Ross Odell

Analysis of growth kinetics by division tracking

Cell division tracking using fluorescent dyes, such as carboxyfluorescein diacetate succinimidyl ester, provides a unique opportunity for analysis of cell growth kinetics. The present review article presents new methods for enhancing resolution of division tracking data as well as derivation of quantities that characterize growth from time‐series data. These include the average time between successive divisions, the proportion of cells that survive and the proliferation per division. The physical significance of these measured quantities is interpreted by formulation of a two‐compartment model of cell cycle transit characterized by stochastic and deterministic cell residence times, respectively. The model confirmed that survival is directly related to the proportion of cells that enter the next cell generation. The proportion of time that cells reside in the stochastic compartment is directly related to the proliferation per generation. This form of analysis provides a starting point for more sophisticated physical and biochemical models of cell cycle regulation.

Flux enhancement in crossflow microfiltration using a collapsible-tube pulsation generator

Abstract A dilute suspension of silica particles in water was microfiltered in an unbaffled tubular ceramic membrane. The crossflow was varied periodically at frequencies inducing fluid-dynamic unsteadiness using a novel device for this application, namely a flexible tube compressed to non-circular cross-section by external pressure. This arrangement is unstable and leads to self-sustained oscillations which can be used to add a pulsatile component to a steady flow. With this arrangement, and a time-averaged crossflow sufficient for turbulence in the membrane filter, filtrate flux increases due to pulsation of up to 60% were demonstrated, without systematic optimisation of the pulse parameters. The gain persisted over the entire range of mean transmembrane pressure and crossflow velocity investigated.

Comparison between the clonogenic, MTT, and SRB assays for determining radiosensitivity in a panel of human bladder cancer cell lines and a ureteral cell line

Using a series of human bladder cancer cell lines and an immortalised normal ureteral cell line, radiosensitivities measured by three different methods after a single dose of X-radiation are compared. Clear differences between cell survival curves obtained using the clonogenic, microtetrazoline (MTT) and sulforhodamine B (SRB) assays are shown. The most sensitive of the assays investigated was the clonogenic assay. The MTT and SRB assays were found to be relatively insensitive especially at lower radiation levels, suggesting that these assays may not be suitable for predicting therapeutic dose schedules in vivo, but will be important for investigating radio-sensitivity in cell lines with very low plating efficiencies. Each assay discriminated between a range of sensitivities in the cell lines examined, and with some minor differences, the ordering of sensitivities using the three assays was similar. Possible explanations for the differences between results obtained with the three assays are discussed.

Zirconium: biomedical and nephrological applications.

Recent years have witnessed a rapid increase in the use of zirconium (Zr)-containing compounds in artificial internal organs. Examples include dental implants and other restorative practices, total knee and hip replacement, and middle-ear ossicular chain reconstruction. In nephrological practice, Zr-containing sorbents have been used in hemofiltration, hemodialysis, peritoneal dialysis, and in the design and construction of wearable artificial kidneys. Zr compounds continue to be widely and extensively used in deodorant and antiperspirant preparations. In the public health arena, Zr compounds have been studied or used in controlling phosphorus pollution and in the reclamation of poison and bacteria-contaminated water. Experimental and clinical studies support the general consensus that Zr compounds are biocompatible and exhibit low toxicity. Reports on possible Zr-associated adverse reactions are rare and, in general, have not rigorously established a cause-and-effect relationship. Although publications on the use of Zr compounds have continued to increase in recent years, reports on Zr toxicity have virtually disappeared from the medical literature. Nevertheless, familiarity with, and continued vigilant monitoring of, the use of these compounds are warranted. This article provides an updated review on the biomedical use of Zr compounds.

Analysis of cell differentiation by division tracking cytometry

We propose a quantitative method to characterize growth and differentiation dynamics of multipotent cells from time series carboxyfluorescein diacetate, succinimidyl ester (CFDA‐SE) division tracking data. The dynamics of cell proliferation and differentiation was measured by combining (CFDA‐SE) division tracking with phenotypic analysis. We define division tracking population statistics such as precursor cell frequency, generation time and renewal rate that characterize growth of various phenotypes in a heterogeneous culture system. This method is illustrated by study of the divisional recruitment of cord blood CD34+ cells by hematopoietic growth factors. The technical issue of assigning the correct generation number to cells was addressed by employing high‐resolution division tracking methodology and daily histogram analysis. We also quantified division‐tracking artifacts such as CFDA‐SE degeneration and cellular auto‐fluorescence. Mitotic activation of cord blood CD34+ cells by cytokines commenced after 2 days of cytokine stimulation. Mean generation number increased linearly thereafter, and it was conclusively shown that CD34+ cells cycle slower than CD34− cells. Generation times for CD34+ and CD34− cells were 24.7 ± 0.8 h and 15.1 ± 0.9 h (±SD, n = 5), respectively. The 20‐fold increase in CD34+ cell numbers at Day 6 could be attributed to a high CD34+ cell renewal rate (91% ± 2% per division). Although cultures were initiated with highly purified CD34+ cells (∼96%), CD34− numbers had expanded rapidly by Day 6. This rapid expansion could be explained by their short generation time as well as a small fraction of CD34+ cells (∼5%) that differentiated into CD34− cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics.

