On Two Models of the Light Pulse Delay in a Saturable Absorber

A comparative analysis of two approaches to description of the light modulation pulse delay in a saturable absorber is presented. According to the simplest model, the delay of the optical pulse is a result of distortion of its shape due to absorption self-modulation in the nonlinear medium. The second model of the effect, proposed at the beginning of our century, connects the pulse delay with the so-called "slow light" resulting from the group velocity reduction under conditions of the coherent population oscillations. It is shown that all the known experimental data on the light pulse delay in saturable absorbers can be comprehensively described in the framework of the simplest model of saturable absorber and do not require invoking the effect of coherent population oscillations with spectral hole-burning and anomalous modifications of the light group velocity. It is concluded that the effect of group velocity reduction under conditions of coherent population oscillations has not received so far any experimental confirmation, and the assertions about real observation of the "slow light" based on this mechanism are groundless.Comment: Regretfully, the journal version of the paper (in Optics and Spectroscopy) appeared to be strongly corrupted due to ignorant editing. In particular, "coherent population oscillations" (CPO) was replaced by "population coherent oscillations" (PCO), "bleaching" - by "clearing", and "bleachable absorber " - by "clearable absorber". Here we present original version of the pape

Liquidus Tracking: Controlled Rate Vitrification for the Cryopreservation of Larger Volumes and Tissues

BACKGROUND: Vitrification of cells or tissue at controlled cooling rates suitable for larger volumes, and with reduced cryoprotectant toxicity. OBJECTIVE: To set out the current understanding of the LiquidusTracking (LT) vitrification technique, and to discuss the challenges and benefits of translating the method into laboratory protocols more generally applicable to meet requirements of large volume and 3-D cryo-banking in the era of regenerative medicine. METHODS: By adding small amounts of cryoprotectants at each step and subsequently cooling the sample just above its freezing point before further increasing CPA concentration, cryoprotectant toxicity is minimized. RESULT: CPA toxicity can be reduced by lowering the temperature. Different manual approaches to LT were evaluated and further improved. CONCLUSIONS: Manual liquidus tracking is complicated and exhibits potential high variability. Nevertheless, this approach offers the possibility of testing several conditions simultaneously and could be used to pre-test conditions prior to automatic LT development

Decisional tool for cost of goods analysis of bioartificial liver devices for routine clinical use

BACKGROUND AIMS: Bioartificial liver devices (BALs) are categorized as advanced therapy medicinal products (ATMPs) with the potential to provide temporary liver support for liver failure patients. However, to meet commercial demands, next-generation BAL manufacturing processes need to be designed that are scalable and financially feasible. The authors describe the development and application of a process economics decisional tool to determine the cost of goods (COG) of alternative BAL process flowsheets across a range of industrial scales. METHODS: The decisional tool comprised an information database linked to a process economics engine, with equipment sizing, resource consumption, capital investment and COG calculations for the whole bioprocess, from cell expansion and encapsulation to fluidized bed bioreactor (FBB) culture to cryopreservation and cryorecovery. Four different flowsheet configurations were evaluated across demands, with cell factories or microcarriers in suspension culture for the cell expansion step and single-use or stainless steel technology for the FBB culture step. RESULTS: The tool outputs demonstrated that the lowest COG was achieved with microcarriers and stainless steel technology independent of the annual demand (1500-30 000 BALs/year). The analysis identified the key cost drivers were parameters impacting the medium volume and cost. CONCLUSIONS: The tool outputs can be used to identify cost-effective and scalable bioprocesses early in the development process and minimize the risk of failing to meet commercial demands due to technology choices. The tool predictions serve as a useful benchmark for manufacturing ATMPs

Liquidus Tracking: Large scale preservation of encapsulated 3-D cell cultures using a vitrification machine

Currently, cryo-banking of multicellular structures such as organoids, especially in large volumes at clinical scale >1 L, remains elusive for reasons such as insufficient dehydration and cryoprotectant additive (CPA(1)) penetration, slow cooling and warming rates and devitrification processes. Here we introduce the concept of Liquidus Tracking (LT) using a semi-automated process for liquid volumes of up to 450 ml including 130 ml of alginate encapsulated liver cells (AELC) that archived controlled and reversible vitrification with minimized toxicity. First a CPA solution with optimal properties for LT was developed by employing different small scale test systems. Combining sugars such as glucose and raffinose with Me2SO improved post-exposure (at +0.5 °C) viabilities from 6% ±3.6 for Me2SO alone up to 58% ±6.1 and 65% ±14.2 respectively (p < 0.01). Other permeating CPAs (e.g. ethylene glycol, propylene glycol, methanol) were investigated as partial replacements for Me2SO. A mixture of Me2SO, ethylene glycol and glucose (ratio 4:2:1- termed LTdeg) supported glass-forming tendencies with appropriate low viscosities and toxicities required for LT. When running the full LT process, using Me2SO alone, no viable cells were recovered; using LTdeg, viable recoveries were improved to 40% ±8 (p<0.001%). Further refinements of improved mixing technique further improved recovery after LT. Recoveries of specific liver cell functions such as synthesis of albumin and alpha-fetoprotein (AFP) were retained in post thaw cultures. In summary: By developing a low-toxicity CPA solution of low viscosity (LTdeg) suitable for LT and by improving the stirring system, post-warming viability of AELC of up to 90% and a AFP secretion of 89% were reached. Results show that it may be possible to develop LT as a suitable cryogenic preservation process for different cell therapy products at large scale

