Sumona Sarkar

The support of bone marrow stromal cell differentiation by airbrushed nanofiber scaffolds

Nanofiber scaffolds are effective for tissue engineering since they emulate the fibrous nanostructure of native extracellular matrix (ECM). Although electrospinning has been the most common approach for fabricating nanofiber scaffolds, airbrushing approaches have also been advanced for making nanofibers. For airbrushing, compressed gas is used to blow polymer solution through a small nozzle which shears the polymer solution into fibers. Our goals were 1) to assess the versatility of airbrushing, 2) to compare the properties of airbrushed and electrospun nanofiber scaffolds and 3) to test the ability of airbrushed nanofibers to support stem cell differentiation. The results demonstrated that airbrushing could produce nanofibers from a wide range of polymers and onto a wide range of targets. Airbrushing was safer, 10-fold faster, 100-fold less expensive to set-up and able to deposit nanofibers onto a broader range of targets than electrospinning. Airbrushing yielded nanofibers that formed loosely packed bundles of aligned nanofibers, while electrospinning produced un-aligned, single nanofibers that were tightly packed and highly entangled. Airbrushed nanofiber mats had larger pores, higher porosity and lower modulus than electrospun mats, results that were likely caused by the differences in morphology (nanofiber packing and entanglement). Airbrushed nanofiber scaffolds fabricated from 4 different polymers were each able to support osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs). Finally, the differences in airbrushed versus electrospun nanofiber morphology caused differences in hBMSC shape where cells had a smaller spread area and a smaller volume on airbrushed nanofiber scaffolds. These results highlight the advantages and disadvantages of airbrushing versus electrospinning nanofiber scaffolds and demonstrate that airbrushed nanofiber scaffolds can support stem cell differentiation.

Fabrication of a layered microstructured polycaprolactone construct for 3-D tissue engineering.

Successful artificial tissue scaffolds support regeneration by promoting cellular organization as well as appropriate mechanical and biological functionality. We have previously shown in vitro that 2-D substrates with micrometer-scale grooves (5 μm deep, 18 μm wide, with 12 μm spacing) can induce cell orientation and ECM alignment. Here, we have transferred this microtopography onto biodegradable polycaprolactone (PCL) thin films. We further developed a technique to layer these cellularized microtextured scaffolds into a 3-D tissue construct. A surface modification technique was used to attach photoreactive acrylate groups on the PCL scaffold surface onto which poly(ethylene glycol)-diacrylate (PEG-DA) gel could be photopolymerized. PEG-DA serves as an adhesive layer between PCL scaffolds, resulting in a VSMC-seeded layered 3-D composite structure that is highly organized and structurally stable. The PCL surface modification chemistry was confirmed via XPS, and the maintenance of cell number and orientation on the modified PCL scaffolds was demonstrated using colorimetric and imaging techniques. Cell number and orientation were also investigated after cells were cultured in the layered 3-D configuration. Such 3-D tissue mimics fabricated with precise cellular organization will enable systematic testing of the effects of cellular orientation on the functional and mechanical properties of tissue-engineered blood vessels.

Strategies for Achieving Measurement Assurance for Cell Therapy Products

The cell therapy industry has identified the inability to reliably characterize cells as possibly its greatest challenge and has called for standards and reference materials to provide assurance for measurements of cell properties. The challenges in characterization of cell therapy products can be largely addressed with systematic approaches for assessing sources of uncertainty and improving confidence in key measurements. This article presents the many strategies that can be used to ensure measurement confidence and discusses them in terms of how they can be applied to characterization of cell therapy products. Application of these strategies to cell measurements will help to establish qualified assays for cell characterization, which may help streamline regulatory approval and enable more efficient development of cell therapy products.

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

The development of standards for the field of regenerative medicine has been noted as a high priority by several road-mapping activities. Additionally, the U.S. Congress recognizes the importance of standards in the 21st Century Cure Act. Standards will help to accelerate and streamline cell and gene therapy product development, ensure the quality and consistency of processes and products, and facilitate their regulatory approval. Although there is general agreement for the need of additional standards for regenerative medicine products, a shared understanding of standards is required for real progress toward the development of standards to advance regenerative medicine. Here, we describe the roles of standards in regenerative medicine as well as the process for standards development and the interactions of different entities in the standards development process. Highlighted are recent coordinated efforts between the U.S. Food and Drug Administration and the National Institute of Standards and Technology to facilitate standards development and foster science that underpins standards development.

3D Cellular Morphotyping of Scaffold Niches

There is currently no method for assessing the nature of the cell niche provided by 3D biomaterial scaffolds. Analyzing human bone marrow stromal cell (hBMSC) 3D cell shape in response to different biomaterial scaffolds allowed the 3D cell niche promoted by biomaterial scaffolds to be evaluated. Primary hBMSCs (p5) were seeded (5,000 cells/cm2) in 10 different biomaterial scaffolds and cultured for 24 h. Samples were fixed and stained for actin and nucleus, imaged with confocal microscopy to obtain a 3D volume (z-stack), and 3D cell shape was analyzed with computational approaches. Over 100 cells were imaged per scaffold group (10 scaffold groups, ~1250 cells total), resulting in the largest known 3D stem cell dataset (~135,000 files, ~135 GB) and enabling a high degree of statistical rigor. The images were segmented using an automated algorithm and a final dataset of 969 well-segmented cells were analyzed with 79 shape metrics, which enabled 3D cellular morphotyping of scaffold niches. The variety of scaffolds studied promoted different cell morphologies during culture and there were significant differences in shape metrics, particularly for cell depth, surface area, and volume. This study demonstrated a quantitative approach to analyze 3D cell shape and morphotype and is the largest known study analyzing 3D cell shape in response to a variety of biomaterial scaffolds. The dataset is publically accessible with an online 3D viewer. These results could inform the selection of prospective scaffolds for applications based on 3D cell shape in the tissue of interest.