Understanding In Vitro Tissue Culture-Induced Variation Phenomenon in Microspore System.

In vitro tissue culture plant regeneration is a complicated process that requires stressful conditions affecting the cell functioning at multiple levels, including signaling pathways, transcriptome functioning, the interaction between cellular organelles (retro-, anterograde), compounds methylation, biochemical cycles, and DNA mutations. Unfortunately, the network linking all these aspects is not well understood, and the available knowledge is not systemized. Moreover, some aspects of the phenomenon are poorly studied. The present review attempts to present a broad range of aspects involved in the tissue culture-induced variation and hopefully would stimulate further investigations allowing a better understanding of the phenomenon and the cell functioning.

Automation of Organoid Cultures: Current Protocols and Applications.

Organoids are three-dimensional, functional structures that mimic in vivo organs. They offer new opportunities for the modeling of cancer and infectious and rare hereditary diseases. Furthermore, the advent of organoid biobanks opens new avenues for drug screening in a personalized fashion and holds much promise for personalized regenerative medicine. Thus, there is a need for reproducible, large-scale organoid generation with minimal variability, making manual approaches impracticable. Here, we review the current use of automation in organoid culture and analysis, using cerebral and retinal organoids as illustrations of current applications. An increased demand for automated organoid platforms is anticipated.

Diagnostic Accuracy of Bone Culture Versus Biopsy in Diabetic Foot Osteomyelitis.

OBJECTIVE: To compare the diagnostic accuracy of bone culture (microbiology) and biopsy (histology) in patients with acute or chronic diabetic foot osteomyelitis (DFO). METHODS: This cross-sectional study involved patients for whom providers had a clinical suspicion of DFO. Two bone samples were taken: one for microbiologic testing and another for histologic testing. The sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio were calculated for bone culture results in relation to the probability of DFO diagnosis. RESULTS: Fifty-two patients were included; 69% had positive bone culture results, and 90.4% had positive histology results (P = .013), and of those 90.4%, 25.5% had acute and 74.5% had chronic DFO. The sensitivity of the microbiologic bone culture result was 0.70, the specificity was 0.40, the positive predictive value was 0.92, and the negative predictive value was 0.13. CONCLUSIONS: Histology provides more accurate diagnosis of DFO than microbiology, especially for patients with chronic DFO. These patients could be underdiagnosed because of false-negative results provided by bone culture. Providers should perform both tests to confirm the presence of DFO.

Validation of the Media Fill Method for Solid Tissue Processing in Good Manufacturing Practice-Compliant Cell Production.

Over the past few years, a large number of clinical studies for advanced therapy medicinal products have been registered and/or conducted for treating various diseases around the world and many have generated very exciting outcomes. Media fill, the validation of the aseptic manufacturing process, is the simulation of medicinal product manufacturing using nutrient media. The purpose of this study is to explain the media fill procedure stepwise in the context of cellular therapy medicinal products. The aseptic preparation of patient individual cellular product is simulated by using tryptic soy broth as the growth medium, and sterile vials as primary packaging materials.

Increasing the availability of quality human tissue for research.

Advances in 3D and other in vitro tissue model platforms have led to fundamental improvements in research on human disease, development of novel therapies, and safety testing. In addition, histological and cellular investigations of human tissues continue to serve as keystones in understanding disease and health processes. In recognition of the importance of human tissues in research, the Physicians Committee for Responsible Medicine held a workshop. Working closely with key stakeholders from the research community, regulatory agencies, and organ procurement organizations, the goal was to explore, understand, and address the barriers to increased use of human organs, tissues, and cells in research. Workshop participants were tasked with identifying the challenges of accessing and qualifying tissues for research purposes and creating a strategy to help meet the needs of the research communities to increase the availability and quality of human tissues in biomedical and translational research. Break-out groups identified significant challenges in the areas of policy, scientific development, and public engagement with respect to the provision and application of tissues and cells for scientific advancement. Following working group recommendations, stakeholders concluded that there is a need to facilitate the availability and quality of human tissues for the research community, as well as provide a framework for education of the public, medical professionals, and researchers to foster donation and utilization for research in place of animal models. The success of these new initiatives will facilitate greater access to high-quality human tissues for biomedical and translational research and help ensure the transition away from the dependence on animal models.

