NIST and FDA collaboration on standards development activities and laboratory programs supporting translation of regenerative medicine products

Standards are a powerful force in the protection of public health and safety, the development and commercialization of new technologies, and facilitation of national and international commerce. In the field of regenerative medicine, the need for standards has emerged as a common gap from various workshops, meetings, and discussion forums. Here we provide an update on multiple collaborative efforts between the U.S. Food & Drug Administration (FDA) and the National Institute of Standards and Technology (NIST), to support standards development for regenerative medicine R&D, manufacturing, and translation. Collaborative efforts include coordinated workshops, leadership and participation in standards development efforts within relevant SDOs, and joint research projects. These collaborations leverage NIST expertise in measurement sciences to address specific analytical scientific challenges as well as leveraging FDA regulatory expertise in regenerative medicine products to ensure that the science and standards developed address significant regulatory challenges that recur across the field. This talk will focus on joint FDA-NIST efforts to develop standards within ISO/TC 276: Biotechnology, and several collaborative projects on cell counting and cell viability to support the development of standards. Cell viability is an important quality attribute often used as a manufacturing in-process control or a release criterion for cell therapy products. Assurance for cell viability measurements has been particularly difficult due to ambiguities in the definition of viable cells as well as measurement challenges associated with quantifying cell health. The joint FDA-NIST project in cell viability aims to provide strategies for establishing validation protocols for cell viability measurements

Synthesis and characterization of a chondroitin sulfate based hybrid bio/synthetic biomimetic aggrecan macromolecule

Lower back pain resulting from intervertebral disc degeneration is one of the leading musculoskeletal disorders confronting our health system. In order to mechanically stabilize the disc early in the degenerative cascade and prevent the need for spinal fusion surgeries, we have proposed the development of a hybrid-bio/synthetic biomimetic proteoglycan macromolecule for injection into the disc in the early stages of degeneration. The goal of this thesis was to incorporate natural chondroitin sulfate (CS) chains into bottle brush polymer synthesis strategies for the fabrication of CS-macromolecules which mimic the proteoglycan structure and function while resisting enzymatic degradation. Both the “grafting-to” and “grafting-through” techniques of bottle brush synthesis were explored. CS was immobilized via a terminal primary amine onto a model polymeric backbone (polyacrylic acid) for investigation of the “grafting-to” strategy and an epoxy-amine step-growth polymerization technique was utilized for the “grafting-through” synthesis of CS-macromolecules with polyethylene glycol backbone segments.Incorporation of a synthetic polymeric backbone at the terminal amine of CS was confirmed via biochemical assays, 1H-NMR and FTIR spectroscopy, and CS-macromolecule size was demonstrated to be higher than that of natural CS via gel permeation chromatography, transmission electron microscopy and viscosity measurements. Further analysis of CS-macromolecule functionality indicated maintenance of natural CS properties such as high fixed charge density, high osmotic potential and low cytotoxicity with nucleus pulposus cells.These studies are the first attempt at the incorporation of natural CS into biomimetic bottle brush structures. CS-macromolecules synthesized via the methods developed in these studies may be utilized in the treatment and prevention of debilitating back pain as well as act as mimetics for other proteoglycans implicated in cartilage, heart valve, and nervous system tissue function.Ph.D., Biomedical Engineering -- Drexel University, 201

Evaluating The Quality Of Cell Counting Measurements Using Experimental Design And Statistical Analysis

Cell counting measurements are critical in the research, development, and manufacturing of cell therapy products where they support decision-making in product testing and release. Evaluating cell quantity with accuracy and precision has remained a challenge for many specific applications or purposes. While new measurement platforms have been developed with increased measurement throughput and improved precision, discrepancies between cell counts acquired via various measurement processes are still pervasive. In addition, the industry as a whole has recognized that complex biological properties, as well as operator, equipment, and procedure variations can greatly affect the measurement quality, and the development of a single reference material or reference measurement is impractical to address broad counting needs. Here, we describe an experimental design and statistical analysis approach to evaluate the quality of a given cell counting measurement process. The experimental design uses a dilution series study with replicate samples as well as procedures to reduce pipetting error, and operator and temporal bias. The statistical analysis methods generate a set of metrics for evaluating measurement quality in terms of accuracy and precision, where accuracy is based on deviation from proportionality. In this design, a proportional response to dilution fraction serves as an internal control, where deviation from a proportional response is indicative of a systematic or non-systematic bias in the measurement process. The utility of this approach was demonstrated in the counting of human mesenchymal stem cells (hMSC) via automated or manual counting methods, where the automated method performed better in terms of both precision and proportionality. These results enabled a transition from the labor intensive and often imprecise manual counting method to the automated counting method. The experimental design and statistical method presented here is agnostic to the cell type and analytical platform, thus suitable as a horizontal approach to evaluate the quality of cell counting measurements with respect to method selection, optimization, and validation, thereby facilitating subsequent decision making. We are also working closely with industry partners and Standards Development Organizations (SDOs) to develop cell counting standards using this and other strategies

Assessing the suitability of cell counting methods during different stages of a cell processing workflow using an ISO 20391-2 guided study design and analysis

