3D extracellular matrix derived model of alveolar rhabdomyosarcoma

INTRODUCTION: Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood, among the subtypes the Alveolar (ARMS) is the more aggressive with a higher tendency to metastasize [1]. Integrins are a class of transmembrane adhesion molecules that mediate survival, differentiation, migration and differentiation [2]. Here we investigate the role of integrins in ARMS metastatic migration in an engineered 3D scaffold. METHODS: ARMS xenografts are obtained from subcutaneous injection of RH30 cells in immunodeficient mice. Composition of the ECM is determined by proteomic analysis. The main components of the ECM are used to enrich a 3D collagen scaffold cultured in a perfusion bioreactor. Cells are analyzed by qPCR for the expression of a panel of integrins. Presence of the protein is confirmed by flow cytometry immunofluorescence. MMPs expression is evaluated by zymography. RESULTS: Verified the expression of human and ARMS marker and typical tumor morphology in xenografts, they are processed for proteomic analysis. Proteomic data analysis is currently under investigation. Preliminary data culturing RH30 cells in 3D bioreactor show upregulation of ITG5 and CXCR4 receptor compared to 2D condition. Localization and quantification at protein level will be assessed respectively by immunofluorescence and cytofluorimetry. Expression of MMP-9 and MMP-2 has been assessed by zymography comparing the expression of these MMPs in 2D vs 3D bioreactor and RH30 isolated from the xenograft. DISCUSSION & CONCLUSIONS: Preliminary data on ITG expression show that in 3D scaffold the expression of ITG5 and CXCR4 is upregulated. In parallel the active form of MMP-2 is more present in 3D models compared to 2D. Other groups reported a mechanical interaction between ITG5 and MMP-2 [3]. This interaction will be studied in a more representative engineered 3D scaffold to shed light on the complex interaction between ECM and metastatic progression

Validation of analytical methods in compliance with good manufacturing practice: A practical approach

BACKGROUND: The quality and safety of cell therapy products must be maintained throughout their production and quality control cycle, ensuring their final use in the patient. We validated the Lymulus Amebocyte Lysate (LAL) test and immunophenotype according to International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, considering accuracy, precision, repeatability, linearity and range. METHODS: For the endotoxin test we used a kinetic chromogenic LAL test. As this is a limit test for the control of impurities, in compliance with International Conference on Harmonization Q2 Guidelines and the EU Pharmacopoeia, we evaluated the specificity and detection limit. For the immunophenotype test, an identity test, we evaluated specificity through the Fluorescence Minus One method and we repeated all experiments thrice to verify precision. The immunophenotype validation required a performance qualification of the flow cytometer using two types of standard beads which have to be used daily to check cytometer reproducibly set up. The results were compared together. Collected data were statistically analyzed calculating mean, standard deviation and coefficient of variation percentage (CV%). RESULTS: The LAL test is repeatable and specific. The spike recovery value of each sample was between 0.25 EU/ml and 1 EU/ml with a CV% < 10%. The correlation coefficient (≥ 0.980) and CV% (< 10%) of the standard curve tested in duplicate showed the test's linearity and a minimum detectable concentration value of 0.005 EU/ml. The immunophenotype method performed thrice on our cell therapy products is specific and repeatable as showed by CV% inter -experiment < 10%. CONCLUSIONS: Our data demonstrated that validated analytical procedures are suitable as quality controls for the batch release of cell therapy products. Our paper could offer an important contribution for the scientific community in the field of CTPs, above all to small Cell Factories such as ours, where it is not always possible to have CFR21 compliant software