Implant Sonication versus Tissue Culture for the Diagnosis of Spinal Implant Infection.

MINI: We compared the sensitivity and specificity of peri-implant tissue culture to the vortexing-sonication technique for the diagnosis of spinal implant infection (SII). Lower thresholds of sonicate fluid culture positivity showed increased sensitivity with maintained specificity. We recommend a threshold of 20 CFU/10 mL for sonicate culture positivity for the diagnosis of SII. STUDY DESIGN: This is a retrospective study comparing the diagnosis of spinal implant infection (SII) by peri-implant tissue culture to vortexing-sonication of retrieved spinal implants. OBJECTIVE: We hypothesized that vortexing-sonication would be more sensitive than peri-implant tissue culture. SUMMARY OF BACKGROUND DATA: We previously showed implant vortexing-sonication followed by culture to be more sensitive than standard peri-implant tissue culture for diagnosing of SII. In this follow-up study, we analyzed the largest sample size available in the literature to compare these two culture methods and evaluated thresholds for positivity for sonicate fluid for SII diagnosis. METHODS: We compared peri-implant tissue culture to the vortexing-sonication technique which samples bacterial biofilm on the surface of retrieved spinal implants. We evaluated different thresholds for sonicate fluid positivity and assessed the sensitivity and specificity of the two culture methods for the diagnosis of SII. RESULTS: A total of 152 patients were studied. With more than 100 colony forming units (CFU)/10 mL as a threshold for sonicate fluid culture positivity, there were 46 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 65.2% and 79.6%; the specificities were 88.7% and 93.4%, respectively. With more than 50 CFU/10 mL as a threshold, there were 50 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 68.0% and 76.0%; the specificities were 92.2% for both methods. Finally, with more than or equal to 20 CFU/10 mL as a threshold, there were 52 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 69.2% and 82.7%; the specificities were 94.0% and 92.0%, respectively. CONCLUSION: Implant sonication followed by culture is a sensitive and specific method for the diagnosis of SII. Lower thresholds for defining sonicate fluid culture positivity allow for increased sensitivity with a minimal decrease in specificity, enhancing the clinical utility of implant sonication. LEVEL OF EVIDENCE: 4.

This is a retrospective study comparing the diagnosis of spinal implant infection (SII) by peri-implant tissue culture to vortexing­sonication of retrieved spinal implants. We hypothesized that vortexing­sonication would be more sensitive than peri-implant tissue culture. We previously showed implant vortexing­sonication followed by culture to be more sensitive than standard peri-implant tissue culture for diagnosing of SII. In this follow-up study, we analyzed the largest sample size available in the literature to compare these two culture methods and evaluated thresholds for positivity for sonicate fluid for SII diagnosis. We compared peri-implant tissue culture to the vortexing­sonication technique which samples bacterial biofilm on the surface of retrieved spinal implants. We evaluated different thresholds for sonicate fluid positivity and assessed the sensitivity and specificity of the two culture methods for the diagnosis of SII. A total of 152 patients were studied. With more than 100 colony forming units (CFU)/10 mL as a threshold for sonicate fluid culture positivity, there were 46 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 65.2% and 79.6%; the specificities were 88.7% and 93.4%, respectively. With more than 50 CFU/10 mL as a threshold, there were 50 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 68.0% and 76.0%; the specificities were 92.2% for both methods. Finally, with more than or equal to 20 CFU/10 mL as a threshold, there were 52 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 69.2% and 82.7%; the specificities were 94.0% and 92.0%, respectively. Implant sonication followed by culture is a sensitive and specific method for the diagnosis of SII. Lower thresholds for defining sonicate fluid culture positivity allow for increased sensitivity with a minimal decrease in specificity, enhancing the clinical utility of implant sonication. Level of Evidence: 4.

