Biology Methods and Protocols最新文献

Cell replacement in aganglionic intestines is a promising, yet merely experimental tool for the therapy of congenital dysganglionosis of the enteric nervous system like Hirschsprung disease. While the injection of single cells or neurospheres to a defined and very restricted location is trivial, the translation to the clinical application, where large aganglionic or hypoganglionic areas need to be colonized (hundreds of square centimetres), afford a homogeneous distribution of multiple neurospheres all over the affected tissue areas. Reaching the entire aganglionic area in vivo is critical for the restoration of peristaltic function. The latter mainly depends on an intact nervous system that extends throughout the organ. Intra-arterial injection is a common method in cell therapy and may be the key to delivering cells or neurospheres into the capillary bed of the colon with area-wide distribution. We describe an experimental method for monitoring the distribution of a defined number of neurospheres into porcine recta ex vivo, immediately after intra-arterial injection. We designed this method to localize grafting sites of single neurospheres in precise biopsies which can further be examined in explant cultures. The isolated perfused porcine rectum allowed us to continuously monitor the perfusion pressure. A blockage of too many capillaries would lead to an ischaemic situation and an increase of perfusion pressure. Since we could demonstrate that the area-wide delivery of neurospheres did not alter the overall vascular resistance, we showed that the delivery does not significantly impair the local circulation.

Organoid generation from pluripotent stem cells is a cutting-edge technique that has created new possibilities for modelling human organs in vitro, as well as opening avenues for regenerative medicine. Here, we present a protocol for generating skin organoids (SKOs) from human-induced pluripotent stem cells (hiPSCs) via direct embryoid body formation. This method provides a consistent start point for hiPSC differentiation, resulting in SKOs with complex skin architecture and appendages (e.g. hair follicles, sebaceous glands, etc.) across hiPSC lines from two different somatic sources.

Age-related lens opacification (cataract) remains the leading cause of visual impairment and blindness worldwide. In low- and middle-income countries, utilization of cataract surgical services is often limited despite community-based outreach programmes. Community-led research, whereby researchers and community members collaboratively co-design intervention is an approach that ensures the interventions are locally relevant and that their implementation is feasible and socially accepted in the targeted contexts. Community-led interventions have the potential to increase cataract surgery uptake if done appropriately. In this study, once the intervention is co-designed it will be implemented through a cluster-randomized controlled trial (cRCT) with ward as a unit of randomization. This study will utilise both the qualitative methods for co-designing the intervention and the quantitative methods for effective assessment of the developed community-led intervention through a cRCT in 80 rural wards of Dodoma region, Tanzania (40 Intervention). The 'intervention package' will be developed through participatory community meetings and ongoing evaluation and modification of the intervention based on its impact on service utilization. Leask's four stages of intervention co-creation will guide the development within Rifkin's CHOICE framework. The primary outcomes are two: the number of patients attending eye disease screening camps, and the number of patients accepting cataract surgery. NVivo version 12 will be used for qualitative data analysis and Stata version 12 for quantitative data. Independent and paired t-tests will be performed to make comparisons between and within groups. P-values less than 0.05 will be considered statistically significant.