Vitrification within a nanoliter volume : oocyte and embryo cryopreservation within a 3D photopolymerized device

Open Access funding enabled and organized by CAUL and its Member Institutions. KRD is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58–2019). KD acknowledges funding from the UK Engineering and Physical Sciences Research Council (grants EP/P030017/1). This study was funded by the Australian Research Council (ARC) Centre of Excellence for Nanoscale BioPhotonics (CE140100003).Purpose Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. Methods The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). Results Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. Conclusion The Pod and Garage system minimized the volume of cryoprotectant at vitrification—by ~ 1000-fold—improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.Publisher PDFPeer reviewe

Pre-Mortem Cryopreservation: Recognizing a Patient’s Right to Die in Order to Live

I. Introduction II. The Science of Cryonic Preservation ... A. History of Cryonics ... B. The Process ... C. Current Science ... 1. Cryobiology ... a. Successful Births with Once-Frozen Embryos ... b. If You Can Thaw a Frozen Dog ... c. Surgeries Performed during Suspended Animation ... d. Successful Revival after Three Hours of Clinical Death ... 2. Advancements in Nanotechnology ... 3. Obstacles to Overcome III. Relevant Law ... A. Laws Governing Cryonics ... B. Right to Die and Assisted Suicide ... C. States’ Interests Are Not Compelling ... 1. Interest in Preserving Life and Preventing Suicide ... 2. Interest in Maintaining Medical Ethical Standards ... 3. Interest in Protecting Vulnerable Persons and Preventing Abuse ... 4. Are There Health Care Cost Savings to Consider? IV. Conclusio

