A versatile platform for three-dimensional dynamic suspension culture applications

In the last decades, the rapid upgrading in cell biological knowledge has bumped the interest in using cell-based therapeutic approaches as well as cell-based model systems for the treatment of diseases. Given the rapid translation towards cell-based clinical treatments and the consequent increasing demand of cell sources, three-dimensional (3D) suspension cultures have demonstrated to be an advantageous alternative to monolayer techniques for large scale expansion of cells and for the generation of three-dimensional model systems in a scale-up perspective. In this scenario, a versatile bioreactor platform suitable for 3D dynamic suspension cell culture under tuneable shear stress conditions is developed and preliminarily tested in two different biotechnological applications. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits a laminar hydrodynamics, enabling dynamic cell suspension in an environment favourable to mass transport. Technically, the bioreactor is conceived to produce dynamic suspension cell culture under tuneable shear stress conditions without the use of moving components (from ultralow to moderate shear stress). A multiphysics computational modelling strategy is applied for the development and optimization of the suspension bioreactor platform. The in silico modelling is used to support the design and optimization phase of the bioreactor platform, providing a comprehensive analysis of its operating principles, also supporting the development/optimization of culture protocols directly in silico, and thus minimizing preliminary laboratory tests. After the technical assessment of the functionality of the device and a massive number of in silico simulations for its characterization, the bioreactor platform has been employed for two preliminary experimental applications, in order to determine the suitability of the device for culturing human cells under dynamic suspension. In detail, the bioreactor platform has been used to culture lung cancer cells for spheroid formation (Calu-3 cell line) under ultralow shear stress conditions, and for human induced pluripotent stem cell (hiPSC) dynamic suspension culture. The use of the bioreactor platform for the formation of cancer cell spheroids under low shear stress conditions confirms the suitability of the device for its use as dynamic suspension bioreactor. In fact, compared to static cell suspension, after 5 days of dynamic suspension culture the bioreactor platform preserves morphological features, promotes intercellular connection, increases the number of cycling cells, and reduces double strand DNA damage. Calu-3 cells form functional 3D spheroids characterized by more functional adherence junctions between cells. Moreover, the computational model has been used as a tool for assisting the setup of the experimental framework with the extraction of the fluid dynamic features establishing inside the bioreactor culture chamber. As second proof of concept application, the bioreactor platform has been tested for the dynamic suspension of hiPSCs. Starting from the ‘a priori’ knowledge gained by the development of the in silico culture protocol, the agglomeration of human induced pluripotent stem cells has been modulated by means of the combination of moderate intermittent shear stress and free-fall transport within the bioreactor culture chamber. The inoculation of single cells suspensions inside the bioreactor chamber promotes cell-cell interaction and consequently the formation of human induced pluripotent stem cell aggregates. In conclusion, the impeller-free functioning principle characterizing the proposed bioreactor platform demonstrates to be promising for human cell dynamic suspension culture. In the future, this bioreactor platform will be further optimized for the realization of impeller-free dynamic suspension bioreactors dedicated and optimized to specific applications in stem cell and cancer cell culture

Culture-Derived Human Platelets for Clinical Transfusion

Platelets are a vital component of human blood due to their role in clotting. Despite the incredible importance of platelets for human survival, there is very little supply of platelets to meet the clinical demand. Currently, platelets are only supplied from donations, whether through whole blood or apheresis. Therefore, there is a need for ex vivo means of producing platelets at a price that is competitive with the current market price. This project seeks to meet 65% of the demand for platelets in Philadelphia by producing platelets ex vivo in a process that utilizes shear stress to induce platelet formation from megakaryocytes. To begin this batch process, induced pluripotent stem cells derived from bone marrow are proliferated over the course of 10 days. During a 20-day differentiation phase that occurs in 2 parallel 2000 L stirred single-use bioreactors, a number of growth factors are supplied to the stem cell culture to induce differentiation of the cells into megakaryocytes. The megakaryocytes, once mature, are exposed to shear stress in a specially developed microfluidic device, inspired by the work done in Dr. Daeyeon Lee’s lab, to induce platelet formation. Once platelets have been generated, they are processed over the course of 5 days to be separate, washed, and concentrated in a resuspension solution via a series of centrifugation and mixing steps, after which they will be packaged into platelet units and transported to surrounding hospitals for clinical transfusion. This process design results a yearly production of 69,550 300 mL platelet units each year, with a platelet count of 5.5x1010 platelets in each unit. For this process, the IRR is 28.99%, the NPV is 14.4 million in the year 2030, and ROI is 23.21%

Numerical study of fluid flow, mass transfer and cell growth in a three-dimensional bioreactor for bone marrow culture

Imperial Users onl

Bioreactor-Based Bone Tissue Engineering

The aim of this chapter is to describe the main issues of bone tissue engineering. Bone transplants are widely used in orthopedic, plastic and reconstructive surgery. Current technologies like autologous and allogenic transplantation have several disadvantages making them relatively unsatisfactory, like donor site morbidity, chronic pain, and immunogenicity and risk hazard from infectious disease. Therefore, regenerative orthopedics seeks to establish a successful protocol for the healing of severe bone damage using engineered bone grafts. The optimization of protocols for bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries. We discuss mesenchymal stem cells as the most suitable cell type for bone tissue engineering. They can be isolated from a variety of mesenchymal tissues and can differentiate into osteoblasts when given appropriate mechanical support and osteoinductive signal. Mechanical support can be provided by different cell scaffolds based on natural or synthetic biomaterials, as well as combined composite materials. Three-dimensional support is enabled by bioreactor systems providing several advantages as mechanical loading, homogeneous distribution of cells and adequate nutrients/waste exchange. We also discuss the variety of osteoinductive signals that can be applied in bone tissue engineering. The near future of bone healing and regeneration is closely related to advances in tissue engineering. The optimization of protocols of bone graft production using autologous mesenchymal stem cells loaded on appropriate scaffolds, exposed to osteogenic inducers and mechanical force in bioreactor, should be able to solve the current limitations in managing bone injuries

Introducing monitoring and automation in cartilage tissue engineering, toward controlled clinical translation

The clinical application of tissue engineered products requires to be tightly connected with the possibility to control the process, assess graft quality and define suitable release criteria for implantation. The aim of this work is to establish techniques to standardize and control the in vitro engineering of cartilage grafts. The work is organized in three sub-projects: first a method to predict cell proliferation capacity was studied, then an in line technique to monitor the draft during in vitro culture was developed and, finally, a culture system for the reproducible production of engineered cartilage was designed and validated. Real-time measurements of human chondrocyte heat production during in vitro proliferation. Isothermal microcalorimetry (IMC) is an on-line, non-destructive and high resolution technique. In this project we aimed to verify the possibility to apply IMC to monitor the metabolic activity of primary human articular chondrocytes (HAC) during their in vitro proliferation. Indeed, currently, many clinically available cell therapy products for the repair of cartilage lesions involve a process of in vitro cell expansion. Establishing a model system able to predict the efficiency of this lengthy, labor-intensive, and challenging to standardize step could have a great potential impact on the manufacturing process. In this study an optimized experimental set up was first established, to reproducible acquire heat flow data; then it was demonstrated that the HAC proliferation within the IMC-based model was similar to proliferation under standard culture conditions, verifying its relevance for simulating the typical cell culture application. