Mechanism of action, potency and efficacy: considerations for cell therapies.

One of the most challenging aspects of developing advanced cell therapy products (CTPs) is defining the mechanism of action (MOA), potency and efficacy of the product. This perspective examines these concepts and presents helpful ways to think about them through the lens of metrology. A logical framework for thinking about MOA, potency and efficacy is presented that is consistent with the existing regulatory guidelines, but also accommodates what has been learned from the 27 US FDA-approved CTPs. Available information regarding MOA, potency and efficacy for the 27 FDA-approved CTPs is reviewed to provide background and perspective. Potency process and efficacy process charts are introduced to clarify and illustrate the relationships between six key concepts: MOA, potency, potency test, efficacy, efficacy endpoint and efficacy endpoint test. Careful consideration of the meaning of these terms makes it easier to discuss the challenges of correlating potency test results with clinical outcomes and to understand how the relationships between the concepts can be misunderstood during development and clinical trials. Examples of how a product can be "potent but not efficacious" or "not potent but efficacious" are presented. Two example applications of the framework compare how MOA is assessed in cell cultures, animal models and human clinical trials and reveals the challenge of establishing MOA in humans. Lastly, important considerations for the development of potency tests for a CTP are discussed. These perspectives can help product developers set appropriate expectations for understanding a product's MOA and potency, avoid unrealistic assumptions and improve communication among team members during the development of CTPs.

Modeling, validation and verification of three-dimensional cell-scaffold contacts from terabyte-sized images.

BACKGROUND: Cell-scaffold contact measurements are derived from pairs of co-registered volumetric fluorescent confocal laser scanning microscopy (CLSM) images (z-stacks) of stained cells and three types of scaffolds (i.e., spun coat, large microfiber, and medium microfiber). Our analysis of the acquired terabyte-sized collection is motivated by the need to understand the nature of the shape dimensionality (1D vs 2D vs 3D) of cell-scaffold interactions relevant to tissue engineers that grow cells on biomaterial scaffolds. RESULTS: We designed five statistical and three geometrical contact models, and then down-selected them to one from each category using a validation approach based on physically orthogonal measurements to CLSM. The two selected models were applied to 414 z-stacks with three scaffold types and all contact results were visually verified. A planar geometrical model for the spun coat scaffold type was validated from atomic force microscopy images by computing surface roughness of 52.35 nm ±31.76 nm which was 2 to 8 times smaller than the CLSM resolution. A cylindrical model for fiber scaffolds was validated from multi-view 2D scanning electron microscopy (SEM) images. The fiber scaffold segmentation error was assessed by comparing fiber diameters from SEM and CLSM to be between 0.46% to 3.8% of the SEM reference values. For contact verification, we constructed a web-based visual verification system with 414 pairs of images with cells and their segmentation results, and with 4968 movies with animated cell, scaffold, and contact overlays. Based on visual verification by three experts, we report the accuracy of cell segmentation to be 96.4% with 94.3% precision, and the accuracy of cell-scaffold contact for a statistical model to be 62.6% with 76.7% precision and for a geometrical model to be 93.5% with 87.6% precision. CONCLUSIONS: The novelty of our approach lies in (1) representing cell-scaffold contact sites with statistical intensity and geometrical shape models, (2) designing a methodology for validating 3D geometrical contact models and (3) devising a mechanism for visual verification of hundreds of 3D measurements. The raw and processed data are publicly available from https://isg.nist.gov/deepzoomweb/data/ together with the web -based verification system.

Three-dimensional, label-free cell viability measurements in tissue engineering scaffolds using optical coherence tomography.

In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5 min) image large sample volumes (1 mm3 ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.

Measuring dimensionality of cell-scaffold contacts of primary human bone marrow stromal cells cultured on electrospun fiber scaffolds.