Validation of automated pipetting systems for cell culture seeding, exposure and bio-analytical assays in sulfur mustard toxicology.

In vitro cell culture experiments are highly important techniques to accelerate drug discovery, conduct safety testing and reduce the need for animal studies. Therefore, automatization may help to enhance the technical precision, reduce external (including operator's) influence on the data and thus improve reliability. Prior to application in scientific studies, validation of automated systems is absolutely necessary. In this study we present the validation of two combined automated pipetting systems to conduct toxicity studies in HaCaT cells consisting of cell seeding, noxious agent exposure and several assays to assess cell survival, apoptosis and interleukin production. After initial validation of pipetting accuracy, we compared homogeneity after automated seeding to plates seeded by expert laboratory technicians. Moreover, automated dispensing of a potentially unstable noxious agent was analyzed in terms of speed and consistency. We found a 2 % technical imprecision for the cell survival assay and 4.5-6 % for the other assays, bioluminescent and ELISA techniques. Thus, we could demonstrate the excellent technical precision of our assays. In a final step, we found that intraday variations, though acceptable, were much larger than technical variations and had to assume an intraday biological variability between different wells of the same experimental group.

Standardization of in vitro nervous tissue culture for honeybee: A high specificity toxicological approach.

Bees are important pollinators that help to maintain the biodiversity of wild and cultivated plants. However, the increased and inappropriate use of agrochemicals has caused an imbalance in the populations of these insects visiting flowers for pollen and nectar collection. Therefore, new research methods for understanding the mechanisms of action of pesticides and their impacts on the brains of bees, such as neurotoxicity and cellular changes, in response to different active characteristics and dosages of insecticides are necessary. Thus, with the aim of developing tests with greater specificity at the level of cells or tissues, this study sought to standardize a method for the in vitro culture of the nervous tissue of Apis mellifera. For this purpose, the brains of six foragers bees were transferred to three different insect cell culture media and it supplementation with 10% foetal bovine serum (FBS): Grace, Schneider, Leibovitz, Grace + FBS, Schneider + FBS and Leibovitz + FBS media for each collection time. Nervous tissue was collected after 1, 6, 12 and 24 h of incubation in a humidified CO2 incubator at 32 °C, and histological sections of the organs were analysed. The results showed that Leibovitz medium and Leibovitz medium + serum are potential culture media for the cultivation of nervous tissue, since they resulted in less tissue spacing and tissue disarrangement. Therefore, additional supplements are necessary to obtain an ideal medium for the cultivation of A.mellifera nervous tissue.

Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.

Experimental models of the human brain are needed for basic understanding of its development and disease1. Human brain organoids hold unprecedented promise for this purpose; however, they are plagued by high organoid-to-organoid variability2,3. This has raised doubts as to whether developmental processes of the human brain can occur outside the context of embryogenesis with a degree of reproducibility that is comparable to the endogenous tissue. Here we show that an organoid model of the dorsal forebrain can reliably generate a rich diversity of cell types appropriate for the human cerebral cortex. We performed single-cell RNA-sequencing analysis of 166,242 cells isolated from 21 individual organoids, finding that 95% of the organoids generate a virtually indistinguishable compendium of cell types, following similar developmental trajectories and with a degree of organoid-to-organoid variability comparable to that of individual endogenous brains. Furthermore, organoids derived from different stem cell lines show consistent reproducibility in the cell types produced. The data demonstrate that reproducible development of the complex cellular diversity of the central nervous system does not require the context of the embryo, and that establishment of terminal cell identity is a highly constrained process that can emerge from diverse stem cell origins and growth environments.

Sphere­forming assay vs. organoid culture: Determining long­term stemness and the chemoresistant capacity of primary colorectal cancer cells.

