Identification of optimal flow rate for culture media, cell density, and oxygen toward maximization of virus production in a fed-batch baculovirus-insect cell system.

In recent times, it has been realized that novel vaccines are required to combat emerging disease outbreaks, and faster optimization is required to respond to global vaccine demands. Although, fed-batch operations offer better productivity, experiment-based optimization of a new fed-batch process remains expensive and time-consuming. In this context, we propose a novel computational framework that can be used for process optimization and control of a fed-batch baculovirus-insect cell system. Since the baculovirus expression vector system (BEVS) is known to be widely used platforms for recombinant protein/vaccine production, we chose this system to demonstrate the identification of optimal profile. Toward this, first, we constructed a mathematical model that captures the time course of cell and virus growth in a baculovirus-insect cell system. Second, the proposed model was used for numerical analysis to determine the optimal operating profiles of control variables such as culture media, cell density, and oxygen based on a multiobjective optimal control formulation. Third, a detailed comparison between batch and fed-batch culture was perfromed along with a comparison between various alternatives of fed-batch operation. Finally, we demonstrate that a model-based quantification of controlled feed addition in fed-batch culture is capable of providing better productivity as compared to a batch culture. The proposed framework can be utilized for the estimation of optimal operating regions of different control variables to achieve maximum infected cell density and virus yield while minimizing the substrate/media, uninfected cell, and oxygen consumption.

Nonviral Platform for Expression of Recombinant Protein in Insect Cells.

Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free methods for protein expression require fewer steps for obtaining protein expression by eliminating virus amplification and measuring the infectivity of the virus. The nonviral method uses a nonlytic plasmid to transfect the gene of interest into the insect cells instead of using baculovirus, a lytic system. In this chapter, we describe one of the transfection methods, which uses polyethyleneimine (PEI) as a DNA delivery material into the insect cells to express the recombinant protein in both adherent and suspension cells.

Tissue Oxygenation Measurements to Aid Scalpel Debridement Removal in Patients With Diabetes.

BACKGROUND: Callus formation in the diabetic foot increases the risk of ulcer onset. It is standard procedure to remove these dead tissue layers to reduce rising pressures. In a surgical procedure known as scalpel debridement, or chiropody the callus tissue is removed up to the epidermal layer. Factors may influence the outcome of this surgical process such as clinician inexperience. In an effort to standardize the debridement process, tissue oxygenation (TO) measurements are obtained before and after to study the effect of debridement on callus tissue. METHODS: Fifteen debridement cases were analyzed using near infrared (NIR) imaging to study changes in TO. The NIR-based device used in this study estimates effective changes in TO in terms of oxy-, deoxy-, total hemoglobin, and oxygen saturation. Weber contrasts between callus tissue and the surrounding normal tissue were compared following debridement for all TO parameters. In a secondary analysis, callus tissue was segmented into quadrants and a percent of significance (in terms of total TO change) was calculated using a t-test. RESULTS: Results show majority of cases displayed greater than 80% as the significant change in TO following debridement, except in cases with the presence of blood clot (a common precursor for ulceration). In cases where incomplete debridement was suspected, a significant change in TO was still observed. CONCLUSIONS: With extensive systematic studies in the future, NIR imaging technique to measure changes in TO may be implemented as a low-cost hand-held imaging device useful for objectively assessing the effectiveness of the scalpel debridement process.

Breath-Hold Paradigm to Assess Variations in Oxygen Flow in Diabetic Foot Ulcers Using a Noncontact Near-Infrared Optical Scanner.

Objective: Diabetic foot ulcers (DFUs) occur in almost 25% of all patients with diabetes in their lifetime, with oxygen being the key limiting factor in healing. Identifying regions of compromised oxygenated flow can help clinicians cater the wound treatment process, possibly reducing wound healing time. Herein, a handheld, noncontact near-infrared optical scanner (NIROS) was developed and used to measure temporal changes in hemoglobin concentrations in response to a breath-hold (BH) paradigm. Approach: Noncontact imaging studies were carried out on DFU subjects and control subjects in response to a 20-s BH paradigm. Continuous-wave-based multiwavelength diffused reflective signals were acquired to generate effective oxy-hemoglobin, deoxy-hemoglobin, total hemoglobin, and oxygen saturation concentration maps using modified Beer-Lambert's law. Pearson's correlation analysis was carried out to determine variations in oxygen flow from hemoglobin concentration maps and the extent of variation observed in controls versus DFU subjects. Results: Temporal changes in hemoglobin concentration maps were observed in controls and DFU subjects. However, the oxygen flow in response to BH varied within 10% in all controls but significantly varied between wound and background regions in subjects with DFUs. Innovation: A method to assess variations in oxygen supply in and around DFUs was demonstrated using NIROS. This approach has potential to better cater DFU treatment process. Conclusion: Changes in all hemoglobin parameters due to 20 s of BH was observed. Pearson's analysis indicates that oxy-hemoglobin, deoxy-hemoglobin, and oxygen saturation fluctuations are synchronous in controls. In DFUs, changes are asynchronous with blood flow between the wound region and background region being significantly different.