Protocol for drug screening of patient-derived tumor organoids using high-content fluorescent imaging.

High-content imaging of tumor organoids (TOs) treated with therapeutic agents provides detailed cell viability readouts at the organoid level. In contrast, most used protocols provide one number per well. While requiring the use of inverted microscopy with an automated stage, this protocol can provide critical information about heterogeneous responses of TOs to various treatments. This protocol describes a technique for culturing and drug testing TOs using fluorescent indicators of cell viability with high reproducibility. For complete details on the use and execution of this protocol, please refer to Larsen et al. (2021).

Causative Role of Anoxic Environment in Bacterial Regulation of Human Intestinal Function.

Introduction: Life on Earth depends on oxygen; human tissues require oxygen signaling, whereas many microorganisms, including bacteria, thrive in anoxic environments. Despite these differences, human tissues and bacteria coexist in close proximity to each other such as in the intestine. How oxygen governs intestinal-bacterial interactions remains poorly understood. Methods: To address to this gap, we created a dual-oxygen environment in a microfluidic device to study the role of oxygen in regulating the regulation of intestinal enzymes and proteins by gut bacteria. Two-layer microfluidic devices were designed using a fluid transport model and fabricated using soft lithography. An oxygen-sensitive material was integrated to determine the oxygen levels. The intestinal cells were cultured in the upper chamber of the device. The cells were differentiated, upon which bacterial strains, a facultative anaerobe, Escherichia coli Nissle 1917, and an obligate anaerobe, Bifidobacterium Adolescentis, were cultured with the intestinal cells. Results: The microfluidic device successfully established a dual-oxygen environment. Of particular importance in our findings was that both strains significantly upregulated mucin proteins and modulated several intestinal transporters and transcription factors but only under the anoxic-oxic oxygen gradient, thus providing evidence of the role of oxygen on bacterial-epithelial signaling. Conclusions: Our work that integrates cell and molecular biology with bioengineering presents a novel strategy to engineer an accessible experimental system to provide tailored oxygenated environments. The work could provide new avenues to study intestine-microbiome signaling and intestinal tissue engineering, as well as a novel perspective on the indirect effects of gut bacteria on tissues including tumors. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00735-x.

A pan-cancer organoid platform for precision medicine.

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

Plasma steroid profiles and ovarian response in llamas treated with eCG for superovulation combined with exogenous progesterone during early luteal phase.

The objective of this study was to evaluate the effects of plasma progesterone (P4) concentrations during eCG-ovarian follicular superstimulatory treatment performed in early luteal phase and estradiol concentrations during peri-ovulatory period on ovarian response, number and embryo quality. On Day -2, females (n = 75) having a follicle ≥7 mm were treated with GnRH to induce ovulation. On Day 0, females that had ovulations (n = 54) were treated with 1000 IU eCG and were assigned to one of two treatments: (1) intravaginal device (ID) containing 0.5 g P4 (P4 group) and (2) no ID (Control group). On Day 5, females were administered PGF2α and the ID was removed. On Day 7 and 8, females were mated and embryo recovery was performed 7 or 8 days later. Blood samples were collected from Day 0 to 9. Number (± SD) of follicles ≥7 mm on day of mating was greater (P =  0.04) in the control (9.7 ± 4.2) than P4-treated (6.7 ±â€¯4.9) group; number of corpora lutea did not differ (5.5 ±â€¯3.1 and 5.2 ±â€¯3.4 respectively). Ovulation rate was greater (P <  0.01) in the P4-group (77.4%; 130/168) than control group (53.3%; 135/253). Number of embryos with an excellent grade (grade 1) tended to be greater (P =  0.07) in the P4-group (82.4%; 42/51) than control group (65.4%; 36/55). It was concluded that supplementation with exogenous P4 during eCG treatment in early luteal phase inhibits excessive follicular growth, increases ovulation rate and improves embryo quality.

Real-time measurement of cholesterol secreted by human hepatocytes using a novel microfluidic assay.