The properties and structure of the cellular microenvironment can influence cell behavior. Sites of cell adhesion to the extracellular matrix (ECM) initiate intracellular signaling that directs cell functions such as proliferation, differentiation, and apoptosis. Electrospun fibers mimic the fibrous nature of native ECM proteins and cell culture in fibers affects cell shape and dimensionality, which can drive specific functions, such as the osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs), by. In order to probe how scaffolds affect cell shape and behavior, cell-fiber contacts were imaged to assess their shape and dimensionality through a novel approach. Fluorescent polymeric fiber scaffolds were made so that they could be imaged by confocal fluorescence microscopy. Fluorescent polymer films were made as a planar control. hBSMCs were cultured on the fluorescent substrates and the cells and substrates were imaged. Two different image analysis approaches, one having geometrical assumptions and the other having statistical assumptions, were used to analyze the 3D structure of cell-scaffold contacts. The cells cultured in scaffolds contacted the fibers in multiple planes over the surface of the cell, while the cells cultured on films had contacts confined to the bottom surface of the cell. Shape metric analysis indicated that cell-fiber contacts had greater dimensionality and greater 3D character than the cell-film contacts. These results suggest that cell adhesion site-initiated signaling could emanate from multiple planes over the cell surface during culture in fibers, as opposed to emanating only from the cell's basal surface during culture on planar surfaces.

Biglycan regulates bone development and regeneration.

Endochondral bone development and regeneration relies on activation and proliferation of periosteum derived-cells (PDCs). Biglycan (Bgn), a small proteoglycan found in extracellular matrix, is known to be expressed in bone and cartilage, however little is known about its influence during bone development. Here we link biglycan with osteoblast maturation starting during embryonic development that later affects bone integrity and strength. Biglycan gene deletion reduced the inflammatory response after fracture, leading to impaired periosteal expansion and callus formation. Using a novel 3D scaffold with PDCs, we found that biglycan could be important for the cartilage phase preceding bone formation. The absence of biglycan led to accelerated bone development with high levels of osteopontin, which appeared to be detrimental to the structural integrity of the bone. Collectively, our study identifies biglycan as an influencing factor in PDCs activation during bone development and bone regeneration after fracture.

Quantitative, traceable determination of cell viability using absorbance microscopy.

Cell viability, an essential measurement for cell therapy products, lacks traceability. One of the most common cell viability tests is trypan blue dye exclusion where blue-stained cells are counted via brightfield imaging. Typically, live and dead cells are classified based on their pixel intensities which may vary arbitrarily making it difficult to compare results. Herein, a traceable absorbance microscopy method to determine the intracellular uptake of trypan blue is demonstrated. The intensity pixels of the brightfield images are converted to absorbance images which are used to calculate moles of trypan blue per cell. Trypan blue cell viability measurements, where trypan blue content in each cell is quantified, enable traceable live-dead classifications. To implement the absorbance microscopy method, we developed an open-source AbsorbanceQ application that generates quantitative absorbance images. The validation of absorbance microscopy is demonstrated using neutral density filters. Results from four different microscopes demonstrate a mean absolute deviation of 3% from the expected optical density values. When assessing trypan blue-stained Jurkat cells, the difference in intracellular uptake of trypan blue in heat-shock-killed cells using two different microscopes is 3.8%. Cells killed with formaldehyde take up ~50% less trypan blue as compared to the heat-shock-killed cells, suggesting that the killing mechanism affects trypan blue uptake. In a test mixture of approximately 50% live and 50% dead cells, 53% of cells were identified as dead (±6% standard deviation). Finally, to mimic batches of low-viability cells that may be encountered during a cell manufacturing process, viability was assessed for cells that were 1) overgrown in the cell culture incubator for five days or 2) incubated in DPBS at room temperature for five days. Instead of making live-dead classifications using arbitrary intensity values, absorbance imaging yields traceable units of moles that can be compared, which is useful for assuring quality for biomanufacturing processes.

Applying the Cytocentric Principles to Regenerative Medicine for Reproducibility.

