2 research outputs found
Monitoring biological functions of cultured tissues using microdialysis
Continuous monitoring during tissue culture is important for the success of engineered tissue
development. It is also challenging due to lack of suitable established monitoring techniques. In
this study, microdialysis, a sampling technique for measuring the unbound solute concentrations
in the tissues and organs of the living body, was adopted to monitor functional tissue growth in a
bioreactor with explanted bovine caudal intervertebral discs (IVD) as the test tissue.
Apart from cell metabolic activities, cell and tissue biological functions were
investigated for the development of microdialysis for monitoring purposes. Methodologies of
microdialysis with large pore size membrane probes for sampling macromolecular
bio-functional markers were established. The effects of pumping methods, including 'push',
'pull' or 'push-and-pull', and the effect of the resulting transmembrane pressure on the fluid
balance, and the relative recovery of small molecules and of macromolecules (proteins) were
experimentally studied. The validity of the internal reference in-situ calibration was examined
in detail. It was concluded that a push-and-pull system was the only effective method to
eliminate fluid loss or gain. The relative recovery of small solutes was hardly affected by the
applied pumping methods; however the relative recovery of macromolecules was
significantly influenced by them. The in situ calibration technique using Phenol Red can
provide reliable results for small molecules including glucose and lactic acid. Using lOkDa
and 70kDa fluorescent dextrans as the internal standard for in situ calibration of large
molecules of similar size, it was found that the pull pump system did not work well but that
the push-and-pull pumping method did work well.
A novel bioreactor system for in vitro IVD culture with static load and microdialysis
monitoring was developed. Explanted IVDs were cultured under three different loads for up
to 7 days. A single microdialysis probe with 3000 kDa membrane was inserted into each
of the IVDs at a defined location. The in situ calibration technique was proved valid in
the experiments and membrane fouling was not significant. The tissue metabolism and
extracellular matrix turnover during 7 day culture were continuously monitored to
investigate the effect of different loads. Microdialysis proved to be a feasible and efficient
method for multi-parameter monitoring of tissue culture.
Substantial effort was directed towards the identification of functional macromolecular markers
in conjunction with microdialysis sampling. Amongst several proteins sampled, chitinase-3-like
protein 1 (CHI3L1), a major soluble protein secreted by cultured IVD cells in alginate beads and by
cultured IVD explants was identified following its successful isolation. Then it was established as a
suitable functional marker. The effect of physico-chemical and mechanical stimuli (e.g. osmolarity,
pH, oxygen tension and mechanical load) on secretion of CHI3L1 by cultured IVD cells and
chondrocytes in alginate beads and by cultured IVD explant were investigated. CHI3L1 release was
sensitive to physico-chemical stimulation. The production of CHI3L1 was directly correlated with the
cell metabolism and this could be readily monitored with microdialysis.</p
Pseudorapidity densities of charged particles with transverse momentum thresholds in pp collisions at √ s = 5.02 and 13 TeV
The pseudorapidity density of charged particles with minimum transverse momentum (pT) thresholds of 0.15, 0.5, 1, and
2 GeV/c is measured in pp collisions at the center of mass energies of √s=5.02 and 13 TeV with the ALICE detector. The study is carried out for inelastic collisions with at least one primary charged particle having a pseudorapidity (η) within 0.8pT larger than the corresponding threshold. In addition, measurements without pT-thresholds are performed for inelastic and nonsingle-diffractive events as well as for inelastic events with at least one charged particle having
|η|2GeV/c), highlighting the importance of such measurements for tuning event generators. The new measurements agree within uncertainties with results from the ATLAS and CMS experiments obtained at √s=13TeV.