Simulating microgravity using a random positioning machine for inducing cellular responses to mechanotransduction in human osteoblasts.

The mechanotransduction pathways that mediate cellular responses to contact forces are better understood than those that mediate response to distance forces, especially the force of gravity. Removing or reducing gravity for significant periods of time involves either sending samples to space, inducing diamagnetic levitation with high magnetic fields, or continually reorienting samples for a period, all in a manner that supports cell culturing. Undesired secondary effects due to high magnetic fields or shear forces associated with fluid flow while reorienting must be considered in the design of ground-based devices. We have developed a lab-friendly and compact random positioning machine (RPM) that fits in a standard tissue culture incubator. Using a two-axis gimbal, it continually reorients samples in a manner that produces an equal likelihood that all possible orientations are visited. We contribute a new control algorithm by which the distribution of probabilities over all possible orientations is completely uniform. Rather than randomly varying gimbal axis speed and/or direction as in previous algorithms (which produces non-uniform probability distributions of orientation), we use inverse kinematics to follow a trajectory with a probability distribution of orientations that is uniform by construction. Over a time period of 6 h of operation using our RPM, the average gravity is within 0.001 23% of the gravity of Earth. Shear forces are minimized by limiting the angular speed of both gimbal motors to under 42 °/s. We demonstrate the utility of our RPM by investigating the effects of simulated microgravity on adherent human osteoblasts immediately after retrieving samples from our RPM. Cytoskeletal disruption and cell shape changes were observed relative to samples cultured in a 1 g environment. We also found that subjecting human osteoblasts in suspension to simulated microgravity resulted in less filamentous actin and lower cell stiffness.

Towards a bioengineered uterus: bioactive sheep uterus scaffolds are effectively recellularized by enzymatic preconditioning.

Uterine factor infertility was considered incurable until recently when we reported the first successful live birth after uterus transplantation. However, risky donor surgery and immunosuppressive therapy are factors that may be avoided with bioengineering. For example, transplanted recellularized constructs derived from decellularized tissue restored fertility in rodent models and mandate translational studies. In this study, we decellularized whole sheep uterus with three different protocols using 0.5% sodium dodecyl sulfate, 2% sodium deoxycholate (SDC) or 2% SDC, and 1% Triton X-100. Scaffolds were then assessed for bioactivity using the dorsal root ganglion and chorioallantoic membrane assays, and we found that all the uterus scaffolds exhibited growth factor activity that promoted neurogenesis and angiogenesis. Extensive recellularization optimization was conducted using multipotent sheep fetal stem cells and we report results from the following three in vitro conditions; (a) standard cell culturing conditions, (b) constructs cultured in transwells, and (c) scaffolds preconditioned with matrix metalloproteinase 2 and 9. The recellularization efficiency was improved short-term when transwells were used compared with standard culturing conditions. However, the recellularization efficiency in scaffolds preconditioned with matrix metalloproteinases was 200-300% better than the other strategies evaluated herein, independent of decellularization protocol. Hence, a major recellularization hurdle has been overcome with the improved recellularization strategies and in vitro platforms described herein. These results are an important milestone and should facilitate the production of large bioengineered grafts suitable for future in vivo applications in the sheep, which is an essential step before considering these principles in a clinical setting.

Protocols for Rat Uterus Isolation and Decellularization: Applications for Uterus Tissue Engineering and 3D Cell Culturing.

Sophisticated culturing conditions are required to grow cells in a three-dimensional (3D) environment. Cells then require a type of scaffold rich in proteins, growth factors, and signaling molecules that simulates their natural environment. Tissues from all species of animals have an organ-specific extracellular matrix (ECM) structure that plays a key role in cell proliferation and migration. Hence, the scaffold composition plays a significant role for any successful 3D cell culturing system. We developed a whole rat uterus ECM scaffold by the perfusion of detergents and ionic solutions through the vascular system of an isolated normal rat uterus in a process termed "decellularization." The generated rat uterus scaffolds consist of a cell-free ECM structure similar to that of the normal rat uterus, and are thus excellent platforms on to which new cells can be added. Rat uterus 3D cell culturing systems based on these scaffolds could become valuable to decidual differentiation- and embryo implantation studies, or for investigating invasion mechanisms of endometrial cancer cells. They could also be used for the creation of tissue engineered uterine tissue, for partial or whole organogenesis developed for transplantation applications to treat absolute uterine infertility. This is a condition affecting about 1 in 500 women, and is only treatable by a uterus transplantation. This article provides valuable troubleshooting notes and describes in detail how to generate rat uterus scaffolds, including the delicate surgery required to isolate the uterus with an intact vascular tree which facilitates vascular perfusion and re-transplantation.