Purpose of Review: Cell and tissue products do not just reflect their present conditions; they are the culmination of all they have encountered over time. Currently, routine cell culture practices subject cell and tissue products to highly variable and non-physiologic conditions. This article defines five cytocentric principles that place the conditions for cells at the core of what we do for better reproducibility in Regenerative Medicine. Recent Findings: There is a rising awareness of the cell environment as a neglected, but critical variable. Recent publications have called for controlling culture conditions for better, more reproducible cell products. Summary: Every industry has basic quality principles for reproducibility. Cytocentric principles focus on the fundamental needs of cells: protection from contamination, physiologic simulation, and full-time conditions for cultures that are optimal, individualized, and dynamic. Here, we outline the physiologic needs, the technologies, the education, and the regulatory support for the cytocentric principles in regenerative medicine.

Tissue Engineering Measurands.

A metrological perspective for thinking about the characterization of tissue engineered medical products (TEMPs) may help improve communication between researchers. During the development lifecycle of a TEMP, many product properties are measured over the long path to a product release. The selection of each measurement is designed to establish that the product is safe and efficacious (i.e., successful). However, there is often miscommunication during discussions of product characterization. The miscommunication stems from inherent assumptions that are made about the measurements. A "measurand chart" can help clarify these assumptions to enable a more coherent discussion of the value of each measurement. A measurand is defined as "the quantity or property intended to be measured". Tissue engineering measurands are discussed in terms of three case studies including "cell viability in a scaffold", "potency", and "biocompatibility". Topics including a measurement model, defining tissue engineering measurands and definitional uncertainty, are discussed to further refine thinking about tissue engineering measurands. Awareness of these concepts while discussing product characterization can enhance communication and strategic thinking so that the resulting plan is clear and purposeful.

Workshop on the characterization of fiber-based scaffolds: Challenges, progress, and future directions.

A critical component of many tissue-engineered medical products (TEMPs) is the scaffold or biomaterial. The industry's understanding of scaffold properties and their influence on cell behavior has advanced, but our technical capability to reliably characterize scaffolds requires improvement, especially to enable large-scale manufacturing. In response to the key findings from the 2013 ASTM International Workshop of Standards and Measurements for Tissue Engineering Scaffolds, the National Institute of Standards and Technology (NIST), ASTM International, BiofabUSA, and the Standards Coordinating Body (SCB) organized a workshop in 2018 titled, "Characterization of Fiber-Based Scaffolds". The goal was to convene a group of 40 key industry stakeholders to identify major roadblocks in measurements of fiber-based scaffold properties. This report provides an overview of the findings from this collaborative workshop. The four major consensus findings were that (a) there is need for a documentary standard guide that would aid developers in the selection of test methods for characterizing fiber-based scaffolds; (b) there is a need for a strategy to assess the quality of porosity and pore size measurements, which could potentially be ameliorated by the development of a reference material; (b) there are challenges with the lexicon used to describe and assess scaffolds; and (d) the vast array of product applications makes it challenging to identify consensus test methods. As a result of these findings, a working group was formed to develop an ASTM Standard Guide for Characterizing Fiber-Based Constructs that will provide developers guidance on selecting measurements for characterizing fiber-based scaffolds.

Deep learning predicts function of live retinal pigment epithelium from quantitative microscopy.

Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and noninvasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to noninvasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell-derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine-learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate noninvasive cell therapy characterization can be achieved with QBAM and machine learning.

Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation.

The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (beta-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins generally lack bioactive peptides for enhancing cell functions. Thus, a synthetic spider silk, spidroin, containing two RGD cell adhesive sequences (RGD-spidroin) was engineered. RGD-spidroin was blended with different ratios of fibroin and spun coat into films on glass coverslips. beta-Sheet formation, contact angle, surface topography and RGD surface presentation were characterized and correlated with cell behavior. We found that the amount of beta-sheet formation was directly related to the RGD-spidroin content of the blends after annealing, with the pure RGD-spidroin demonstrating the highest amount of beta-sheet content. The increased beta-sheet content improved film stability under culture conditions. A new visualization technique demonstrated that the RGD presentation on the film surface was affected by both the RGD-spidroin content and annealing conditions. It was determined that 10mass% RGD-spidroin was necessary to improve film stability and to achieve osteoblast attachment and differentiation.