Towards automated phenotyping in plant tissue culture

Plant in vitro culture techniques comprise important fundamental methods of modern plant research, propagation and breeding. Innovative scientific approaches to further develop the cultivation process, therefore, have the potential of far-reaching impact on many different areas. In particular, automation can increase efficiency of in vitro propagation, a domain currently con-strained by intensive manual labor. Automated phenotyping of plant in vitro culture bears the potential to extend the evaluation of in vitro plants from manual destructive endpoint measurements to continuous and objective digital quantification of plant traits. Consequently, this can lead to a better understanding of crucial developmental processes and will help to clarify the emergence of physiological disorders of plant in vitro cultures. The aim of this dissertation was to investigate and exemplify the potential of optical sensing methods and machine learning in plant in vitro culture from an interdisciplinary point of view. A novel robotic phenotyping system for automated, non-destructive, multi-dimensional in situ detection of plant traits based on low-cost sensor technology was con-ceptualized, developed and tested. Various sensor technologies, including an RGB camera, a laser distance sensor, a micro spectrometer, and a thermal camera, were applied partly for the first time under these challenging conditions and evaluated with respect to the resulting data quality and feasibility. In addition to the development of new dynamic, semi-automated data processing pipelines, the automatic acquisition of multisensory data across an entire subculture passage of plant in vitro cultures was demonstrated. This allowed novel time series images of different developmental processes of plant in vitro cultures and the emergence of physiological disorders to be captured in situ for the first time. The digital determination of relevant parameters such as projected plant area, average canopy height, and maximum plant height, was demonstrated, which can be used as critical descriptors of plant growth performance in vitro. In addition, a novel method of non-destructive quantification of media volume by depth data was developed which may allow monitoring of water uptake by plants and evaporation from the culture medium. The phenotyping system was used to investigate the etiology of the physiological growth anomaly hyperhydricity. Therefore, digital monitoring of the morphology and along with spectro-scopic studies of reflectance behavior over time were conducted. The new optical characteristics identified by classical spectral analysis, such as reduced reflectance and major absorption peaks of hyperhydricity in the SWIR region could be validated to be the main discriminating features by a trained support vector machine with a balanced accuracy of 84% on test set, demonstrating the feasibility of a spectral detection of hyperhydricity. In addition, an RGB image dataset was used for automated detection of hyperhydricity using deep neural networks. The high-performance metrics with precision of 83.8% and recall of 95.7% on test images underscore the presence of for detection sufficient number of discriminating features within the spatial RGB data, thus a second approach is proposed for automatic detection of hyperhydricity based on RGB images. The resulting multimodal sensor data sets of the robotic phenotyping system were tested as a supporting tool of an e-learning module in higher education to increase the digital skills in the field of sensing, data processing and data analysis, and evaluated by means of a student survey. This proof-of-concept study revealed an overall high level of acceptance and advocacy by students with 70% good to very good rating. However, with increased complexity of the learning task, stu-dents experienced excessive demands and rated the respective session lower. In summary, this study is expected to pave the way for increased use of automated sensor-based phenotyping in conjunction with machine learning in plant research and commercial mi-cropropagation in the future.Die pflanzliche In-vitro-Kultur umfasst wichtige grundlegende Methoden der modernen Pflanzenforschung, -vermehrung und -züchtung. Innovative wissenschaftliche Ansätze zur Wei-terentwicklung des Kultivierungsprozess können daher weitreichenden Einfluss auf viele unter-schiedliche Bereiche haben. Insbesondere die Automatisierung kann die Effizienz der In-vitro-Vermehrung steigern, die derzeit durch die intensive manuelle Arbeit beschränkt wird. Automa-tisierte Phänotypisierung von In-vitro-Kulturen ermöglicht es, die Erfassung von manuellen de-struktiven Endpunktmessungen auf eine kontinuierliche, objektive und digitale Quantifizierung der Pflanzenmerkmale auszuweiten. Dies kann zu einem besseren Verständnis entscheidender Entwicklungsprozesse führen und die Entstehung physiologischer Störungen zu klären. Ziel dieser Dissertation war es, das Potential optischer Erfassungsmethoden und des maschinellen Lernens für die pflanzliche In-vitro-Kultur unter interdisziplinären Gesichtspunk-ten zu untersuchen und exemplarisch aufzuzeigen. Ein neuartiger Phänotypisierungsroboter zur automatisierten, zerstörungsfreien, mehrdimensionalen In-situ-Erfassung von Pflanzenmerkmalen wurde auf Basis kostengünstiger Sensortechnik entwickelt. Unterschiedliche Sensortechnologien, darunter eine RGB-Kamera, ein Laser-Distanzsensor, ein Mikrospektrometer und eine Wärmebildkamera, wurden teils zum ersten Mal unter diesen schwierigen Bedingungen eingesetzt und im Hinblick auf die resultierende Datenqualität und Realisierbarkeit bewertet. Neben der Entwicklung dynamischer, halbautomatischer Datenverarbeitungspipelines, wurde die automatische Erfassung multisensorischer Daten über eine gesamte Subkulturpassage der In-vitro-Kulturen demonstriert. Dadurch konnte erstmals Zeitrafferaufnahmen verschiedener Ent-wicklungsprozesse von pflanzlichen In-vitro-Kulturen und das Auftreten von physiologischen Störungen in situ erfasst werden. Die digitale Bestimmung relevanter Kenngrößen wie der proji-zierten Pflanzenfläche, der durchschnittlichen Bestandshöhe und der maximalen Pflanzenhöhe wurde demonstriert, die als wichtige Deskriptoren für das pflanzliche Wachstum dienen können. Darüber hinaus konnte eine neue Methode für die Pflanzenwissenschaften entwickelt werden, um die Wasseraufnahme von Pflanzen und die Verdunstung von Kulturmedien auf der Grundlage einer zerstörungsfreien Quantifizierung des Medienvolumens zu überwachen. Der Phänotypisierungsroboter wurde zur Untersuchung der Entstehung der Wachs-tumsanomalie Hyperhydrizität eingesetzt. Hierfür wurden ein digitales Monitoring der Morpho-logie der Explantate mit begleitenden spektroskopischen Untersuchungen des Reflexionsverhal-tens im Zeitverlauf durchgeführt. Die durch Spektralanalyse identifizierten optischen Merkmale, wie den reduzierter Reflexionsgrad und die Hauptabsorptionspeaks der Hyperhydrizität in der SWIR-Region, konnten als die wichtigsten Unterscheidungsmerkmale durch ein Support-Vektor-Maschine-Model mit einer Genauigkeit von 84% auf dem Testsatz validiert werden und damit Machbarkeit der spektrale Identifizierung von Hyperhydrizität aufzeigen. Darüber wurde für die automatische Detektion der Hyperhydrizität auf Basis von RGB-Bildern ein neuronales Netz trainiert. Die hohen Kennzahlen im Testdatensatz wie die Präzision von 83,8 % und einem Recall von 95,7 % unterstreichen das Vorhandensein einer für die Erkennung ausreichenden Anzahl von Unterscheidungsmerkmalen innerhalb der räumlichen RGB-Daten. Somit konnte ein zweiter An-satz der automatischen Detektion von Hyperhydrizität durch RGB-Bilder präsentiert werden. Die resultierenden Sensordatensätze des Phänotypisierungsroboters wurden als unter-stützendes Werkzeug eines E-Learning Moduls zur Steigerung digitaler Kompetenzen im Bereich Sensortechnik, Datenverarbeitung und -auswertung in der Hochschulausbildung erprobt und an-hand der Befragung von Studierenden evaluiert. Diese Machbarkeitsstudie ergab eine insgesamt hohe Akzeptanz durch die Studierenden mit 70% guten bis sehr guten Bewertungen. Mit zuneh-mender Komplexität der Lernaufgabe fühlten sich die Studierenden jedoch überfordert und bewerteten die jeweilige Session schlechter. Zusammenfassend zielt diese Arbeit darauf ab den Weg für einen verstärkten Einsatz der automatisierten, sensorbasierten Phänotypisierung in Kombination mit den Techniken des ma-schinellen Lernens der Forschung und der kommerziellen Mikrovermehrung zukünftig zu ebnen.Bundesministerium für Ernährung und Landwirtschaft (BMEL)/Digitale Experimentierfelder/28DE103F18/E

