Bioprocess optimization for cell‐based therapies is a resource heavy activity. To reduce the associated cost and time, process development may be carried out in small volume systems, with the caveat ...that such systems be predictive for process scale‐up. The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, kLa, has been identified as a critical parameter for predictive process scale‐up. Here, we describe the development of a 96‐well microplate with integrated Redbud Posts to provide mixing and enhanced kLa. Mixing in the microplate is characterized by observation of dyes and analyzed using the relative mixing index (RMI). The kLa is measured via dynamic gassing out method. Actuating Redbud Posts are shown to increase rate of planar homogeneity (2 min) verse diffusion alone (120 min) and increase oxygenation, with increasing stirrer speed (3500‐9000 rpm) and decreasing fill volume (150‐350 μL) leading to an increase in kLa (4‐88 h−1). Significant increase in Chinese Hamster Ovary growth in Redbud Labs vessel (580,000 cells mL‐1) versus the control (420,000 cells mL‐1); t(12.814) = 8.3678, p ≤ .001), and CD4+ Naïve cell growth in the microbioreactor indicates the potential for this technology in early stage bioprocess development and optimization.
In this research article, we describe a novel microbioreactor that is agitated via surface attached microposts and demonstrate increased oxygen transfer and mixing capabilities compared to standard static cultures. This microbioreactor is demonstrated to promote increased mammalian cell growth. Since the microreactor environment more closely monitors that of stirred tank reactors, there are potential applications in small‐scale process development.
Microscale fermentation systems are important high throughput tools in clone selection, and bioprocess set up and optimization, since they provide several parallel experiments in controlled ...conditions of pH, temperature, agitation, and gas flow rate. In this work we evaluated the performance of biotechnologically relevant strains with different respiratory requirements in the micro‐Matrix microbioreactor. In particular Escherichia coli K4 requires well aerated fermentation conditions to improve its native production of chondroitin‐like capsular polysaccharide, a biomedically attractive polymer. Results from batch and fed‐batch experiments demonstrated high reproducibility with those obtained on 2 L reactors, although highlighting a pronounced volume loss for longer‐term experiments. Basfia succiniciproducens and Actinobacillus succinogenes need CO2 addition for the production of succinic acid, a building block with several industrial applications. Different CO2 supply modes were tested for the two strains in 24 h batch experiments and results well compared with those obtained on lab‐scale bioreactors. Overall, it was demonstrated that the micro‐Matrix is a useful scale‐down tool that is suitable for growing metabolically different strains in simple batch process, however, a series of issues should still be addressed in order to fully exploit its potential.
Adoptive T‐cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing ...process remains a challenge. Here, a single‐use 24‐well microbioreactor (micro‐Matrix) was assessed for its use as a high‐throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion‐mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions. Agitation was shown to play a fundamental role in the proliferation of T cells. A shaking speed of 200 rpm drastically improved the final viable cell concentration (VCC), while the viability was maintained above 90% throughout the cultivation. VCCs reached a maximum of 9.22 × 106 cells/ml. The distribution of CD8+ central memory T cells (TCM), was found to be largely unaffected by the shaking speed. A clear interaction between pH and DO (p < .001) was established for the cell growth and the optimal culture conditions were identified for a combination of 200 rpm, 25% DO, and pH of 7.4. The combination of microbioreactor technology and Design of Experiment methodology provides a powerful tool to rapidly gain an understanding of the design space of the T‐cell manufacturing process.
Adoptive T‐cell therapy has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia, yet lack of process control and characterisation remain major challenges in the manufacturing process. Here, a single‐use 24‐well microbioreactor was used in conjunction with Design of Experiment methodology to investigate the effect of varying key process parameters on the growth and differentiation of primary T cells in a perfusion‐mimic process and an optimal parameter combination was identified within the investigated design space.
