A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis.

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.

Human Brain Organoids as an In Vitro Model System of Viral Infectious Diseases.

Brain organoids, or brainoids, have shown great promise in the study of central nervous system (CNS) infection. Modeling Zika virus (ZIKV) infection in brain organoids may help elucidate the relationship between ZIKV infection and microcephaly. Brain organoids have been used to study the pathogenesis of SARS-CoV-2, human immunodeficiency virus (HIV), HSV-1, and other viral infections of the CNS. In this review, we summarize the advances in the development of viral infection models in brain organoids and their potential application for exploring mechanisms of viral infections of the CNS and in new drug development. The existing limitations are further discussed and the prospects for the development and application of brain organs are prospected.

Ultrasound-based cell sorting with microbubbles: A feasibility study.

The isolation and sorting of cells is an important process in research and hospital labs. Most large research and commercial labs incorporate fluorescently or magnetically labeled antibodies adherent to cell surface antigens for cell identification and separation. In this paper, a process is described that merges biochemical labeling with ultrasound-based separation. Instead of lasers and fluorophore tags, or magnets and magnetic particle tags, the technique uses ultrasound and microbubble tags. Streptavidin-labeled microbubbles were mixed with a human acute lymphoblastic leukemia cell line, CCL 119, conjugated with biotinylated anti-CD7 antibodies. Tagged cells were forced under ultrasound, and their displacement and velocity quantified. Differential displacement in a flow stream was quantified against erythrocytes, which showed almost no displacement under ultrasound. A model for the acoustic radiation force on the conjugated pairs compares favorably with observations. This technology may improve on current time-consuming and costly purification procedures.

Micropropagation in the Twenty-First Century.

Despite more than a century of research on effective biotechnological methods, micropropagation continues to be an important tool for the large-scale production of clonal plantlets of several important plant species that retain genetic fidelity and are pest-free. In some cases, micropropagation is the only technique that supports the maintenance and promotes the economic value of specific agricultural species. The micropropagation of plants solved many phytosanitary problems and allowed both the expansion and access to high-quality plants for growers from different countries and economic backgrounds, thereby effectively contributing to an agricultural expansion in this and the last century. The challenges for micropropagation in the twenty-first century include cost reduction, enhanced efficiency, developing new technologies, and combining micropropagation with other systems/propagation techniques such as microcuttings, hydroponics, and aeroponics. In this chapter, we discuss the actual uses of micropropagation in this century, its importance and limitations, and some possible techniques that can effectively increase its wider application by replacing certain conventional techniques and technologies.

A Customizable, Low-Cost Perfusion System for Sustaining Tissue Constructs.

The fabrication of engineered vascularized tissues and organs requiring sustained, controlled perfusion has been facilitated by the development of several pump systems. Currently, researchers in the field of tissue engineering require the use of pump systems that are in general large, expensive, and generically designed. Overall, these pumps often fail to meet the unique demands of perfusing clinically useful tissue constructs. Here, we describe a pumping platform that overcomes these limitations and enables scalable perfusion of large, three-dimensional hydrogels. We demonstrate the ability to perfuse multiple separate channels inside hydrogel slabs using a preprogrammed schedule that dictates pumping speed and time. The use of this pump system to perfuse channels in large-scale engineered tissue scaffolds sustained cell viability over several weeks.

A Cost-Effective Culture System for the In Vitro Assembly, Maturation, and Stimulation of Advanced Multilayered Multiculture Tubular Tissue Models.