Pre-Mortem Cryopreservation: Recognizing a Patient’s Right to Die in Order to Live

I. Introduction II. The Science of Cryonic Preservation ... A. History of Cryonics ... B. The Process ... C. Current Science ... 1. Cryobiology ... a. Successful Births with Once-Frozen Embryos ... b. If You Can Thaw a Frozen Dog ... c. Surgeries Performed during Suspended Animation ... d. Successful Revival after Three Hours of Clinical Death ... 2. Advancements in Nanotechnology ... 3. Obstacles to Overcome III. Relevant Law ... A. Laws Governing Cryonics ... B. Right to Die and Assisted Suicide ... C. States’ Interests Are Not Compelling ... 1. Interest in Preserving Life and Preventing Suicide ... 2. Interest in Maintaining Medical Ethical Standards ... 3. Interest in Protecting Vulnerable Persons and Preventing Abuse ... 4. Are There Health Care Cost Savings to Consider? IV. Conclusio

Decision points for individualized hormonal stimulation with recombinant gonadotropins for treatment of women with infertility

It is essential that fertility treatment is individualized based on a thorough diagnostic work-up, with treatment tailored to the patients' requirements. This individualization should be kept in mind during the main decision points that occur before and during treatment. Treatment customization must include consideration of both the woman and her partner involved in the process together, including their collective treatment goals. Once treatment goals have been agreed and diagnostic evaluations performed, personalization based on patient characteristics, together with an understanding of treatment goals and patient preferences, enables the selection of appropriate treatments, protocols, products and their dosing. Following treatment initiation, monitoring and adaptation of product and dose can then ensure optimal outcomes. Currently, it is not possible to base treatment decisions on every characteristic of the patient and personalization is based on biomarkers that have been identified as the most relevant. However, in the future, the use of artificial intelligence coupled with continuous monitoring should enable greater individualization and improve outcomes. This review considers the current state-of-the-art related to decision points during individualized treatment of female infertility, before looking at future developments that might further assist in making individualized treatment decisions, including the use of computer-assisted decision making. ispartof: GYNECOLOGICAL ENDOCRINOLOGY vol:35 issue:12 pages:1027-1036 ispartof: location:England status: publishe

Design of Vitrification Machine

Cryopreservation of living cells and biological material by vitrification requires the expertise of a skilled lab technician and a large amount of time. Vitrification must be performed one sample at a time on tiny subjects, which makes for a tedious and unreliable process. Moreover, there is a lack of standardization in the methods for preparing cells and biological material for the vitrification process. The purpose of the Vitrification Machine is to greatly simplify the process by making it faster, more efficient, cheaper and more reliable. The machine will be capable of handling several subjects at a time and will completely automate the most tedious portions of the vitrification process. This ease of use will allow researchers to experiment with new vitrification preparation methods on a larger number of samples more quickly and reliably. The applications of the Vitrification Machine are wide, but it is specifically being developed to automate the vitrification of human oocytes and eventually embryos. Female patients diagnosed with diseases whose treatments are detrimental to the reproductive process (such as chemotherapy), can have their oocytes preserved for use after their treatment is complete. The automation of vitrification by the Vitrification Machine will make this process faster, more reliable, more affordable, and therefore more available to patients. There currently is no known product on the market that fills all these needs or has the potential to drive down the cost of this portion of the fertility preservation process. Since the Vitrification Machine will be useful to clinical In-Vitro Fertilization (IVF) laboratories, animal science research and fertility specialists in both research and clinical settings, the market potential of the product is very large

A Contactless and Biocompatible Approach for 3D Active Microrobotic Targeted Drug Delivery

