCs ameliorate the RR-EAE course, suggesting that they could modulate disease progression. Moreover, their features studied are fully comparable with the standardized Ad-MSCs obtained from the C57BL/6 mouse strain, which strengthens their use in cell therapy. CONCLUSION: SJL-AdMSCs might be a suitable source of Ad-MSCs for studies related to the properties of MSCs and their application as promising therapeutic tools in autologous transplants in experimental medicine.

Killer cell immunoglobulin-like receptor genes in Spanish multiple sclerosis patients.

Killer cell immunoglobulin-like receptors (KIRs) are regulators of cytolytic activity of natural killer and certain T cells through interactions with human leukocyte antigen (HLA) class I ligands. KIRs have been shown to contribute to the pathogenesis of several autoimmune diseases, but their role in multiple sclerosis (MS) is still unclear. Here we determined the influence of KIR genes and their HLA class I ligands on susceptibility to MS and on the response to interferon-beta treatment in a Spanish population. KIR and HLA genotyping were performed in 200 MS patients and 200 controls. Significantly higher frequencies were found for KIR2DL5 and KIR3DS1 genes in MS patients and the carriage of the KIR2DL1 gene was associated with a higher progression index. Moreover, the frequency of the HLA-Bw4 motif was significantly reduced in MS patients. The KIR2DL1 and HLA-C2 matches were more frequent in MS patients, whereas the KIR3DL1 and HLA-Bw4 matches were more frequent in healthy controls. Nevertheless, non significant associations were found between all the KIR genes and therapeutic response to interferon-beta. Our results confirm that the carriage of HLA-Bw4 is a protective factor in MS and suggest that KIR2DL5 and KIR3DS1 may have a predisposing role in the disease.

Kinetics and incidence of anti-natalizumab antibodies in multiple sclerosis patients on treatment for 18 months.

Natalizumab is a monoclonal antibody shown to be highly effective in the treatment of relapsing-remitting multiple sclerosis (RRMS). Patients treated with natalizumab can develop antibodies directed against this agent that may affect the efficacy and safety of the drug. In this observational study, the kinetics of the appearance and the incidence of anti-natalizumab antibodies were followed prospectively for 18 months in a cohort of 64 consecutive patients treated with natalizumab for relapsing MS. Blood samples were drawn immediately before starting natalizumab therapy and each month afterwards. The presence of antibodies against natalizumab was assessed by enzyme-linked immunosorbent assay (ELISA) in all patients. Anti-natalizumab antibodies were detected in nine (14.1%) natalizumab-treated patients, three (4.68%) of whom were transiently positive while six (9.37%) were persistently positive (these patients discontinued natalizumab). All positive titres were observed during the first 4 months of treatment. One patient with a hypersensitivity reaction also had persistent antibodies. We conclude that antibodies against natalizumab develop early, within the first 6 months of therapy with natalizumab. Although no antibodies were detected after 4 months of therapy in this particular study, this does not rule out their development later on in exceptional cases.