Application of a simple unstructured kinetic and cost of goods models to support T-cell therapy manufacture

Manufacturing of cell therapy products requires sufficient understanding of the cell culture variables and associated mechanisms for adequate control and risk analysis. The aim of this study was to apply an unstructured ordinary differential equation-based model for prediction of T-cell bioprocess outcomes as a function of process input parameters. A series of models were developed to represent the growth of T-cells as a function of time, culture volumes, cell densities, and glucose concentration using data from the Ambr®15 stirred bioreactor system. The models were sufficiently representative of the process to predict the glucose and volume provision required to maintain cell growth rate and quantitatively defined the relationship between glucose concentration, cell growth rate, and glucose utilization rate. The models demonstrated that although glucose is a limiting factor in batch supplied medium, a delivery rate of glucose at significantly less than the maximal specific consumption rate (0.05 mg 1 × 10⁶ cell h⁻¹) will adequately sustain cell growth due to a lower glucose Monod constant determining glucose consumption rate relative to the glucose Monod constant determining cell growth rate. The resultant volume and exchange requirements were used as inputs to an operational BioSolve cost model to suggest a cost-effective T-cell manufacturing process with minimum cost of goods per million cells produced and optimal volumetric productivity in a manufacturing settings. These findings highlight the potential of a simple unstructured model of T-cell growth in a stirred tank system to provide a framework for control and optimization of bioprocesses for manufacture.     

Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications

This article describes the development history of packed-bed bioreactors (PBRs) used for the culture of mammalian cells. It further reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocess and biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities (>10(8) cells ml(-1)) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is their relatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-fold would be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselves as compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for a minimum of 1 to 2 months. Achieving this will greatly enhance the usefulness of PBR technology in clinical practice.     

Performance evaluation of a novel conceptual bioprocess for clinically-required mass production of hematopoietic cells

Objective

The novel engineered bioprocess, which was designed and modeled to provide the clinically relevant cell numbers for different therapies in our previous work (Kaleybar et al. Food Bioprod Process 122:254–268, https://doi.org/10.1016/j.fbp.2020.04.012, 2020), was evaluated by using U937 as hematopoietic model cells.

Results

The culture system showed a 30-fold expansion of U937 cells in one-step during a 10-day culture period. The cell growth profile, the substrate and oxygen consumptions, and byproduct formations were all in agreement with the model predications during 7 days. The cell proliferation decrease after 7 days was attributed to optional oxygen limiting condition in the last days of culture. The bioreactor culture system revealed also a slight enhancement of lactate dehydrogenase (LDH) production as compared to the 2D conventional culture system, indicating the low impact of shear stress on cellular damage in the dynamic system.

Conclusions

The results demonstrated that the conceptual bioprocess for suspended stem cell production has a great potential in practice although additional experiments are required to improve the system.

     

Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Retrovirus mediated gene transduction of human T-cell subsets

Purpose: Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukaemia. Graft versus host disease (GVHD) is a potentially life threatening complication of AlloBMT mediated by the T cells contained within the graft. In order to be able to control GVHD, the allogeneic T cells may be transduced with a suicide gene such as herpes simplex virus thymidine kinase (HSV-tk). For this strategy to be successful, all subsets of T cells should be transduced to a similar extent. Also, the transduction protocol should not induce expression of unwanted homing receptors, nor should it lead to unwanted skewing of the T-cell receptor repertoire. We have studied the transduction efficiency of naïve and memory subsets of CD4+ and CD8+ T cells, and examined the transduced T-cell subsets for possible changes in T-cell receptor (TCR) repertoire and homing receptor expression. Methods: The cells were transduced using a Moloney murine retroviral vector carrying a conjugate of the genes encoding the truncated form of the cell surface marker, low affinity nerve growth factor receptor (LNGFR) and HSV-tk. Transduction efficiency and homing receptor expression were quantified by flow cytometry. TCR repertoire was determined by spectratyping. Results: We obtained a transduction efficiency of 30–50% of the cells, with no difference between the T-cell subsets. Cell surface receptors responsible for homing to skin, gastrointestinal tract or lymph nodes were practically absent at the end of 2 weeks in culture. The activation procedure seemed to favour the expansion of certain T-cell clones over polyclonal populations. However, there was no difference in the TCR repertoire between transduced and non-transduced cells. Conclusion: Changes in the composition of the T-cell subsets at the end of the cell culture were the results of the activation, and not the suicide gene transduction. The transduced T cells did not express unwanted homing receptors.     

Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy

Background aimsCytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in pre-clinical models, and this efficacy has translated to success in several clinical trials. Many early trials were disappointing in large part because of the lack of proliferation and subsequent persistence of transferred cells. Recent investigations have pointed to the importance of delivering highly proliferative cells, whether of naive or early memory phenotypes.MethodsWe investigated the influence of two common cell culturing methods used in early trials and their relationship to T-cell phenotype and pre-clinical efficacy.ResultsWe observed that stimulation with soluble anti-CD3 antibody OKT-3 and high-dose interleukin-2 produces more effector memory-type T cells with shorter average telomeres when compared with cells generated with the use of CD3/CD28 beads. When used in xenograft models of leukemia, bead-stimulated cells proliferated earlier and to a higher degree than those generated with the use of OKT-3/IL2 and resulted in better disease control despite no difference in distribution or migration throughout the mouse. Inclusion of the known successful clinical 4-1BB endodomain in the CAR could not rescue the function of OKT-3/IL-2–cultured cells. T cells isolated from animals that survived long-term (>120 days) retained a central memory–like phenotype and demonstrated a memory response to a large re-challenge of CD19-positive leukemia.ConclusionsIn summary, we confirm that cells with a younger phenotype or higher proliferative capacity perform better in pre-clinical models and that cell culturing influences cell phenotype seemingly independent of the 4-1BB endodomain in the CAR structure.     

Establishing the scalable manufacture of primary human T-cells in an automated stirred-tank bioreactor

Advanced cell and gene therapies such as chimeric antigen receptor T-cell immunotherapies (CAR-T), present a novel therapeutic modality for the treatment of acute and chronic conditions including acute lymphoblastic leukemia and non-Hodgkin lymphoma. However, the development of such immunotherapies requires the manufacture of large numbers of T-cells, which remains a major translational and commercial bottleneck due to the manual, small-scale, and often static culturing systems used for their production. Such systems are used because there is an unsubstantiated concern that primary T-cells are shear sensitive, or prefer static conditions, and therefore do not grow as effectively in more scalable, agitated systems, such as stirred-tank bioreactors, as compared with T-flasks and culture bags. In this study, we demonstrate that not only T-cells can be cultivated in an automated stirred-tank bioreactor system (ambr® 250), but that their growth is consistently and significantly better than that in T-flask static culture, with equivalent cell quality. Moreover, we demonstrate that at progressively higher agitation rates over the range studied here, and thereby, higher specific power inputs (P/M W kg⁻¹), the higher the final viable T-cell density; that is, a cell density of 4.65 ± 0.24 × 10⁶ viable cells ml⁻¹ obtained at the highest P/M of 74 × 10⁻⁴ W kg⁻¹ in comparison with 0.91 ± 0.07 × 10⁶ viable cells ml⁻¹ at the lowest P/M of 3.1 × 10⁻⁴ W kg⁻¹. We posit that this improvement is due to the inability at the lower agitation rates to effectively suspend the Dynabeads®, which are required to activate the T-cells; and that contact between them is improved at the higher agitation rates. Importantly, from the data obtained, there is no indication that T-cells prefer being grown under static conditions or are sensitive to fluid dynamic stresses within a stirred-tank bioreactor system at the agitation speeds investigated. Indeed, the opposite has proven to be the case, whereby, the cells grow better under higher agitation speeds while maintaining their quality. This study is the first demonstration of primary T-cell ex vivo manufacture activated by Dynabeads® in an automated stirred-tank bioreactor system such as the ambr® 250 and the findings have the potential to be applied to multiple other cell candidates for advanced therapy applications.     

Apoptosis: A mammalian cell bioprocessing perspective

Apoptosis is a form of programmed and controlled cell death that accounts for the majority of cellular death in bioprocesses. Cell death affects culture longevity and product quality; it is instigated by several stresses experienced by the cells within a bioreactor. Understanding the factors that cause apoptosis as well as developing strategies that can protect cells is crucial for robust bioprocess development. This review aims to a) address apoptosis from a bioprocess perspective; b) describe the significant apoptotic mechanisms linking them to the most relevant stresses encountered in bioreactors; c) discuss the design of operating conditions in order to avoid cell death; d) focus on industrially relevant cell lines; and e) present anti-apoptosis strategies including cell engineering and model-based optimization of bioprocesses. In addition, the importance of apoptosis in quality-by-design bioprocess development from clone screening to production scale are highlighted.     

Bioprocess development for endospore production by Bacillus coagulans using an optimized chemically defined medium

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%–90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.     

Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns

Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi‐product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 10⁶ cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network. Biotechnol. Bioeng. 2013; 110: 1376–1385. © 2012 Wiley Periodicals, Inc.     