Cell counting is a fundamental measurement for determining viable cell numbers in biomanufacturing processes. The properties of different cell types and the range of intended uses for cell counts within a biomanufacturing process can lead to challenges in identifying suitable counting methods for each potential application. This is further amplified by user subjectivity in identifying the cells of interest and further identifying viable cells. Replacement of traditionally used manual counting methods with automated systems has alleviated some of these issues. However, a single cell type can exhibit different physical properties at various stages of cell processing which is further compounded by process impurities such as cell debris or magnetic beads. These factors make it challenging to develop a robust cell counting method that offers a high level of confidence in the results. Several initiatives from standards development organizations have attempted to address this critical need for standardization in cell counting. This study utilizes flow-based and image-based methods for the quantitative measurement of cell concentration and viability in the absence of a reference material, based on the tools and guidance provided by the International of Standards (ISO) and the US National Institute of Standards and Technology (NIST). Primary cells were examined at different stages of cell processing in a cell therapy workflow. Results from this study define a systematic approach that enables the identification of counting methods and parameters that are best suited for specific cell types and workflows to ensure accuracy and consistency. Cell counting is a foundational method used extensively along various steps of cell and gene therapy. The standard used in this study may be applied to other cell and gene therapy processes to enable accurate measurement of parameters required to guide critical decisions throughout the development and production process. Using a framework that confirms the suitability of the cell counting method used can minimize variability in the process and final product

Sensor technologies for quality control in engineered tissue manufacturing

The use of engineered cells, tissues, and organs has the opportunity to change the way injuries and diseases are treated. Commercialization of these groundbreaking technologies has been limited in part by the complex and costly nature of their manufacture. Process-related variability and even small changes in the manufacturing process of a living product will impact its quality. Without real-time integrated detection, the magnitude and mechanism of that impact are largely unknown. Real-time and non-destructive sensor technologies are key for in-process insight and ensuring a consistent product throughout commercial scale-up and/or scale-out. The application of a measurement technology into a manufacturing process requires cell and tissue developers to understand the best way to apply a sensor to their process, and for sensor manufacturers to understand the design requirements and end-user needs. Furthermore, sensors to monitor component cells’ health and phenotype need to be compatible with novel integrated and automated manufacturing equipment. This review summarizes commercially relevant sensor technologies that can detect meaningful quality attributes during the manufacturing of regenerative medicine products, the gaps within each technology, and sensor considerations for manufacturing

A Bioinformatics 3D Cellular Morphotyping Strategy For Assessing Biomaterial Scaffold Niches

Many biomaterial scaffolds have been advanced to provide synthetic cell niches for tissue engineering and drug screening applications; however, current methods for comparing scaffold niches focus on cell functional outcomes or attempt to normalize materials properties between different scaffold formats. We demonstrate a three-dimensional (3D) cellular morphotyping strategy for comparing biomaterial scaffold cell niches between different biomaterial scaffold formats. Primary human bone marrow stromal cells (hBMSCs) were cultured on 8 different biomaterial scaffolds, including fibrous scaffolds, hydrogels, and porous sponges, in 10 treatment groups to compare a variety of biomaterial scaffolds and cell morphologies. A bioinformatics approach was used to determine the 3D cellular morphotype for each treatment group by using 82 shape metrics to analyze approximately 1000 cells. We found that hBMSCs cultured on planar substrates yielded planar cell morphotypes, while those cultured in 3D scaffolds had elongated or equiaxial cellular morphotypes with greater height. Multivariate analysis was effective at distinguishing mean shapes of cells in flat substrates from cells in scaffolds, as was the metric L1-depth (the cell height along its shortest axis after aligning cells with a characteristic ellipsoid). The 3D cellular morphotyping technique enables direct comparison of cellular microenvironments between widely different types of scaffolds and design of scaffolds based on cell structure-function relationships

A Bioinformatics 3D Cellular Morphotyping Strategy for Assessing Biomaterial Scaffold Niches

Many biomaterial scaffolds have been advanced to provide synthetic cell niches for tissue engineering and drug screening applications; however, current methods for comparing scaffold niches focus on cell functional outcomes or attempt to normalize materials properties between different scaffold formats. We demonstrate a three-dimensional (3D) cellular morphotyping strategy for comparing biomaterial scaffold cell niches between different biomaterial scaffold formats. Primary human bone marrow stromal cells (hBMSCs) were cultured on 8 different biomaterial scaffolds, including fibrous scaffolds, hydrogels, and porous sponges, in 10 treatment groups to compare a variety of biomaterial scaffolds and cell morphologies. A bioinformatics approach was used to determine the 3D cellular morphotype for each treatment group by using 82 shape metrics to analyze approximately 1000 cells. We found that hBMSCs cultured on planar substrates yielded planar cell morphotypes, while those cultured in 3D scaffolds had elongated or equiaxial cellular morphotypes with greater height. Multivariate analysis was effective at distinguishing mean shapes of cells in flat substrates from cells in scaffolds, as was the metric L₁-depth (the cell height along its shortest axis after aligning cells with a characteristic ellipsoid). The 3D cellular morphotyping technique enables direct comparison of cellular microenvironments between widely different types of scaffolds and design of scaffolds based on cell structure–function relationships