Optimal molecular profiling of tissue and tissue components

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.     

Soft tissue vibrations within one soft tissue compartment

The concept of muscle tuning suggests that vibrations of the soft tissue compartments of the leg initiated by impacts are minimized by muscular activity prior to heel-strike of heel-toe running. For the quantification of muscle tuning it has been assumed (1) that the soft tissue compartment acts as one lumped mass and (2) that vibration energy dissipation does occur within one muscle. The purpose of this study was to test these two assumptions. It was hypothesized that (H1) the movement of the soft tissue compartment is not homogeneous, (H2) the vibration frequencies for different muscles within one soft tissue compartment are different and (3) attenuation of vibration movement within one muscle does occur. Soft tissue vibrations were measured using accelerometers on four locations on the quadriceps soft tissue compartment during heel-toe running. There were differences in the peak soft tissue acceleration and time of peak acceleration between accelerometer locations. The dominant frequency was similar throughout the soft tissue compartment, however; there was an attenuation of high-frequency vibration energy between distal and proximal points overlying one muscle. This evidence suggests that accelerometer placement is important when quantifying the acceleration magnitude and timing of peak soft tissue compartment but not when estimating the resonant vibration characteristics of a soft tissue compartment. It also provides initial evidence to support the idea that vibration control through muscle tuning may be achieved through changes in energy dissipating properties within the soft tissue compartment.     

Synthetic tissue biology: tissue engineering meets synthetic biology

We propose the term "synthetic tissue biology" to describe the use of engineered tissues to form biological systems with metazoan-like complexity. The increasing maturity of tissue engineering is beginning to render this goal attainable. As in other synthetic biology approaches, the perspective is bottom-up; here, the premise is that complex functional phenotypes (on par with those in whole metazoan organisms) can be effected by engineering biology at the tissue level. To be successful, current efforts to understand and engineer multicellular systems must continue, and new efforts to integrate different tissues into a coherent structure will need to emerge. The fruits of this research may include improved understanding of how tissue systems can be integrated, as well as useful biomedical technologies not traditionally considered in tissue engineering, such as autonomous devices, sensors, and manufacturing.     

Microgravity tissue engineering

Summary Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter × 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.     

Fixed archival tissue

The labeling of oligonucleotide probes using a fluorescein-labeled nucleotide is described. The reaction is characterized by careful control of the nucleotide and probe molar ratio in order to produce a tail that gives good detection sensitivity without compromising hybridization stringency control of the probe sequence. The labeling reaction can be easily monitored for incorporation of the fluorescent label and the probes can be used in many applications.     

Locating tissue collections in tissue economies--deriving value from biomedical research

This paper examines diverging notions of value in the use of tissue sample collections and other information resources using a case study of hereditary colorectal cancer research in Finland. Recent science and technology policies that emphasize the production of commercial value derived from tissue sample collections are challenged by varying conceptions of value, as well as structural factors that relate to the combination of different public population information systems in the Finnish research system. Such challenges reflect a tension in the economic aspirations of the ideology of the knowledge society in relation to the goals of national health care policies, as well as the role of the state as a mediator of knowledge production and commercial development.     

Ethical tissue: a not-for-profit model for human tissue supply

Following legislative changes in 2004 and the establishment of the Human Tissue Authority, access to human tissues for biomedical research became a more onerous and tightly regulated process. Ethical Tissue was established to meet the growing demand for human tissues, using a process that provided ease of access by researchers whilst maintaining the highest ethical and regulatory standards. The establishment of a licensed research tissue bank entailed several key criteria covering ethical, legal, financial and logistical issues being met. A wide range of stakeholders, including the HTA, University of Bradford, flagged LREC, hospital trusts and clinical groups were also integral to the process.     

A rapid connective tissue stain for glycol methacrylate embedded tissue

No reliable connective tissue stains for GMA sections were available until recently. However, the use of toluidine blue in combination with basic fuchsin appeared to be a rapid and reliable connective tissue stain for glycol methacrylate (GMA) embedded tissue.