Generation of colon cancer–derived tumor-infiltrating T cells (TILs) for adoptive cell therapy

Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2–enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 10⁹ cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3⁺/CD8⁺ effector memory phenotype (CD45RO⁺/CCR7⁻). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay)     

Toward precision manufacturing of immunogene T-cell therapies

Cancer can be effectively targeted using a patient's own T cells equipped with synthetic receptors, including chimeric antigen receptors (CARs) that redirect and reprogram these lymphocytes to mediate tumor rejection. Over the past two decades, several strategies to manufacture genetically engineered T cells have been proposed, with the goal of generating optimally functional cellular products for adoptive transfer. Based on this work, protocols for manufacturing clinical-grade CAR T cells have been established, but these complex methods have been used to treat only a few hundred individuals. As CAR T-cell therapy progresses into later-phase clinical trials and becomes an option for more patients, a major consideration for academic institutions and industry is developing robust manufacturing processes that will permit scaling-out production of immunogene T-cell therapies in a reproducible and efficient manner. In this review, we will discuss the steps involved in cell processing, the major obstacles surrounding T-cell manufacturing platforms and the approaches for improving cellular product potency. Finally, we will address the challenges of expanding CAR T-cell therapy to a global patient population.     

Bioprocess parameters of cell growth and human μ opioid receptor expression in recombinant Drosophila S2 cell cultures in a bioreactor

The paper describes a recombinant Schneider 2 (rS2) cell culture and protein expression in a bioreactor. S2 cells were transfected with a plasmid containing a fusion protein (human μ opioid receptor, hMOR, and green fluorescent protein, EGFP) under the control of inducible metallothionein promoter. A bioprocess in a bioreactor with 5% dissolved oxygen, 27°C and 120 rpm enabled the cell culture to attain 5.3×10⁷ viable cells/mL at 96 h. The induction decreased the cell multiplication (2.5×10⁷ viable cells/mL at 72 h). Glutamine and glucose and low levels of lactate were consumed. A fast recombinant protein synthesis took place and, at 6 h of induction, 2×10⁴ receptors/cell could be detected by a functional binding assay. Fluorescence measurements showed a progressive increase of recombinant protein expression with a maximal value of 1.26×10⁵ fluo counts/s at 24 h of induction. The data shown in this paper indicate a practical and scaleable cell culture bioprocess procedure for the preparation of recombinant proteins expressed in S2 cells.     

Stem-like memory T cells are generated during hollow fiber perfusion-based expansion and enriched after cryopreservation in an automated modular cell therapy manufacturing process

Background aimsModular automation is a flexible and reliable option to build the foundation of a new or evolving process or to introduce automation to a process that is already established. Herein the authors demonstrate that modular automation provides both high-quality and high-yield T-cell products.MethodsCells from three individual donors collected on an automated continuous flow centrifugation system were successfully expanded in a functionally closed, automated, perfusion-based hollow fiber bioreactor. These cells were then prepared for cryopreservation in an automated closed-system device that maintains temperature and aliquots a mixed cell product and cryoprotectant into product bags. Cell product bags were thawed and expanded in flasks. Samples taken throughout this manufacturing process were analyzed for cell phenotype, exhaustion markers and functionality. The proportion of CD4+ and CD8+ T cells was maintained through each step, from pre-expansion and post-expansion to immediately after thaw and 24 h after thaw.ResultsInterestingly, phenotypic markers such as CD45RO, CD45RA and CCR7 evolved throughout the process and stem-like memory T cells emerged as the predominant phenotype in the clinically relevant 24-h post-thaw sample.ConclusionsModular automation supported the generation of stem-like memory T cells that were not terminally exhausted and were able to produce effector cytokines upon restimulation.     

Extracellular vesicles concentration is a promising and important parameter for industrial bioprocess monitoring

Extracellular vesicles (EVs) are membrane vesicles that are produced by cells to be released into their microenvironment. In this study, we present the EV concentration as a new factor for optimization of industrial bioprocess control. The release of EVs depends on many cell properties, including cell activation and stress status, and cell death. Therefore, the EV concentration might provide a readout for identification of the cell state and the conditions during a bioprocess. Our data show that the EV concentration increased during the bioprocess, which indicated deteriorating conditions in the bioreactor. This increase in EV concentration in the fermentation broth was the consequence of two different processes: cell activation, and cell death. However, the release of EVs from activated living cells had a much weaker impact on EV concentration in the bioreactor than those released during cell death. EVs and cells in the bioprocess environment were quantified by flow cytometry. The most accurate data were obtained directly from unprocessed samples, making the monitoring of the EV concentration a rapid, easy, and cheap method. These EV concentrations reflect the conditions in the bioreactor and provide new information regarding the state of the bioprocess. Therefore, we suggest EV concentration as a new and important parameter for the monitoring of industrial bioprocesses.     

