Prolongation of Life in Anephric Rats following de novo Renal Organogenesis

One solution to the shortage of human organs available for transplantation envisions growing new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. Allotransplantation of embryonic day 15 metanephroi into the omentum of adult hosts is followed by differentiation, growth, vascularization and function of the implants. Here we show that survival of rats with all native renal mass removed can be increased by prior metanephros transplantation and ureteroureterostomy. Excretion of urine formed by metanephroi is prerequisite for enhanced survival. This is the first demonstration that life can be extended following de novo renal organogenesis.     

A computerized mechanical cell stimulator for tissue culture: Effects on skeletal muscle organogenesis

Summary A tissue culture system has been developed which can mechanically stimulate cells growing on a highly elastic plastic substratum in a 24-well cell growth chamber. The collagen-coated substratum to which the cells attach and grow in the Mechanical Cell Stimulator (Model I) can be repetitively stretched and relaxed by stepper motor with linear accuracy of 30 μm. The activity controlling unit is an Apple IIe computer interfaced with the cell growth chamber via optical data links and is capable of simulating many of the mechanical activity patterns that cells are subjected to in vivo. Primary avian skeletal myoblasts proliferate and fuse into multinucleated myotubes in this set-up in a manner similar to normal tissue culture dishes. Under static culture conditions, the muscle cells differentiate into networks of myotubes which show little orientation. Growing the proliferating muscle cells on a unidirectional stretching substratum causes the developing myotubes to orient parallel to the direction of movement. In contrast, growing the cells on a substratum undergoing continuous stretch-relaxation cycling orients the developing myotubes perpendicular to the direction of movement. Neither type of mechanical activity significantly affects the rate of cell proliferation of the rate of myoblast fusion into myotubes. These results indicate that during in vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous bone elongation and by spontaneous contractions, only bone elongation plays a significant role in proper fiber orientation for subsequent functional work. Supported by grants NS16753, AR36266, and RR05818 from the National Institutes of Health, Bethesda, MD.     

An inexpensive, 3D‐printable breast muscle meter for field ornithologists

The size of the pectoral muscle is an important component of body condition in birds and has been linked to indices of fitness and migratory performance. Bauchinger et al. (2011. Journal of Ornithology 152: 507–514) developed, calibrated, and validated an aluminum “muscle meter” device that estimates the size of pectoral muscles noninvasively. To make this tool more widely available, we created a CAD model from 3D‐scan data of the aluminum muscle meter that can be 3D‐printed in durable plastic for ~ $30 USD. We tested this device on seven species of songbirds in Jamaica, The Bahamas, Cameroon, Equatorial Guinea, and Michigan. We demonstrate that the breast muscle meter measurements are (1) repeatable among users, (2) correlated with a four‐category visual breast muscle scoring system, and (3) correlated with scaled mass index (an index of body condition). Muscle scores from our device outperformed the traditional four‐category muscle scoring system in predicting scaled mass index. Finally, with our device, we quantified the increasing breast muscle size of American Redstarts (Setophaga ruticilla) from March through May as they prepared for spring migration. Given the precision of the 3D‐scanning hardware used to generate our 3D image for printing, we produced a plastic muscle meter that is as precise and useful as the aluminum original, but more cost‐effective and widely available.     

Three-dimensional printing biotechnology for the regeneration of the tooth and tooth-supporting tissues

The tooth and its supporting tissues are organized with complex three-dimensional (3D) architecture, including the dental pulp with a blood supply and nerve tissues, complex multilayer periodontium, and highly aligned periodontal ligament (PDL). Mimicking such 3D complexity and the multicellular interactions naturally existing in dental structures represents great challenges in dental regeneration. Attempts to construct the complex system of the tooth and tooth-supporting apparatus (i.e., the PDL, alveolar bone, and cementum) have made certain progress owing to 3D printing biotechnology. Recent advances have enabled the 3D printing of biocompatible materials, seed cells, and supporting components into complex 3D functional living tissue. Furthermore, 3D bioprinting is driving major innovations in regenerative medicine, giving the field of regenerative dentistry a boost. The fabrication of scaffolds via 3D printing is already being performed extensively at the laboratory bench and in clinical trials; however, printing living cells and matrix materials together to produce tissue constructs by 3D bioprinting remains limited to the regeneration of dental pulp and the tooth germ. This review summarizes the application of scaffolds for cell seeding and biofabricated tissues via 3D printing and bioprinting, respectively, in the tooth and its supporting tissues. Additionally, the key advantages and prospects of 3D bioprinting in regenerative dentistry are highlighted, providing new ideas for dental regeneration.     