The development of tubular engineered tissues is a challenging research area aiming to provide tissue substitutes but also in vitro models to test drugs, medical devices, and even to study physiological and pathological processes. In this work, the design, fabrication, and validation of an original cost-effective tubular multilayered-tissue culture system (TMCS) are reported. By exploiting cellularized collagen gel as scaffold, a simple moulding technique and an endothelialization step on a rotating system, TMCS allowed to easily prepare in 48 h, trilayered arterial wall models with finely organized cellular composition and to mature them for 2 weeks without any need of manipulation. Multilayered constructs incorporating different combinations of vascular cells are compared in terms of cell organization and viscoelastic mechanical properties demonstrating that cells always progressively aligned parallel to the longitudinal direction. Also, fibroblast compacted less the collagen matrix and appeared crucial in term of maturation/deposition of elastic extracellular matrix. Preliminary studies under shear stress stimulation upon connection with a flow bioreactor are successfully conducted without damaging the endothelial monolayer. Altogether, the TMCS herein developed, thanks to its versatility and multiple functionalities, holds great promise for vascular tissue engineering applications, but also for other tubular tissues such as trachea or oesophagus.

Laboratory Workflow Analysis of Culture of Periprosthetic Tissues in Blood Culture Bottles.

Culture of periprosthetic tissue specimens in blood culture bottles is more sensitive than conventional techniques, but the impact on laboratory workflow has yet to be addressed. Herein, we examined the impact of culture of periprosthetic tissues in blood culture bottles on laboratory workflow and cost. The workflow was process mapped, decision tree models were constructed using probabilities of positive and negative cultures drawn from our published study (T. N. Peel, B. L. Dylla, J. G. Hughes, D. T. Lynch, K. E. Greenwood-Quaintance, A. C. Cheng, J. N. Mandrekar, and R. Patel, mBio 7:e01776-15, 2016, https://doi.org/10.1128/mBio.01776-15), and the processing times and resource costs from the laboratory staff time viewpoint were used to compare periprosthetic tissues culture processes using conventional techniques with culture in blood culture bottles. Sensitivity analysis was performed using various rates of positive cultures. Annualized labor savings were estimated based on salary costs from the U.S. Labor Bureau for Laboratory staff. The model demonstrated a 60.1% reduction in mean total staff time with the adoption of tissue inoculation into blood culture bottles compared to conventional techniques (mean ± standard deviation, 30.7 ± 27.6 versus 77.0 ± 35.3 h per month, respectively; P < 0.001). The estimated annualized labor cost savings of culture using blood culture bottles was $10,876.83 (±$337.16). Sensitivity analysis was performed using various rates of culture positivity (5 to 50%). Culture in blood culture bottles was cost-effective, based on the estimated labor cost savings of $2,132.71 for each percent increase in test accuracy. In conclusion, culture of periprosthetic tissue in blood culture bottles is not only more accurate than but is also cost-saving compared to conventional culture methods.

A simple short term method to study thyroid disruption using a fetal rat thyroid culture.

INTRODUCTION: Thyroid modulation activity has not been investigated for many chemical substances. Due to ethical, practical and financial reasons, in vivo evaluation of a large number of compounds is not feasible. It has been proposed that an in vitro mechanism-based strategy could be more adequate for the identification of thyroid hormone disrupting chemicals. Here we describe a simple and mostly inexpensive, short term culture assay to study thyroid disruption. METHODS: Fetal thyroids collected from gestation day 20.5 were cultured up to 24h in Hank's saline solution, at 37°C with oxygenation at 0 and 12h. Viability of the cultured explants was evaluated by the MTT assay. Positive (thyroid stimulating hormone, TSH) and negative (6-propyl-2-thiouracil, PTU) modulation of cultured thyroids was assessed with morphometrical analysis of H & E stained gland sections. Thyroxine expression was evaluated by immunohistochemistry. RESULTS: Viability was shown to increase with time of culture with higher metabolic activity being achieved at 24h as compared to shorter periods of incubation. Follicular epithelial cells exhibited a statistically significant dependence on thyrotropin concentration, although more evident in the inner than in the outer portion of the glands. As expected, TSH induced expression of thyroxin while PTU inhibited it. DISCUSSION: GD20.5 fetal thyroids may be cultured up to 24h under relatively simple laboratory conditions during which viability and function of the gland are preserved showing that it is possible to reproduce in vivo response under in vitro conditions. This culture could be a suitable short term assay to study mechanism of thyroid disruption.