Network structure and macromolecular drug release from poly(vinyl alcohol) hydrogels fabricated via two crosslinking strategies

Injectable hydrogels have potential biomedical applications ranging from tissue fillers to drug delivery vehicles. This study focussed on evaluating the structure of poly(vinyl alcohol) (PVA) hydrogels of variable solid content and high molecular weight model drug release from the networks formed via either conventional photo-polymerization compared with chemical initiation of polymerization using an oxidation-reduction (redox) reaction. Swelling behaviour was characterised in water to assess the structural properties. Model drugs, FITC-Dextran, 20 kDa (FD20) and 4 kDa (FD4) were loaded in the hydrogels prior to curing and drug release studies conducted. Redox-cured hydrogels were more swollen than UV-cured systems, lost approximately 20% of their polymer mass compared to only 5% from UV-cured hydrogels and subsequently exhibited networks of larger mesh sizes. Also, networks of variable solid contents showed different structural properties with systems of higher polymer concentration exhibiting a smaller mesh size. The difference in structural properties of the networks affected release of FD20, being faster in redox-cured than UV-cured hydrogels, and slower from systems of higher solid content. Release of FD4 was faster than FD20 from networks of same solid content. This study suggested that PVA hydrogels can be cured by redox-initiation to function as a controlled delivery system for macromolecular drugs.

Multi-type branching models to describe cell differentiation programs.

Cell proliferation and differentiation is described by a multi-type branching process, a probability model that defines the inheritance of cell type. Cell type is defined by (i) a repression index related to the time required for S-phase entry and (ii) phenotype as determined by cell markers and division history. The inheritance of cell type is expressed as the expected number and type of progeny cells produced by a mother cell given her type. Expressions for the expected number and type of cells produced by a multi-cellular (bulk culture) system are derived from the general model by making the simplifying assumption that cell generation times are independent. The multi-type Smith-Martin model (MSM) makes the further assumption that cell generation times are lag-exponentially distributed with phenotype transitions occurring just before entry into S-phase. The inheritance-modified MSM (IMSM) model includes the influence of generation time memory so that mother and daughter generation times are correlated. The expansion of human cord blood CD34(+) cells by haematopoietic growth factors was division tracked in bulk culture using carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE). The MSM model was fitted to division tracking data to identify cell cycle length, and the rates of CD34 antigen down-regulation and apoptosis. The IMSM model was estimated for mouse granulocyte-macrophage progenitors using live cell imaging data. Multi-type branching models describe cell differentiation dynamics at both single- and multi-cell scales, providing a new paradigm for systematic analysis of stem and progenitor cell development.

Acute cellular interaction with textured surfaces in blood contact

Textured blood-contacting surfaces can promote the formation of a blood-compatible pseudo-neointima. We hypothesized that by controlling the surface texturing, the pseudo-neointima thickness could be controlled. The hypothesis was tested experimentally by fabricating the polyurethane textured surfaces with three different fiber lengths, and exposing them simultaneously to the flowing blood in an ovine ex vivo carotid-jugular series shunt for periods up to 4 h. The textured surface consisted of regularly spaced tapered micro-fibers of defined length on a smooth base-plane surface. Because of the simple surface topography, detailed computational fluid-dynamic modeling of the surface could be obtained as a parallel study. Experimental results showed that white cell was the predominant cell type deposited on the textured surfaces, whereas macroscopic thrombus formation occurred only in one of nine blood-contacting experiments. White cell density on the textured base-plane surface was subsequently quantified by image-analyzing the electron micrographs of blood-contacted textured surfaces. The statistical analysis of cell densities on individual textured surfaces showed effects of wall shear stress on the textured base plane (which was obtained from the fluid-dynamic modeling), the longitudinal position of the test section in the series shunt, and blood-contact time.

β2-microglobulin kinetics in an ovine model

The kinetics of 125I-beta 2 microglobulin (beta 2M) and 51Cr-EDTA were measured in normal and nephrectomized sheep. In normal sheep the beta 2M clearance was similar to that of EDTA (i.e., glomerular filtration rate), as were the distribution volumes. In anephric sheep the beta 2M clearance was 7.7 +/- 1.4 ml/min.

Quantifying prosthetic gait deviation using simple outcome measures

AIM To develop a subset of simple outcome measures to quantify prosthetic gait deviation without needing three-dimensional gait analysis (3DGA). METHODS Eight unilateral, transfemoral amputees and 12 unilateral, transtibial amputees were recruited. Twenty-eight able-bodied controls were recruited. All participants underwent 3DGA, the timed-up-and-go test and the six-minute walk test (6MWT). The lower-limb amputees also completed the Prosthesis Evaluation Questionnaire. Results from 3DGA were summarised using the gait deviation index (GDI), which was subsequently regressed, using stepwise regression, against the other measures. RESULTS Step-length (SL), self-selected walking speed (SSWS) and the distance walked during the 6MWT (6MWD) were significantly correlated with GDI. The 6MWD was the strongest, single predictor of the GDI, followed by SL and SSWS. The predictive ability of the regression equations were improved following inclusion of self-report data related to mobility and prosthetic utility. CONCLUSION This study offers a practicable alternative to quantifying kinematic deviation without the need to conduct complete 3DGA.