An open hardware 3-D printed device for measuring tensile properties of thermoplastic filament polymers at cryogenic temperatures.

With the emerging recognition of open scientific hardware, rapid prototyping technology such as three-dimensional (3-D) printing is becoming widely available for fields such as cryobiology, and cryopreservation, where material selection for instruments and hardware has traditionally been problematic due to extreme low temperatures. A better understanding of the mechanical properties of 3-D printing thermoplastics at cryogenic temperatures is essential to material selection, part design, and printing optimization. The goal of the present study was to explore the feasibility of development for a 3-D printed device ('CryoTensileDevice') to hold a test specimen in liquid nitrogen and be mounted in standard mechanical testing systems to evaluate 3-D printing material behaviors at cryogenic temperatures. The CryoTensileDevice was prototyped with flexible filaments with a per-unit material cost of < US$5 and a printing time of < 5 h. The commonly used printing filament polylactic acid (PLA) was selected to evaluate the utility of the CryoTensileDevice. At room temperature, the CryoTensileDevice did not significantly (P > 0.05) affect PLA tensile measurements such as Young's modulus, yield stress, yield strain, stress at break, or strain at break. With the CryoTensileDevice, specimens 3-D printed with PLA at 50%, 75%, and 100% infill rates had comparable tensile properties when tested at room and liquid nitrogen temperatures. The PLA showed superior performance in tensile properties in comparison to acrylonitrile butadiene styrene (ABS). This device can assist characterization of 3-D printing approaches for cryogenic work, and opens a pathway for future innovations to create a variety of 3-D printed devices to study a wide range of material properties for cryogenic applications.

3-D Printed Customizable Vitrification Devices for Preservation of Genetic Resources of Aquatic Species.

Sperm vitrification as an alternative approach to conventional cryopreservation (equilibrium freezing) allows quick and low-cost sample preservation and is suitable for small-bodied aquatic species with miniscule testis, fieldwork at remote locations, and small-scale freezing for research purposes. The goal of this present study was to develop operational prototypes of 3-dimensional (3-D) printed vitrification devices with innovative components that can provide comprehensive functionalities for practical repository development for aquatic species. The design featured an elongated loop to suspend a thin film of sperm sample in cryoprotectant, a retractable sleeve to protect the vitrified samples and allow permanent labeling, a handle to facilitate processing and storage, and a shaft with annular grooves to guide positioning of the protective retractable sleeve. To span a wide range of sample capacities and configurations, a total of 39 different configurations (3 loop lengths ×13 loop heights) were fabricated by 3-D printing with the thermoplastics polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS). A total of 86 devices were fabricated with ABS filament with a print failure rate of 9%, and 97 devices were fabricated with PLA filament with a failure rate of 20%. Major types of printing failures included disconnected loops, insufficient build surface adhesion, stringing, and inconsistent extrusion. The sample volume capacity ranged from 1-47 µL and had linear relationships to the loop lengths and layer numbers. Vitrified samples were observed in 10-mm and 15-mm loops fabricated with PLA and ABS but not in 20-mm loops. This study demonstrated the feasibility of development of standardized low-cost ($0.05 material cost) devices fabricated by 3-D printing with practical functions including vitrification, volume control, labeling, protection, and storage within conventional systems. These prototypes can be further developed, standardized, and used to assist development of germplasm repositories to protect the genetic resources of aquatic species by user groups such as breeders, hatcheries, aquariums, and researchers.

Three-dimensional printing with polylactic acid (PLA) thermoplastic offers new opportunities for cryobiology.

Development of devices through design, prototyping, testing, and fabrication is especially necessary for enhancement of research and eventual application in cryobiology. The advent of 3-dimensional printing offers unique opportunities for this process, given that the materials involved are suitable for use in cryogenic temperatures. We report herein that 3-D printing with polylactic acid (PLA) thermoplastic is ideally suited for cryobiology device development. Devices that are designed and standardized in open-source fashion can be electronically distributed and created locally on increasingly affordable 3-D printers, and can accelerate cryobiology findings and improve reproducibility of results.

Initial assessment of the toxicologic effects of leachates from 3-dimensional (3-D) printed objects on sperm quality in two model fish species.

