1.1 This practice describes how to conduct a non destructive proliferation test for mammalian cells based on metabolic activity that can be used to assess the number of viable cells within 3D-scaffolds for regenerative medicine and in tissue engineered medical products (TEMPs). 1.2 This practice provides a detailed explanation of the resazurin cell metabolic activity method in terms of reagent concentrations, incubation times, cell culture media composition, calibration curve, assay linearity and limitations of the assay. 1.3 This practice describes factors that can interfere with accurate cell proliferation assessment. 1.4 This document does not propose acceptance criteria for a cell-based product based on the application of a cell proliferation test method. 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and to determine the applicability of regulatory limitations prior to use.
Keywordscell proliferation; cell counting; stem cells; cell dose; tissue regeneration; resazurin; non-destructive method
Aim of this practice is to provide a protocol to quantify cell proliferation of viable cells within or on biomaterial scaffolds used in regenerative medicine and in tissue engineered medical products. Cell proliferation on a cell-biomaterial construct is one of the most important processes to be monitored in vitro in order to predict the in vivo performance of the construct before releasing the construct on the market for both research and clinical applications. By monitoring the cell proliferation over time it is possible to obtain important understandings on cellular response to the scaffold, on the compatibility of the biomaterial, on the scaffold ability to support the cell growth, interactions between cell and scaffold, influences of the biomaterial composition on the cell growth or cell differentiation and finally on the efficacy of the cell-scaffold construct. For such a reasons reliability and high reproducibility are necessary intra and inter laboratories. However, there is a lack of standardization in this field. Cell proliferation assessment is typically performed by destructive tests inducing modification or perturbation of the cell growth conditions, by cell detachment and direct cell counting. However, to follow the proliferation over time and understand how a specific 3D cell culture is growing, it is necessary to monitor the same sample, the same scaffold over a long period of time, typically 3 weeks. Non-destructive methods are strongly required. The practice claims the need for standardization of a resazurin-based method for cell dose estimation within a non-destructive process of cell proliferation monitoring. This practice provides a protocol for a non destructive proliferation test for cells: it can be used to assess the proliferation of viable cells within 3D-scaffolds for regenerative medicine and in tissue engineered medical products. Users will be both researchers and clinicians in applied research centers and preclinical and clinical research hospitals.
The title and scope are in draft form and are under development within this ASTM Committee.Back to Top
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