Three­dimensional (3D) cultures are indispensable for capturing tumor heterogeneity in colorectal cancer (CRC) in vitro. Although 3D cultures (such as sphere­forming assay and organoid culture) can partially preserve the morphological and molecular characteristics of primary CRC, whether these 3D cultures maintain the long­term stemness of cancer stem cells (CSCs) remains largely unknown. In the present study, spheres and organoids were generated side by side using individual primary CRC specimens, then respectively processed as serial passages. The results revealed that during serial passages, the percentage of CSCs (such as cluster of differentiation­133+ and Wnt+ cells) in organoids and the tumor­initiating capacity of organoid­derived cells were constant, while they gradually increased in the sphere­derived cells. Furthermore, during serial passages, resistance to chemotherapeutic agents (including 5­fluorouracil and oxaliplatin) in sphere­ and organoid­derived cells was evaluated. The results indicated that the percentage of chemoresistant cells was constant in serial organoid cultures; however, it gradually increased in the serial sphere­forming assays. Taken together, the results of the present study comprehensively demonstrate that, with regard to long­term culture in vitro, organoid culture may be useful in maintaining tumor heterogeneity and the levels of chemoresistant cells, while the sphere formation assay enriches for CSCs and chemoresistant cells.

Vitrification and in vitro culture had no adverse effect on the follicular development and gene expression of stimulated human ovarian tissue.

AIM: The study assesses the effect of the vitrification procedure on the integrity, morphology, follicular development and gene expression of stimulated human ovarian tissue after warming and two weeks of in vitro culture. METHODS: Ovarian specimens were divided into non-vitrified and vitrified groups and were cultured for two weeks. Morphological analysis and immunohistochemistry were performed. The 17-ß estradiol and anti-Müllerian hormone levels in collected media were assessed. Gene expression was analyzed using real-time reverse transcription polymerase chain reaction. RESULTS: The morphology and immunohistochemistry of bcl-2-like protein 4 and B-cell lymphoma 2 of human stimulated ovarian tissue were similar in both groups. There was no significant difference in the percentage of normal follicles between the groups before and after in vitro culture. In spite of an increase in the percentage of growing follicles in cultured tissues compared to the non-cultured groups, the rate of normal follicles was significantly decreased in both cultured groups (P < 0.05). Gene expression was no different in vitrified tissues compared to the control; however, the expression of growth differentiation factor 9 and follicle stimulating hormone receptor genes were increased and factor in germ line alpha and kit ligand genes were decreased during in vitro culture (P < 0.05). In the two cultured groups, the level of 17-ß estradiol was increased (P < 0.05), but the anti-Müllerian hormone concentration was not statistically altered. CONCLUSIONS: These results showed that the integrity of stimulated human ovarian tissue after vitrification/warming was well preserved; however, the in vitro culture condition needs improvement.

Basic Techniques in Mammalian Cell Tissue Culture.

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc.

Better science with human cell-based organ and tissue models.

At present, animal-based models are the major test systems for assessing the tolerability and safety of chemical substances for regulatory purposes, and also for pivotal efficacy testing in pharmaceutical development. In spite of the high genetic similarity between many laboratory animals and humans, animal models are very poor predictors of human health effects and pathophysiological processes. Thus, models and testing strategies that are more relevant to human biology, are needed for these purposes. The best predictability is achieved with human organotypic models that mimic the microenvironment of human tissues. During their development, such models have to be characterised at the structural, genetic and functional levels, and compared to the respective human tissues. Their predictivity should be confirmed by using known reference chemicals with corresponding human data. The use of these methods in safety assessment and biomedical research, combined with the knowledge gained of the underlying biological processes on gene and protein expression, as well as on cellular signalling, will ultimately lead to better human science and animal welfare.

[Issues of large scale tissue culture of medicinal plant].

In order to increase the yield and quality of the medicinal plant and enhance the competitive power of industry of medicinal plant in our country, this paper analyzed the status, problem and countermeasure of the tissue culture of medicinal plant on large scale. Although the biotechnology is one of the most efficient and promising means in production of medicinal plant, it still has problems such as stability of the material, safety of the transgenic medicinal plant and optimization of cultured condition. Establishing perfect evaluation system according to the characteristic of the medicinal plant is the key measures to assure the sustainable development of the tissue culture of medicinal plant on large scale.