Three-dimensional printing can provide customizable probes for sensing and monitoring in cryobiology applications

Cryopreservation has been recognized as a powerful tool for long-term preservation of genetic resources. However, the outcomes of cryopreservation by different user groups often vary due to inconsistency in procedures and freezing equipment. Herein, we report on the feasibility of providing customizable sensing probes with three-dimensional (3-D) printing to monitor cryopreservation phenomena. The objectives were to: 1) introduce 3-D printing as a fabrication method for developing customizable probes to be used in cryogenic applications; 2) design and fabricate an example of a 3-D printed sensing probe and multiplexer capable of detecting phase-change phenomena based on quantitative data regarding sample electrical resistance and temperature, and 3) demonstrate the sensing platform in cryopreservation conditions and in combination with a custom-made 3-D printed freezing device. The sensing probe developed was designed to fit within standard 0.5-ml French straws. Phase-transition phenomena were detected by analyzing electrical resistance changes. The quantitative data from this device in conjugation with a 3-D printed freezer rack provided cryopreservation capability with high reproducibility and offered an alternative to expensive programmable freezers. The use of 3-D printing provided flexibility to develop new sensing probes or modify existing designs based on specific needs. After initial prototyping, fabrication, and testing of 3-D printed sensing probes, particularly useful designs can lead to the reduction of variation in performing standardized cryopreservation protocols.     

3D printing in biotechnology—An insight into miniaturized and microfluidic systems for applications from cell culture to bioanalytics

Since its invention in the 1980s, 3D printing has evolved into a versatile technique for the additive manufacturing of diverse objects and tools, using various materials. The relative flexibility, straightforwardness, and ability to enable rapid prototyping are tremendous advantages offered by this technique compared to conventional methods for miniaturized and microfluidic systems fabrication (such as soft lithography). The development of 3D printers exhibiting high printer resolution has enabled the fabrication of accurate miniaturized and microfluidic systems—which have, in turn, substantially reduced both device sizes and required sample volumes. Moreover, the continuing development of translucent, heat resistant, and biocompatible materials will make 3D printing more and more useful for applications in biotechnology in the coming years. Today, a wide variety of 3D‐printed objects in biotechnology—ranging from miniaturized cultivation chambers to microfluidic lab‐on‐a‐chip devices for diagnostics—are already being deployed in labs across the world. This review explains the 3D printing technologies that are currently used to fabricate such miniaturized microfluidic devices, and also seeks to offer some insight into recent developments demonstrating the use of these tools for biotechnological applications such as cell culture, separation techniques, and biosensors.     

3-D printing provides a novel approach for standardization and reproducibility of freezing devices

Cryopreservation has become an important and accepted tool for long-term germplasm conservation of animals and plants. To protect genetic resources, repositories have been developed with national and international cooperation. For a repository to be effective, the genetic material submitted must be of good quality and comparable to other submissions. However, due to a variety of reasons, including constraints in knowledge and available resources, cryopreservation methods for aquatic species vary widely across user groups which reduces reproducibility and weakens quality control. Herein we describe a standardizable freezing device produced using 3-dimensional (3-D) printing and introduce the concept of network sharing to achieve aggregate high-throughput cryopreservation for aquatic species. The objectives were to: 1) adapt widely available polystyrene foam products that would be inexpensive, portable, and provide adequate work space; 2) develop a design suitable for 3-D printing that could provide multiple configurations, be inexpensive, and easy to use, and 3) evaluate various configurations to attain freezing rates suitable for various common cryopreservation containers. Through this approach, identical components can be accessed globally, and we demonstrated that 3-D printers can be used to fabricate parts for standardizable freezing devices yielding relevant and reproducible cooling rates across users. With standardized devices for freezing, methods and samples can harmonize into an aggregated high-throughput pathway not currently available for aquatic species repository development.     