Batch, fed-batch, and microcarrier cultures with CHO cell lines in a pressure-cycle driven miniaturized bioreactor

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage-dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed-batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 10(7) /mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed-batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale-up.     

Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control

It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 x 10(6) cells/mL after 9 days of differentiation. Approximately 30%-40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (alphaMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 x 10(9) cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications.     

Features of T-cell subset composition in a D-galactose-induced senescence mouse model

Long-term administration of D-galactose induces oxidative stress and accelerates normal age-related changes. Hence, the D-galactose-treated rodent model has been widely used for aging research. In this study, we examined the immunological characteristics, especially CD4⁺ T-cell subset composition, of D-galactose-induced aging model mice to evaluate the model’s utility in immunosenescence studies. The spleens of aging model mice subjected to repeated subcutaneous injections of D-galactose exhibited significant increases in T cells with the memory phenotype (CD62L^low CD44^high) and individual T-cell subsets (Th1, Th2, Th17 and T_reg). Furthermore, cells with the phenotype of T follicular helper (Tfh) cells were spontaneously increased. The features of T-cell subset composition in D-galactose-treated mice were in close agreement with those observed in normal aged mice and appeared to mimic the currently known normal aging processes associated with T-cell homeostasis. Our results suggest that D-galactose-induced aging models would be useful for immunosenescence studies focusing on T-cell homeostasis and give valuable insight into age-related immune system dysregulation.     

Profound protection against respiratory challenge with a lethal H7N7 influenza A virus by increasing the magnitude of CD8(+) T-cell memory

The recall of CD8(+) T-cell memory established by infecting H-2(b) mice with an H1N1 influenza A virus provided a measure of protection against an extremely virulent H7N7 virus. The numbers of CD8(+) effector and memory T cells specific for the shared, immunodominant D(b)NP(366) epitope were greatly increased subsequent to the H7N7 challenge, and though lung titers remained as high as those in naive controls for 5 days or more, the virus was cleared more rapidly. Expanding the CD8(+) memory T-cell pool (<0.5 to >10%) by sequential priming with two different influenza A viruses (H3N2-->H1N1) gave much better protection. Though the H7N7 virus initially grew to equivalent titers in the lungs of naive and double-primed mice, the replicative phase was substantially controlled within 3 days. This tertiary H7N7 challenge caused little increase in the magnitude of the CD8(+) D(b)NP(366)(+) T-cell pool, and only a portion of the memory population in the lymphoid tissue could be shown to proliferate. The great majority of the CD8(+) D(b)NP(366)(+) set that localized to the infected respiratory tract had, however, cycled at least once, though recent cell division was shown not to be a prerequisite for T-cell extravasation. The selective induction of CD8(+) T-cell memory can thus greatly limit the damage caused by a virulent influenza A virus, with the extent of protection being directly related to the number of available responders. Furthermore, a large pool of CD8(+) memory T cells may be only partially utilized to deal with a potentially lethal influenza infection.     

Computational modeling for the optimization of a cardiogenic 3D bioprocess of encapsulated embryonic stem cells

We present a computational fluid dynamics (CFD)-based model aimed at the identification of optimized culture conditions promoting efficient cardiogenesis of hydrogel-bead-encapsulated embryonic stem cells (ESCs) within a rotating bioreactor. The numerical approach, integrating diffusion, convection, and multiphase fluid dynamics calculations, allowed to evaluate (i) the microgravity motion of the floating beads, (ii) the O₂ delivery to the cells, also (iii) taking into account the cellular O₂ consumption, as a function of different rotation speeds of the breeding chamber. According to our results, a 25?rpm rotation (i) enhances an adequate mixing of the cell carriers, avoiding sedimentation and excessive packing, also maintaining a quite homogeneous distribution of the suspended beads and (ii) imparts a proper cellular O₂ supply, providing cells close to a normoxia condition. The bioreactor working conditions derived from the numerical analysis allowed the attainment of in vitro long-term cell viability maintenance, supporting efficient large-scale generation of ESC-derived cardiomyocytes (ESC-DCs) through a chemical-based conditioning bioprocess. In conclusion, we demonstrated the feasibility of using CFD-based tools, as a reliable and cost-effective strategy to assist the design of a 3D cardiogenic bioprocess.