The use of microfluidics has become widespread in recent years because of the use of lesser resources such as small size, low volume of reagents, and physiological representation of mammalian cells. One of the advantages of microfluidic-based cell culture is the ability to perfuse culture media which tends to improve cellular health and function. Although measurement of cellular function conventionally is carried out using well-plates and plate readers, these approaches are insufficient to carry out in-line analysis of perfused cell cultures because of mismatch between volumes and sensitivity. We report the development of a novel microfluidic device and assay that is carried out under perfusion, in-line to measure the cholesterol secreted from a human hepatocyte tissue-chip. The heart of the assay is the unique implementation of enzymatic chemistry that is carried out on a polystyrene bead. Using this approach, we successfully measured cholesterol secreted by the perfused human hepatocytes.

Effect of GnRH analogue administration on Day 7 after natural mating on formation accessory corpus luteum, progesterone concentration and conception rate in llamas (Lama glama).

The objectives of the present study were to determine the effects of exogenous GnRH administered 7 days after breeding on the formation of an accessory corpus luteum (ACL), plasma progesterone (P4) concentrations and pregnancy rates. Adult females (n = 71) having a follicle ≥ 7 mm in diameter in the ovary were naturally mated (Day 0). On Day 7, ultrasonic examination was performed to confirm the occurrence of ovulation as evidenced by presence of an induced corpus luteum (ICL). Females with an ICL plus a dominant follicle ≥ 7 mm (n = 56) were treated with saline solution (SS, n = 29) or GnRH analogue (n = 27). On Day 14, the formation of an ACL was observed by ultrasonography. Blood samples were collected on Days 7 and 14 to quantify plasma P4 concentrations. On Day 14, 21 of 27 (77.8%) females in the GnRH group developed an ACL, whereas females in the SS group did not. Progesterone concentrations on Day 7 and 14 in those llamas diagnosed as pregnant on Day 30 were not different (P > 0.05) between groups. In addition, P4 concentration was similar for GnRH-treated females having two CL to those with a single CL. Pregnancy rates were similar (P > 0.05) between SS and GnRH groups (55.2% compared with 74.1% respectively) and the pregnancy rate for the GnRH group was not affected (P > 0.05) by the number of CL observed at Day 14 (66.6% and 75.6% for females with one and two CL respectively). In conclusion, GnRH administration on Day 7 after breeding leads to ACL formation; however, neither the plasma P4 concentration nor pregnancy rate was affected by having an ACL.

Revisión de los sistemas de medición de intervenciones de enfermería utilizados en Unidades de Cuidado Intensivo / Analysis of nursing scoring system used in Intensive Care Units

Las Unidades de Cuidados Intensivos (UCI) otorgan atención a un número limitado de pacientes de alta complejidad, con costos elevados, lo que hace necesario introducir criterios de eficacia, efectividad y eficiencia. La utilización de recursos y carga laboral, puede determinarse por varios sistemas de medición de intervenciones de Enfermería, como: Intensive Care Nursing Scoring System (ICNSS), Nine Equivalents of Nursing Manpower (NEMS) y Therapeutic Intervention Scoring System (TISS); siendo este último altamente desarrollado, convirtiéndose en un criterio ideal internacional para carga laboral. Esta revisión bibliográfica tiene como propósito reconocer los beneficios de la utilización de sistemas de medición de intervenciones de Enfermería para las diferentes funciones del rol profesional que desempeñan las enfermeras en las UCIs. La aplicación o utilización de sistemas de medición de intervenciones de Enfermería genera un continuo de múltiples y variados beneficios, los cuales no son exclusivos del equipo de Enfermería, a nivel del usuario o cliente se puede mencionar la disminución de complicaciones del paciente, disminución de morbilidad y mortalidad, una menor estadía en UCI y hospitalaria lo que tendrá un impacto en los costos de las hospitalizaciones; para las Unidades de Cuidados Intensivos implica una mejor planificación de los recursos humanos, carga laboral y asignación de personal, todos aspectos que están relacionados con una gestión del cuidado, otorgando así una atención de mejor calidad.