In vitro culture of Safflower L. cv. Bhima: initiation,growth optimization and organogenesis

Callus induction and in vitro plantlet regeneration systems for safflower (Carthamus tinctorius L.) cv. Bhima using root, hypocotyl, cotyledon and leaf explants were optimized by studying the influence on organogenesis of seedling age, media factors, growth regulators and excision orientation. Supplementation of the medium with an auxin: cytokinin ratio < 1 enhanced the growth rate of callus cultures; however, for 2,4-D the ratio was > 1.34–11.41 μM concentrations of growth regulators (IAA, NAA, BA and Kinetin) in the medium were found effective for callus induction and regeneration in all explants. The calli could be maintained over 32 months. BA (4.43 μM) combined with casein hydrolysate (10 mg l-1) yielded the highest rate of shoot production on hypocotyl (3–6) and cotyledon (5–7) explants and cotyledonary derived callus (4–8). More shoots were produced on explants cut from the most basal region of cotyledons from 5 to 7-day-old seedlings than from older seedlings or more distal cut sites. Apolar placement of explants, inhibited shoot regeneration. The shoot regeneration potential remained upto 7 months in calli developed on NAA + BA. Of three media tested, MS was superior to SH-M and B5. Rooting of shoots was not efficient; 42% of the shoots were rooted on MS medium containing sucrose (7–8%) + IAA (2.8–5.7 μM). Capitula induction was observed in both callus mediated shoots on cotyledons and shoots on rooting medium with sucrose, IAA, NAA and IBA. Well developed plantlets were transferred to the field with a 34% success rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fabrication of thin-layer matrigel-based constructs for three-dimensional cell culture

Extracellular matrix-based hydrogels such as Matrigel are easy-to-use, commercially available, and offer environments for three-dimensional (3-D) cell culture that mimic native tissue. However, manipulating small volumes of these materials to produce thin-layer 3-D culture systems suitable for analysis is difficult because of air–liquid-substrate interfacial tension effects and evaporation. Here, we demonstrate two simple techniques that use standard liquid-handling tools and nontreated 96-well plates to produce uniform, thin-layer constructs for 3-D culture of cells in Matrigel. The first technique, the floating 3-D cell culture method, uses phase-separating polymers to form a barrier between the dispensed Matrigel, air, and cultureware surface to generate consistently thin hydrogels from volumes as low as 5 μL. These unanchored gels provide a useful assay for investigating airway smooth muscle cell contraction and may have future applications in studying asthma pathophysiology. The second technique, the fixed 3-D cell culture method, provides an anchored gel system for culturing noncontractile cells (e.g., neurons) where 20 μL of Matrigel is dispensed into the bottom of a well filled with culture medium to form a thin gel containing embedded cells. This technique has potential widespread applications as an accessible 3-D culture platform for high-throughput production of disease models for evaluation of novel drug therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2733, 2019     

Transplantation of engineered bone tissue using a rotary three-dimensional culture system

Bone is a complex, highly structured, mechanically active, three-dimensional (3-D) tissue composed of cellular and matrix elements. We previously published a report on in situ collagen gelation using a rotary 3-D culture system (CG–RC system) for the construction of large tissue specimens. The objective of the current study was to evaluate the feasibility of bone tissue engineering using our CG–RC system. Osteoblasts from the calvaria of newborn Wistar rats were cultured in the CG–RC system for up to 3 wk. The engineered 3-D tissues were implanted into the backs of nude mice and calvarial round bone defects in Wistar rats. Cell metabolic activity, mineralization, and bone-related proteins were measured in vitro in the engineered 3-D tissues. Also, the in vivo histological features of the transplanted, engineered 3-D tissues were evaluated in the animal models. We found that metabolic activity increased in the engineered 3-D tissues during cultivation, and that sufficient mineralization occurred during the 3 wk in the CG–RC system in vitro. One mo posttransplantation, the transplants to nude mice remained mineralized and were well invaded by host vasculature. Of particular interest, 2 mo posttransplantation, the transplants into the calvarial bone defects of rats were replaced by new mature bone. Thus, this study shows that large 3-D osseous tissue could be produced in vitro and that the engineered 3-D tissue had in vivo osteoinductive potential when transplanted into ectopic locations and into bone defects. Therefore, this system should be a useful model for bone tissue engineering.     