A minimal cost micropropagation protocol for Dianthus caryophyllus L.-- a commercially significant venture.

In tissue culture, high production cost of the products restricts their reach. Though tissue culture is a major strength in floriculture it is marred by pricing issues. Hence, we developed a complete regeneration low cost micropropagation protocol for an economically important floriculture crop, carnation (Dianthus caryophyllus L.). Successful regeneration of carnation from nodal explants on cost-efficient medium indicates that psyllium husk, sugar and RO water can effectively replace the conventional medium comprising agar, sucrose and distilled water. The protocol can contribute to increased carnation production at comparatively reduced cost, and there by encourage wide scale adoption by the common growers.

A Lab Assembled Microcontroller-Based Sensor Module for Continuous Oxygen Measurement in Portable Hypoxia Chambers.

BACKGROUND: Hypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O2 concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O2, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup. METHODOLOGY: With the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O2 concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O2 sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions. RESULTS/CONCLUSIONS: We show the kit's utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O2 concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators.

Comparison of human mesenchymal stem cells isolated by explant culture method from entire umbilical cord and Wharton's jelly matrix.

Adult stem cells are of particular importance for applications in regenerative medicine. Umbilical cord was established recently as an alternative source of mesenchymal stem cell (MSC) instead of bone marrow (BM) and is superior to BM and other adult tissues according to several MSC properties. Additionally, for the purpose of cell therapy in clinical scale, steps of cell isolation, expansion and culture required to be precisely adjusted in order to obtain the most cost-effective, least time-consuming, and least labor-intensive method. Therefore, in this study, we are going to compare two simple and cost-effective explant culture methods for isolation of MSCs from human umbilical cord. One of the methods isolates cells from entire cord and the other from Wharton's jelly matrix. Isolated cells then cultured in simple medium without addition of any growth factor. MSCs obtained via both methods display proper and similar characteristics according to morphology, population doubling time, post-thaw survival, surface antigenicity and differentiation into adipocytes, osteocytes, and chondrocytes. MSCs can easily be obtained from the entire cord and Wharton's jelly, and it seems that both tissues are appropriate sources of stem cells for potential use in regenerative medicine. However, from technical large-scale preview, MSC isolation from entire cord piece is less labor-intensive and time-consuming than from Wharton's jelly part of the cord.

Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate.

Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose explant culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by explant culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As explant culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.

Comparison of different methods for separation of haploid embryo induced through irradiated pollen and their economic analysis in Melon (Cucumis melo var. inodorus).

Irradiated pollen technique is the most successful haploidization technique within Cucurbitaceae. After harvesting of fruits pollinated with irradiated pollen, classical method called as "inspecting the seeds one by one" is used to find haploid embryos in the seeds. In this study, different methods were used to extract the embryos more easily, quickly, economically, and effectively. "Inspecting the seeds one by one" was used as control treatment. Other four methods tested were "sowing seeds direct nutrient media," "inspecting seeds in the light source," "floating seeds on liquid media," and "floating seeds on liquid media after surface sterilization." Y2 and Y3 melon genotypes selected from the third backcross population of Yuva were used as plant material. Results of this study show that there is no statistically significant difference among methods "inspecting the seeds one by one," "sowing seeds direct CP nutrient media," and "inspecting seeds in the light source," although the average number of embryos per fruit is slightly different. No embryo production was obtained from liquid culture because of infection. When considered together with labor costs and time required for embryo rescue, the best methods were "sowing seeds directly in the CP nutrient media" and "inspecting seeds in the light source."

A time and cost efficient approach to functional and structural assessment of living neuronal tissue.