As robotic tools are becoming a fundamental part of present day surgical interventions, microrobotic surgery is steadily approaching clinically-relevant scenarios. In particular, minimally invasive microrobotic targeted drug deliveries are reaching the grasp of the current state-of-the-art technology. However, clinically-relevant issues, such as lack of biocompatibility and dexterity, complicate the clinical application of the results obtained in controlled environments. Consequently, in this work we present a proof-of-concept fully contactless and biocompatible approach for active targeted delivery of a drug-model. In order to achieve full biocompatiblity and contacless actuation, magnetic fields are used for motion control, ultrasound is used for imaging, and induction heating is used for active drug-model release. The presented system is validated in a three-dimensional phantom of human vessels, performing ten trials that mimic targeted drug delivery using a drug-coated microrobot. The system is capable of closed-loop motion control with average velocity and positioning error of 0.3 mm/s and 0.4 mm, respectively. Overall, our findings suggest that the presented approach could augment the current capabilities of microrobotic tools, helping the development of clinically-relevant approaches for active in-vivo targeted drug delivery

A longitudinal study of the experiences and psychological well-being of Indian surrogates

Study question: What is the psychological well-being of Indian surrogates during and after the surrogacy pregnancy? Summary answer: Surrogates were similar to a matched group of expectant mothers on anxiety and stress. However, they scored higher on depression during and after pregnancy. What is known already: The recent ban on trans-national commercial surrogacy in India has led to urgent policy discussions regarding surrogacy. Whilst previous studies have reported the motivations and experiences of Indian surrogates no studies have systematically examined the psychological well-being of Indian surrogates, especially from a longitudinal perspective. Previous research has shown that Indian surrogates are motivated by financial payment and may face criticism from their family and community due to negative social stigma attached to surrogacy. Indian surrogates often recruited by agencies and mainly live together in a “surrogacy house.” Study design, size, duration: A longitudinal study was conducted comparing surrogates to a matched group of expectant mothers over two time points: (a) during pregnancy (Phase1: 50 surrogates, 70 expectant mothers) and (b) 4–6 months after delivery (Phase 2: 45 surrogates, 49 expectant mothers). The Surrogates were recruited from a fertility clinic in Mumbai and the matched comparison group was recruited from four public hospitals in Mumbai and Delhi. Data collection was completed over 2 years. Participants/materials, setting, methods: Surrogates and expectant mothers were aged between 23 and 36 years. All participants were from a low socio-economic background and had left school before 12–13 years of age. In-depth faceto-face semi-structured interviews and a psychological questionnaire assessing anxiety, stress and depression were administered in Hindi to both groups. Interviews took place in a private setting. Audio recordings of surrogate interviews were later translated and transcribed into English. Main results and the role of chance: Stress and anxiety levels did not significantly differ between the two groups for both phases of the study. For depression, surrogates were found to be significantly more depressed than expectant mothers at phase 1 (p = 0.012) and phase 2 (p = 0.017). Within the surrogacy group, stress and depression did not change during and after pregnancy. However, a non-significant trend was found showing that anxiety decreased after delivery (p = 0.086). No participants reported being coerced into surrogacy, however nearly all kept it a secret from their wider family and community and hence did not face criticism. Surrogates lived at the surrogate house for different durations. During pregnancy, 66% (N = 33/50) reported their experiences of the surrogate house as positive, 24% (N = 12/50) as negative and 10% (N = 5/50) as neutral. After delivery, most surrogates (66%, N = 30/45) reported their experiences of surrogacy to be positive, with the remainder viewing it as neutral (28%) or negative (4%). In addition, most (66%, N = 30/45) reported that they had felt “socially supported and loved” during the surrogacy arrangement by friends in the surrogate hostel, clinic staff or family. Most surrogates did not meet the intending parents (49%, N = 22/45) or the resultant child (75%, N = 34/45). Limitations, reasons for caution: Since the surrogates were recruited from only one clinic, the findings may not be representative of all Indian surrogates. Some were lost to follow-up which may have produced sampling bias. Wider implications of the findings: This is the first study to examine the psychological well-being of surrogates in India. This research is of relevance to current policy discussions in India regarding legislation on surrogacy. Moreover, the findings are of relevance to clinicians, counselors and other professionals involved in surrogacy. Trial registration number: N/A

Novel Cryopreservation Strategies for Cell‐Therapies and Pre‐Clinical Research

The expanding need for complex biologics for therapeutic applications, in‐vitro pharmacology and toxicology studies and fundamental research demands the production of banks of well‐characterized and safety‐tested stocks of a large number of cell/tissue samples. This implies the development of effective cryopreservation methodologies that can cope with process scalability and automation and must reflect the biological and physical properties of the cells as these can be significantly altered by the process