Advances in 3D printing of composite scaffolds for the repairment of bone tissue associated defects

The conventional methods of using autografts and allografts for repairing defects in bone, the osteochondral bone, and the cartilage tissue have many disadvantages, like donor site morbidity and shortage of donors. Moreover, only 30% of the implanted grafts are shown to be successful in treating the defects. Hence, exploring alternative techniques such as tissue engineering to treat bone tissue associated defects is promising as it eliminates the above-mentioned limitations. To enhance the mechanical and biological properties of the tissue engineered product, it is essential to fabricate the scaffold used in tissue engineering by the combination of various biomaterials. Three-dimensional (3D) printing, with its ability to print composite materials and with complex geometry seems to have a huge potential in scaffold fabrication technique for engineering bone associated tissues. This review summarizes the recent applications and future perspectives of 3D printing technologies in the fabrication of composite scaffolds used in bone, osteochondral, and cartilage tissue engineering. Key developments in the field of 3D printing technologies involves the incorporation of various biomaterials and cells in printing composite scaffolds mimicking physiologically relevant complex geometry and gradient porosity. Much recently, the emerging trend of printing smart scaffolds which can respond to external stimulus such as temperature, pH and magnetic field, known as 4D printing is gaining immense popularity and can be considered as the future of 3D printing applications in the field of tissue engineering.     

Film tomography as a tool for three-dimensional image construction and gene expression studies

In order to observe three-dimensional (3D) expression patterns of genes in whole animals, whole organs, or whole tissues, in situ hybridization (ISH) of many sections must be carried out and then used to construct a 3D image. For this purpose, we have developed an automatic microtome to prepare tissue sections with an adhesive film. We used commercially available film suitable for sectioning and ISH. We constructed a microtome and, after adherence of the film to a paraffin-embedded tissue block, cut the block with a blade to prepare sections on film. Then, the sections-on-film were automatically set in a plastic frame that was the same size as a conventional glass slide. With this automatic microtome, tissue sections can be made for ISH or immunohistochemistry in addition to conventional hematoxylin and eosin staining without specific training. We demonstrate that we can construct 3D images of gene expression patterns obtained by ISH on sections prepared with this automatic microtome. We have designated this method as 'Film Tomography (FITO)'.     

Methods for rapid prototyping novel labware: using CAD and desktop 3D printing in the microbiology laboratory

Although the microbiology laboratory paradigm has increasingly changed from manual to automated procedures, and from functional to molecular methods, traditional culture methods remain vital. Using inexpensive desktop fused filament fabrication 3D printing, we designed, produced and tested rapid prototypes of customised labware for microbial culture namely frames to make dip slides, inoculation loops, multi-pin replicators, and multi-well culture plates for solid medium. These customised components were used to plate out samples onto solid media in various formats, and we illustrate how they can be suitable for many microbiological methods such as minimum inhibitory concentration tests, or for directly detecting pathogens from mastitis samples, illustrating the flexibility of rapid-prototyped culture consumable parts for streamlining microbiological methods. We describe the methodology needed for microbiologists to develop their own novel and unique tools, or to fabricate and customise existing consumables. A workflow is presented for designing and 3D printing labware and quickly producing easy-to-sterilise and re-useable plastic parts of great utility in the microbiology laboratory.     

Rapid generation of three‐dimensional microchannels for vascularization using a subtractive printing technique

The development of tissue‐engineered products has been limited by lack of a perfused microvasculature that delivers nutrients and maintains cell viability. Current strategies to promote vascularization such as additive three‐dimensional printing techniques have limitations. This study validates the use of an ultra‐fast laser subtractive printing technique to generate capillary‐sized channels in hydrogels prepopulated with cells by demonstrating cell viability relative to the photodisrupted channels in the gel. The system can move the focal spot laterally in the gel at a rate of 2500 mm/s by using a galvanometric scanner to raster the in plane focal spot. A Galilean telescope allows z‐axis movement. Blended hydrogels of polyethylene glycol and collagen with a range of optical clarities, mechanical properties and swelling behavior were tested to demonstrate that the subtractive printing process for writing vascular channels is compatible with all of the blended hydrogels tested. Channel width and patterns were controlled by adjusting the laser energy and focal spot positioning, respectively. After treatment, high cell viability was observed at distances greater than or equal to 18 μm from the fabricated channels. Overall, this study demonstrates a flexible technique that has the potential to rapidly generate channels in tissue‐engineered constructs.      