Utilisation of chimeric lyssaviruses to assess vaccine protection against highly divergent lyssaviruses

Lyssaviruses constitute a diverse range of viruses with the ability to cause fatal encephalitis known as rabies. Existing human rabies vaccines and post exposure prophylaxes (PEP) are based on inactivated preparations of, and neutralising antibody preparations directed against, classical rabies viruses, respectively. Whilst these prophylaxes are highly efficient at neutralising and preventing a productive infection with rabies virus, their ability to neutralise other lyssaviruses is thought to be limited. The remaining 15 virus species within the lyssavirus genus have been divided into at least three phylogroups that generally predict vaccine protection. Existing rabies vaccines afford protection against phylogroup I viruses but offer little to no protection against phylogroup II and III viruses. As such, work involving sharps with phylogroup II and III must be considered of high risk as no PEP is thought to have any effect on the prevention of a productive infection with these lyssaviruses. Whilst rabies virus itself has been characterised in a number of different animal models, data on the remaining lyssaviruses are scarce. As the lyssavirus glycoprotein is considered to be the sole target of neutralising antibodies we generated a vaccine strain of rabies using reverse genetics expressing highly divergent glycoproteins of West Caucasian Bat lyssavirus and Ikoma lyssavirus. Using these recombinants, we propose that recombinant vaccine strain derived lyssaviruses containing heterologous glycoproteins may be a suitable surrogate for wildtype viruses when assessing vaccine protection for the lyssaviruses

Applications and optimization of cryopreservation technologies to cellular therapeutics

Delivery of cell therapies often requires the ability to hold products in readiness whilst logistical, regulatory and potency considerations are dealt with and recorded. This requires reversibly stopping biological time, a process which is often achieved by cryopreservation. However, cryopreservation itself poses many biological and biophysical challenges to living cells that need to be understood in order to apply the low temperature technologies to their best advantage. This review sets out the history of applied cryopreservation, our current understanding of the various processes involved in storage at cryogenic temperatures, and challenges for robust and reliable uses of cryopreservation within the cell therapy arena

Impact of Storage at -80°C on Encapsulated Liver Spheroids After Liquid Nitrogen Storage

For many bioengineered tissues to have practical clinical application, cryopreservation for use on demand is essential. This study examined different thermal histories on warming and short holding periods at different subzero temperatures on subsequent functional recoveries of alginate encapsulated liver spheroids (ELS) for use in a bioartificial liver device. This mimicked transport at liquid nitrogen (-196°C) or dry ice (∼-80°C) temperatures. Holding at -80°C on warming after -196°C storage resulted in ELS expressing significant (p < 0.001) damage compared with direct thaw from liquid nitrogen, with viable cell number falling from 74.0 ± 8.4 million viable cells/mL without -80°C storage to 1.9 ± 0.6 million viable cells/mL 72 h post-thaw after 8 days storage at -80°C. Even 1 day at -80°C after -196°C storage resulted in lower viability (down 21% 24 h post-thaw), viable cell count (down 29% 24 h post-thaw), glucose, and alpha-1-fetoprotein production (reduced by 59% and 95% 24 h from 1 day post-thaw, respectively). Storage at -80°C was determined to be harmful only during the warming cycle. Chemical measurements of the alginate component of ELS were unchanged by cryogenic exposure in either condition

Optical studies of strain and defect distribution in semipolar (1(1)over-bar01) GaN on patterned Si substrates

Formation of defects in semipolar ( 11¯01 )-oriented GaN layers grown by metal-organic chemical vapor deposition on patterned Si (001) substrates and their effects on optical properties were investigated by steady-state and time-resolved photoluminescence (PL) and spectrally and spatially resolved cathodoluminescence (CL). Near-band edge emission is found to be dominant in the c +-wings of semipolar ( 11¯01 )GaN, which are mainly free from defect-related emission lines, while the c – wings contain a large number of basal stacking faults. When the advancing c+ and c — fronts meet to coalesce into a continuous film, the existing stacking faults contained in c — wings continue to propagate in the direction perpendicular to the c-axis and, as a result, the region dominated by stacking fault emission is extended to the film surface.Additional stacking faults are observed within the c+ wings, where the growing c+ wings of GaN are in contact with the SiO2 masking layer. Out-diffusion of oxygen/silicon species and concentration of strain near the contact region are considered as possible causes of the stacking fault formation. CL linescans performed along the surface and across the thickness of the non-coalesced and coalesced layers revealed that, while most of the material in the near-surface region of the non-coalesced layers is relaxed, coalescence results in nonuniform strain distribution over the layer surface. Red-shifted near-band-edge emission from the near-surface region indicates tensile stress near the surface of a coalesced layer, reaching a value of 0.3 GPa. The regions near the GaN/AlN/Si(111) interface show slightly blue shifted, broadened near-band-edge emission, which is indicative of a high concentration of free carriers possibly due to incorporation of shallow-donor impurities (Si and/or O) from the substrate or SiO2 mask. Steady-state and time-resolved PL results indicate that semipolar ( 11¯01 )GaN on patterned Si exhibits optical properties (PL intensity and carrier lifetimes) approaching to those of the state-of-the-art c-plane GaN grown using in situ SiNx nanonetwork mask on c-plane sapphire. Long PL lifetimes (∼2 ns) for the ( 11¯01 )GaN layers show that the semipolar material holds promise for light emitting and detecting devices