X-ray imaging optimization of 3D tissue engineering scaffolds via combinatorial fabrication methods.

We have developed a combinatorial method for determining optimum tissue scaffold composition for several X-ray imaging techniques. X-ray radiography and X-ray microcomputed tomography enable non-invasive imaging of implants in vivo and in vitro. However, highly porous polymeric scaffolds do not always possess sufficient X-ray contrast and are therefore difficult to image with X-ray-based techniques. Incorporation of high radiocontrast atoms, such as iodine, into the polymer structure improves X-ray radiopacity but also affects physicochemical properties and material performance. Thus, we have developed a combinatorial library approach to efficiently determine the minimum amount of contrast agent necessary for X-ray-based imaging. The combinatorial approach is demonstrated in a polymer blend scaffold system where X-ray imaging of poly(desaminotyrosyl-tyrosine ethyl ester carbonate) (pDTEc) scaffolds is improved through a controlled composition variation with an iodinated-pDTEc analog (pI(2)DTEc). The results show that pDTEc scaffolds must include at least 9%, 16%, 38% or 46% pI(2)DTEc (by mass) to enable effective imaging by microradiography, dental radiography, dental radiography through 0.75cm of muscle tissue or microcomputed tomography, respectively. Only two scaffold libraries were required to determine these minimum pI(2)DTEc percentages required for X-ray imaging, which demonstrates the efficiency of this new combinatorial approach for optimizing scaffold formulations.

Injectable and strong nano-apatite scaffolds for cell/growth factor delivery and bone regeneration.

OBJECTIVES: Seven million people suffer bone fractures each year in the U.S., and musculoskeletal conditions cost $215 billion/year. The objectives of this study were to develop moldable/injectable, mechanically strong and in situ-hardening calcium phosphate cement (CPC) composite scaffolds for bone regeneration and delivery of osteogenic cells and growth factors. METHODS: Tetracalcium phosphate [TTCP: Ca(4)(PO(4))(2)O] and dicalcium phosphate (DCPA: CaHPO(4)) were used to fabricate self-setting calcium phosphate cement. Strong and macroporous scaffolds were developed via absorbable fibers, biopolymer chitosan, and mannitol porogen. Following established protocols, MC3T3-E1 osteoblast-like cells (Riken, Hirosaka, Japan) were cultured on the specimens and inside the CPC composite paste carrier. RESULTS: The scaffold strength was more than doubled via reinforcement (p<0.05). Relationships and predictive models were established between matrix properties, fibers, porosity, and overall composite properties. The cement injectability was increased from about 60% to nearly 100%. Cell attachment and proliferation on the new composite matched those of biocompatible controls. Cells were able to infiltrate into the macropores and anchor to the bone mineral-like nano-apatite crystals. For cell delivery, alginate hydrogel beads protected cells during cement mixing and setting, yielding cell viability measured via the Wst-1 assay that matched the control without CPC (p>0.1). For growth factor delivery, CPC powder:liquid ratio and chitosan content provided the means to tailor the rate of protein release from CPC carrier. SIGNIFICANCE: New CPC scaffolds were developed that were strong, tough, macroporous and osteoconductive. They showed promise for injection in minimally invasive surgeries, and in delivering osteogenic cells and osteoinductive growth factors to promote bone regeneration. Potential applications include various dental, craniofacial, and orthopedic reconstructions.

Combinatorial cassettes to systematically evaluate tissue-engineered constructs in recipient mice.

Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (µCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs.

Effect of the scaffold microenvironment on cell polarizability and capacitance determined by probabilistic computations.