Establishment of a three-dimensional culture and mechanical loading system for skeletal myoblasts

Establishment of a three-dimensional (3-D) culture and mechanical loading system which simulates the in vivo environment is critical in cytomechanical studies. The present article attempts to do this by integrating porous PLGA scaffolds with a four-point bending strain unit. Three types of PLGA scaffolds with three average pore sizes were synthesized, i.e., type I (60-88 μm), type II (88-100 μm) and type III (100-125 μm). To establish the 3-D mechanical loading system, PLGA membrane was integrated with conventional force-loading plates and the third passage skeletal myoblasts from neonatal Sprague-Dawley (SD) rats were seeded. Small PLGA membranes were put in 24-well plates followed by cell implantation and MTT assay was performed on days 1, 2, 4, 6 and 8 to compare biocompatibility of the three types of scaffolds. After 3 days’ culture, many more cells had grown in type II than in type I or type III under fluorescence microscopy. In the MTT assay, OD of type II was significantly higher (P < 0.05) than the other two, especially at the early stage. As type II proved to be the best among the three, it was used as the scaffold in the preliminary mechanical loading study and 4000 μstrain cyclic uniaxial strain was imposed. The system worked well and it was found that short to median time of stretching enhances while prolonged time of stretching inhibits cell proliferative activity of the 3-D cultured skeletal myoblasts(P < 0.05). It is concluded that the combination of PLGA scaffolds with a four-point bending strain unit provides a satisfactory 3-D mechanical loading system.     

Somatic embryogenesis and organogenesis of Agave victoriae-reginae: Considerations for its conservation

Agave victoriae-reginae somatic embryos were produced through a callus phase from seedling stem segments cultured on MS medium. The optimal treatment was MS medium with 2.26 M 2,4-D. Multiple shoot regeneration was induced from axillary buds from stem segments cultured on MS medium with 2.2–4.4 M BA. Effect of MS and modified MS medium with 50% macronutrient concentration, both containing 2.2 M BA and sucrose at the following concentrations, 20, 30, 45 and 60 g l^–1, resulted in inconsistent multiple shoot formation. Shoots and somatic embryos formed by this indirect pathway could have a multicellular origin, which might lead to genetic variation. The direct development of pre-existent buds occurred on MS basal medium and increased in the presence of BA; this might be a pathway for the rescue of genotypes of endangered species. Embryos and shoots developed and grew roots on MS medium. Complete plantlets were obtained on MS basal medium. A total of 92% per cent of the plantlets survived and grew when transferred to the greenhouse. Agave micropropagation could supply the commercial plant demand, diminishing the gathering of seeds and plants of this endangered species from the wild.     

Somatic embryogenesis and organogenesis in peanut: The role of thidiazuron and N6-benzylaminopurine in the induction of plant morphogenesis

Intact peanut (Arachis hypogaea L.) seeds, incubated on media containing N⁶-benzylaminopurine (BAP) or thidiazuron (TDZ) exhibited de novo regeneration at the hypocotyledonary notch region. Regeneration was observed when seeds were cultured on either TDZ or BAP but the optimal level of media supplementation was 10 mol·L^–1 for TDZ and 50 mol–L^–1 for BAP. Light microscopic observations revealed that the regenerants induced by TDZ were somatic embryos while those induced by BAP were shoots. An alternative approach of exposing the seeds to TDZ was through vacuum infiltration followed by culture on basal media but BAP did not induce regeneration by this method. Although TDZ has often been classified as a synthetic cytokinin, our results clearly demonstrate that seedlings treated with TDZ undergo a different morphological route of development than that induced by purine cytokinins.     

Whole embryo culture and the study of postimplantation mammalian development

In the past decade, striking advances have been made in the field of gene introducing/disrupting technology including generation of transgenic and knockout mice, which have enabled us to elucidate roles of specific genes in development. In this technology, embryos introduced with exogenous genes or chimeric embryos aggregated/injected with embryonic stem (ES) cells carrying targeted genes are allowed to develop in the uterus of foster mothers. The uterus, however, is like a black box for researchers investigating postimplantation development of mammalian embryos. Embryo culture is one of the powerful techniques that can open this black box. In this review, we focus on the applicable aspects of the whole embryo culture in the study of mammalian development and discuss the future possibilities of this technique.     

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.