The advancement of microbioreactor technology in recent years has transformed early‐ and mid‐stage process development. The monitoring and control capabilities of microbioreactors not only promote ...the quick accumulation of process knowledge but has also led to an increased scalability when compared to traditionally used systems such as shake flasks and microtitre plates. This study seeks to establish a framework for the micro‐Matrix microbioreactor (Applikon‐Biotechnology BV) as process development tool. Using the Dual Indicator System for Mixing Time, the system was initially characterized for mixing properties at varying operating conditions, which was found to yield mixing times between 0.9 and 41.8 s. A matched mixing time was proposed as scale‐down criterion for an IgG4 producing GS‐CHO fed‐batch process between a 5 L stirred tank reactor (STR) and the micro‐Matrix microbioreactor. Growth trends, maximum viable cell concentrations, final titre, and glycoprofiles were nearly identical at both scales. The scale‐down model was then employed to optimize a bolus feeding regime using response surface methodology, which led to a 25.4% increase of the space‐time yield and a 25% increase of the final titre. The optimized feeding strategy was validated at the small‐scale and successfully scaled up to the 5 L STR. This work for the first time provides a framework of how the micro‐Matrix microbioreactor can be implemented in a bioprocess development workflow and demonstrates scalability of growth and production kinetics as well as IgG4 glycosylation between the micro‐Matrix and a benchtop‐scale STR system.
Graphical and Lay Summary
Microbioreactor technology has become an essential part of early‐ and mid‐stage bioprocess development. This study provides a framework of how the shaken micro‐Matrix system can be used for cell culture process development of a mammalian CHO cell line producing a monoclonal antibody. Following scale‐down from a 5 L stirred tank bioreactor, the micro‐Matrix was employed for the optimisation of a feeding strategy and the optimised protocol was successfully scaled up.
Advances in molecular biotechnology have resulted in the generation of numerous potential production strains. Because every strain can be screened under various process conditions, the number of ...potential cultivations is multiplied. Exploiting this potential without increasing the associated timelines requires a cultivation platform that offers increased throughput and flexibility to perform various bioprocess screening protocols. Currently, there is no commercially available fully automated cultivation platform that can operate multiple microbial fed‐batch processes, including at‐line sampling, deep freezer off‐line sample storage, and complete data handling. To enable scalable high‐throughput early‐stage microbial bioprocess development, a commercially available microbioreactor system and a laboratory robot are combined to develop a fully automated cultivation platform. By making numerous modifications, as well as supplementation with custom‐built hardware and software, fully automated milliliter‐scale microbial fed‐batch cultivation, sample handling, and data storage are realized. The initial results of cultivations with two different expression systems and three different process conditions are compared using 5 L scale benchmark cultivations, which provide identical rankings of expression systems and process conditions. Thus, fully automated high‐throughput cultivation, including automated centralized data storage to significantly accelerate the identification of the optimal expression systems and process conditions, offers the potential for automated early‐stage bioprocess development.
The work describes the development of a fully automated, high‐throughput microbial fermentation platform for early‐stage bioprocess development. It also includes the implementation of automatic sample handling and integration into a centralized data storage system, with early results demonstrating its potential as a robust and efficient scale‐down platform.
In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more ...interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time‐efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non‐invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed‐batch process is performed in large‐scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed‐batch mode. This workflow was validated against lab‐scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro‐scale fed‐batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab‐scale bioreactor.
Technical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery. Protein secretion can be achieved via general ...secretion (Sec) pathway. Specific sequences, signal peptides (SPs), are necessary to direct the target protein into the translocation machinery. For example, >150 Sec-specific SPs have been identified for Bacillus subtilis alone. As the best SP for a target protein of choice cannot be predicted a priori, screening of homologous SPs has been shown to be a powerful tool for different expression organisms. While SP libraries between closely related species were successfully applied to optimize recombinant protein secretion, this was not investigated for distantly related species. Therefore, in this study a Sec SP library from low-GC firmicutes B. subtilis is investigated to optimize protein secretion in high-GC actinobacterium Corynebacterium glutamicum using cutinase from Fusarium solani pisi as model protein.