In living systems, it is frequently stated that form follows function by virtue of evolutionary pressures on organism development, but in the study of how functions emerge at the cellular level, function often follows form. We study this chicken versus egg problem of emergent structure-property relationships in living systems in the context of primary human bone marrow stromal cells cultured in a variety of microenvironments that have been shown to cause distinct patterns of cell function and differentiation. Through analysis of a publicly available catalog of three-dimensional (3D) cell shape data, we introduce a family of metrics to characterize the 'form' of the cell populations that emerge from a variety of diverse microenvironments. In particular, measures of form are considered that are expected to have direct significance for cell function, signaling and metabolic activity: dimensionality, polarizability and capacitance. Dimensionality was assessed by an intrinsic measure of cell shape obtained from the polarizability tensor. This tensor defines ellipsoids for arbitrary cell shapes and the thinnest dimension of these ellipsoids, P 1, defines a reference minimal scale for cells cultured in a 3D microenvironment. Polarizability governs the electric field generated by a cell, and determines the cell's ability to detect electric fields. Capacitance controls the shape dependence of the rate at which diffusing molecules contact the surface of the cell, and this has great significance for inter-cellular signaling. These results invite new approaches for designing scaffolds which explicitly direct cell dimensionality, polarizability and capacitance to guide the emergence of new cell functions derived from the acquired form.

Premixed calcium phosphate cements: synthesis, physical properties, and cell cytotoxicity.

OBJECTIVES: Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder-liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. METHODS: PCPCs were formulated from CPC powder+non-aqueous liquid+gelling agent+hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC-Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. RESULTS: Setting time (mean+/-S.D.; n=4) for PCPC-Tartaric was 8.2+/-0.8 min, significantly less than the 61.7+/-1.5 min for the Premixed Control developed previously (p<0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5+/-0.8 MPa, higher than 4.5+/-0.8 MPa of Premixed Control (p=0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p=0.126), indicating that the new PCPCs were as cell compatible as a non-premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p>0.1) from that of the non-premixed CPC control. SIGNIFICANCE: PCPCs will eliminate the powder-liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs.

Fabrication of combinatorial polymer scaffold libraries.

We have designed a novel combinatorial research platform to help accelerate tissue engineering research. Combinatorial methods combine many samples into a single specimen to enable accelerated experimentation and discovery. The platform for fabricating combinatorial polymer scaffold libraries can be used to rapidly identify scaffold formulations that maximize tissue formation. Many approaches for screening cell-biomaterial interactions utilize a two-dimensional format such as a film or surface to present test substrates to cells. However, cells in vivo exist in a three-dimensional milieu of extracellular matrix and cells in vitro behave more naturally when cultured in a three-dimensional environment than when cultured on a two-dimensional surface. Thus, we have designed a method for fabricating combinatorial biomaterial libraries where the materials are presented to cells in the form of three-dimensional, porous, salt-leached, polymer scaffolds. Many scaffold variations and compositions can be screened in a single experiment so that optimal scaffold formulations for tissue formation can be rapidly identified. In summary, we have developed a platform technology for fabricating combinatorial polymer scaffold libraries that can be used to screen cell response to materials in a three-dimensional, scaffold format.

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells.

Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl Biomater, 2016. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 989-1001, 2017.

A switchable positive and negative air pressure device for efficient and gentle handling of nanofiber scaffolds.

A scaffold handling device (SHD) has been designed that can switch from gentle suction to positive pressure to lift and place nanofiber scaffolds. In tissue engineering laboratories, delicate fibrous scaffolds, such as electrospun nanofiber scaffolds, are often used as substrates for cell culture. Typical scaffold handling procedures include lifting the scaffolds, moving them from one container to another, sterilization, and loading scaffolds into cell culture plates. Using tweezers to handle the scaffolds can be slow, can damage the scaffolds, and can cause them to wrinkle or fold. Scaffolds may also acquire a static charge which makes them difficult to put down as they cling to tweezers. An SHD has been designed that enables more efficient, gentle lifting, and placement of delicate scaffolds. Most of the parts to make the SHD can be purchased, except for the tip which can be 3D-printed. The SHD enables more reliable handling of nanofiber scaffolds that may improve the consistency of biomanufacturing processes.