In this manuscript, we describe a protocol for capturing both physiological and structural properties of living neuronal tissue. An essential aspect of this method is its flexibility and fast turnaround time. It is a streamlined process that includes recording of electrophysiological neuronal activity, calcium imaging, and structural analysis. This is accomplished by placing intact tissue on a modified Millicell Biopore insert. The Biopore membrane suspends the tissue in the perfusion solution, allowing for complete access to nutrients, oxygen, and pharmacological agents. The ring that holds the membrane ensures its structural stability; forceps can be used to grip the ring without contacting the filter or the tissue, for easy transfer between multiple setups. We show that tissue readily adheres to the surface of the membrane, its entire surface is visible in transmitted light and accessible for recording and imaging, and remains responsive to physiological stimuli for extended periods of time.

A clinical algorithm for efficient, high-resolution cytogenomic analysis of uncultured perinatal tissue samples.

PURPOSE: Cytogenetic analysis of solid tissue is indispensable in perinatal care, reproductive planning, and detection of gestational trophoblastic disease. Unfortunately, methods in common use suffer from drawbacks including culture artifact, low resolution, and high cost. We propose a new diagnostic algorithm based on direct genetic analysis of tissues (without cell culture) using QF-PCR and array CGH. METHODS: Study samples consisted of specimens submitted to the cytogenetics laboratory between January and June of 2011 that were split and analyzed in parallel by our traditional algorithm (culture and G-banding, plus an interphase FISH aneuploidy panel for culture failures) and the proposed "no-culture" algorithm (first line QF-PCR, plus array CGH on normal QF-PCRs). Data on clinical impact, cost, and turnaround time were collected. RESULTS: Forty specimens were included. The algorithms produced results that were fully concordant in 22 cases, partially concordant in 9 cases, and discordant in 9 cases. The no-culture algorithm detected new, clinically-significant abnormalities in 8 of 40 cases (20%), corrected the sex chromosome assortment in 1 case, reduced the analysis failure rate from 10% to 0%, and provided at least one of these three important benefits in 12 of 40 cases (30%). The algorithm also demonstrated a reduced cost per-specimen and per diagnosis, as well as improved turnaround time, with virtually all cases reported per guidelines. CONCLUSION: These striking results favor the "no-culture" algorithm, which may have the potential to replace standard cytogenetic methods in the clinical laboratory.

An inexpensive alternative bath system for electrophysiological characterization of isolated cardiac tissue.

A tissue bath system, to be used as an alternative to complex perfusion chambers, was constructed for use in cardiac electrophysiological studies. This system consists of an acrylic chamber to hold circulating physiological medium such as DMEM, suspended in a water bath warmed by a hot plate. Temperature and pH were controlled to mimic physiological conditions. Rat and porcine cardiac tissues, were used to test viability of the conditions presented in the bath system. Using a cardiac mapping system, the tissues were stimulated and responses recorded. From the recordings we were able to calculate conduction velocities and spatial dispersion of activation indices. The results are comparable to previous in-vivo work, which suggests that the tissue bath system design can maintain tissue viability. This tissue bath system is a relatively simple alternative for ex-vivo testing of cardiac tissues.

Methodology for optimal in vitro cell expansion in tissue engineering.

Expansion of the cell population in vitro has become an essential step in the process of tissue engineering and also the systematic optimization of culture conditions is now a fundamental problem that needs to be addressed. Herein, we provide a rational methodology for searching culture conditions that optimize the acquisition of large quantities of cells following a sequential expansion process. In particular, the analysis of both seeding density and passage length was considered crucial, and their correct selection should be taken as a requisite to establish culture conditions for monolayer systems. This methodology also introduces additional considerations concerning the running cost of the expansion process. The selection of culture conditions will be a compromise between optimal cell expansion and acceptable running cost. This compromise will normally translate into an increase of passage length further away from the optimal value dictated by the growth kinetic of the cells. Finally, the importance of incorporating functional assays to validate the phenotypical and functional characteristics of the expanded cells has been highlighted. The optimization approach presented will contribute to the development of feasible large scale expansion of cells required by the tissue engineering industry.