Technical communication: stability of propofol in polystyrene-based tissue culture plates.

Propofol has been reported to have high stability in glass and relatively high stability up to 24 hours in polyvinyl chloride-based medical plastics. Recent publications have observed the effects of propofol on cells and tissues grown in culture. Many cell culture plastics are formulated from polystyrene but we could find little information on the stability of propofol exposed to these products. We observed very little change in the concentration of propofol diluted in cell culture medium over 24 hours when exposed to glass, but substantial loss of the drug when exposed to 96-well polystyrene cell culture plates. This decrease was most rapid in the first hour but continued until 24 hours. The type of plastic used in cell and tissue culture experiments with propofol may influence the results by increasing the apparent dose required to see an effect.

Effects of bacterial infection on insulin secretory capacity of human adult pancreatic islets.

This study aims to determine the origin of bacterial contamination of pancreatic tissue cultures, as well as its influence on insulin secretory activity (expressed as stimulation index [SI]) of the pancreatic islets. Pancreatic tissue was obtained after pancreatectomy in patients who had chronic pancreatitis or benign tumours. Islets were isolated under aseptic conditions by a manual method. Microbiological analyses were performed by standard procedures and the SI was determined on the first and seventh day of cultivation. In cultures contamminated by Pseudomonas, SI was 1.58 +/- 1.16 on day 1 and 0.22 +/- 0.14 on day 7 (P<0.01). Cultures contaminated by Enterobacter showed an SI of 0.21 +/- 0.1 on day 1, which increased to 1.19 +/- 0.66 on day 7 (P<0.01). In cases of Staphylococcus contamination, SI was 0.07 +/- 0.05 on day 1 and 0.33 +/- 0.21 on day 7 (P<0.01). The study shows that cell culture contamination originates from an original pancreatic tissue infection. The presence of bacteria may reduce or increase insulin secretion in cell culture, depending on the type of microorganism, and this can provoke reduced or elevated levels of insulin secretion in recipients, thus increasing the chances for the onset of diabetes.

Bioreactors in tissue engineering: scientific challenges and clinical perspectives.

In this Chapter we discuss the role of bioreactors in the translational paradigm of Tissue Engineering approaches from basic research to streamlined tissue manufacturing. In particular, we will highlight their functions as: (1) Pragmatic tools for tissue engineers, making up for limitations of conventional manual and static techniques, enabling automation and allowing physical conditioning of the developing tissues; (2) 3D culture model systems, enabling us to recapitulate specific aspects of the actual in vivo milieu and, when properly integrated with computational modeling efforts and sensing and control techniques, to address challenging scientific questions; (3) Tissue manufacturing devices, implementing bioprocesses so as to support safe, standardized, scaleable, traceable and possibly cost-effective production of grafts for clinical use. We will provide evidences that fundamental knowledge gained through the use of well-defined and controlled bio-reactor systems at the research level will be essential to define, optimize, and moreover, streamline the key processes required for efficient manufacturing models.

Bioreactor technology in cardiovascular tissue engineering.

Cardiovascular tissue engineering is a fast evolving field of biomedical science and technology to manufacture viable blood vessels, heart valves, myocardial substitutes and vascularised complex tissues. In consideration of the specific role of the haemodynamics of human circulation, bioreactors are a fundamental of this field. The development of perfusion bioreactor technology is a consequence of successes in extracorporeal circulation techniques, to provide an in vitro environment mimicking in vivo conditions. The bioreactor system should enable an automatic hydrodynamic regime control. Furthermore, the systematic studies regarding the cellular responses to various mechanical and biochemical cues guarantee the viability, bio-monitoring, testing, storage and transportation of the growing tissue.The basic principles of a bioreactor used for cardiovascular tissue engineering are summarised in this chapter.