The use of 3-dimensional (3-D) printing is gaining popularity in life sciences and driving innovation in fields including aquatic sperm cryopreservation. Yet, little is known about the effects leachates from these objects may have on biological systems. In this study, we investigated if exposure to leachates from 3-D printed objects fabricated from different photo-curable resins could affect sperm quality in two model fish species, zebrafish (Danio rerio) and goldfish (Carassius auratus). Leachates were collected following contact periods of 10 min and 22 h with objects manufactured using a mask LCD resin printer and three different commercially available resins (i.e., standard, eco-friendly, and impact-resistant). Sperm cells were exposed to the leachates for 18 min, and parameters related to sperm motility, cell count, and membrane integrity were evaluated. All experiments were blinded. Leachate originating from contact with impact-resistant resin for 10 min significantly reduced the cell count of zebrafish sperm, while leachate originating from contact with standard resin for 22 h significantly increased the beat cross frequency of goldfish sperm. The changes were not observed across species and no adverse effects were recorded in percent motility, velocity, amplitude of lateral head movement, or membrane integrity of sperm. Our findings demonstrate that exposure to leachates from certain 3-D printed resins can affect sperm quality, while other resins may support sperm quality evaluation. Further investigations are warranted to assess other parameters, effects, and their biological relevance for a variety of aquatic species.

Biomechanical and Biological Characterization of XGel, a Human-Derived Hydrogel for Stem Cell Expansion and Tissue Engineering.

Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delivery of cells and drugs. Existing hydrogel classifications include synthetic, recombinant, chemically defined, plant- or animal-based, and tissue-derived matrices. There is a need for materials that can support both human tissue modeling and clinically relevant applications requiring stiffness tunability. Human-derived hydrogels are not only clinically relevant, but they also minimize the use of animal models for pre-clinical studies. This study aims to characterize XGel, a new human-derived hydrogel as an alternative to current murine-derived and synthetic recombinant hydrogels that features unique physiochemical, biochemical, and biological properties that support adipocyte and bone differentiation. Rheology studies determine the viscosity, stiffness, and gelation features of XGel. Quantitative studies for quality control support consistency in the protein content between lots. Proteomics studies reveal that XGel is predominantly composed of extracellular matrix proteins, including fibrillin, collagens I-VI, and fibronectin. Electron microscopy of the hydrogel provides phenotypic characteristics in terms of porosity and fiber size. The hydrogel demonstrates biocompatibility as a coating material and as a 3D scaffold for the growth of multiple cell types. The results provide insight into the biological compatibility of this human-derived hydrogel for tissue engineering.

Nanophotothermolysis of multiple scattered cancer cells with carbon nanotubes guided by time-resolved infrared thermal imaging.

Nanophotothermolysis with long laser pulses for treatment of scattered cancer cells and their clusters is introduced with the main focus on real-time monitoring of temperature dynamics inside and around individual cancer cells labeled with carbon nanotubes. This technique utilizes advanced time- and spatially-resolved thermal radiometry imaging for the visualization of laser-induced temperature distribution in multiple-point absorbing targets. The capability of this approach was demonstrated for monitoring of thermal effects under long laser exposure (from millisecond to seconds, wavelength 1,064 nm, maximum power 1 W) of cervical cancer HeLa cells labeled with carbon nanotubes in vitro. The applications are discussed with a focus on the nanophotothermolysis of small tumors, tumor margins, or micrometastases under the guidance of near-IR and microwave radiometry.

miR-148b-nanoparticle conjugates for light mediated osteogenesis of human adipose stromal/stem cells.

Delivery systems providing spatial and temporal control have the potential to improve outcomes in surgical reconstruction and regenerative medicine by precise modulation of wound healing and tissue repair processes. In this study we describe a synthesis and oligonucleotide functionalization process of silver nanoparticle complexes for photoactivated microRNA (miRNA) delivery. The activity of the PC-miR-148b-SNP construct is demonstrated by light mediated delivery of miR-148b mimic resulting in differentiation of human autologous adipose derived mesenchymal stromal/stem cells (hASCs) into an osteogenic linage. The conjugate, upon photoactivation, increases alkaline phosphatase (ALP) activity in the cell membrane and calcification (mineralization) of hASCs on days 7 and 14 respectively. Additionally, the expression of mRNA for the early, middle and late stage osteogenic markers; ALP, RunX2 and osteocalcin (OCN) respectively, was also significantly upregulated at days 7 and 28, respectively after photoactivation of PC-miR-148b-SNP and release of miR-148b mimics. Additionally, PC-miR-148b-SNP conjugate is readily delivered to the intracellular compartment without the use of transfection vectors commonly required for free oligonucleotides. This technology demonstrates photo-controlled, spatial and temporal modulation of osteogenesis in hASCs.