A homologous SP library (~150 SP) for recombinant cutinase secretion in B. subtilis was successfully transferred to C. glutamicum as alternative secretion host. Cutinase secretion in C. glutamicum was quantified using an automated micro scale cultivation system for online growth monitoring, cell separation and cutinase activity determination. Secretion phenotyping results were correlated to those from a previous study, in which the same SP library was used to optimize secretion of the same cutinase but using B. subtilis as host. Strikingly, behavior of specific SP-cutinase combinations was changed dramatically between B. subtilis and C. glutamicum. Some SPs showed comparable cutinase secretion performances in both hosts, whereas other SPs caused diametrical extracellular cutinase activities.
The optimal production strain for a specific target protein of choice still cannot be designed in silico. Not only the best SP for a target protein has to be evaluated each time from scratch, the expression host also affects which SP is best. Thus, (heterologous) SP library screening using high-throughput methods is considered to be crucial to construct an optimal production strain for a target protein.
In this work we present advances in three dimensional (3D) neuronal cell culture systems based on a reversible assembly of a microbioreactor with a microelectrode array (MEA) to create a MEMS-based ...3D cell culture system for in vitro neuro-electrophysiological recordings. A batch of six molds were milled in poly (methyl methacrylate). The molds were used for soft lithography of polydimethylsiloxane (PDMS). In the center of the PDMS shape, a porous polyethersulfone (PES) cylindrical tube was press-fitted to form a growth barrier between the culture chamber inside the PES tube and the microfluidic channel surrounding the PES tube. A thin layer of partially cured PDMS was used to seal the bottom of the microbioreactor and provide reversible adhesion with the glass surface of a MEA. SH-SY5Y cells were successfully differentiated inside the microbioreactors in Matrigel and demonstrated extended neuronal networks over a height of at least 184 micrometers within the system. In previous microbioreactor designs visibility was limited due to the closed top with the dispensing holes. The new open top design allows for a better evaluation of the cell culture by optical detection methods during the experiment. Electrophysiological activity was recorded within the microbioreactor using human induced pluripotent stem cell-derived cortical neurons cultured in Matrigel, in 3D, up until 21 days in vitro. In summary, we present advances made in the design, the fabrication process and integration of microbioreactors with MEAs. Optical imaging capabilities improved significantly with an open top and the culture time was further extended from 7 to 21 DIV without leakage or degradation thanks to introducing PES as a barrier material and an enhanced assembly procedure. The latter facilitated a sufficient long-term culture for neurons to mature in an environment free from flow-induced stress and provided a proof of principle for the recording of electrophysiological activity of cortical neurons cultured in 3D.
Understanding how microenvironmental cues influence cellular behavior will enable development of efficient and robust pluripotent stem cell differentiation protocols. Unlike traditional cell culture ...dishes, microfluidic bioreactors can provide stable microenvironmental conditions by continuous medium perfusion at a controlled rate. The aim of this study is to investigate whether a microfluidic culture device could be used as a perfused platform for long‐term cell culture processes such as the retinal differentiation of human induced pluripotent stem cells. The perfusion flow rate is established based on the degradation and consumption of growth factors (DKK‐1, Noggin, IGF‐1, and bFGF) and utilizing the Péclet number. The device's performance analyzed by qRT‐PCR show improvements compared to the well‐plate control as characterized by significantly higher expression of the markers Pax6, Chx10, and Crx on Day 5, Nrl on day 10, Crx, and Rhodopsin on day 21. Optimization of perfusion rate is an important operating variable in development of robust processes for differentiation cultures. Result demonstrates convective delivery of nutrients via perfusion has a significant impact upon the expression of key retinal markers. This study is the first continuously perfused long‐term (21 days) retinal differentiation of hiPSCs in a microfluidic device.
Providing stable culture conditions is key in development of efficient and robust pluripotent stem cell differentiation protocols. Microfluidic cell culture devices can provide such conditions by continuous medium perfusion at a controlled rate. This study investigated delivery of the key nutrients to the pluripotent stem cells via perfusion using a microfluidic culture device (MFCD) as a tool and retinal differentiation as a case study. Results indicated significant improvements in expression of key retinal genes compared to the well‐plate control culture. Moreover, it showed optimization of perfusion rate is an important operating variable in development of robust differentiation cultures. This study represents a significant step forward toward the application of microfluidic technology to pluripotent stem cells in the fields of developmental biology, disease modelling, and regenerative medicine process development.
PDF
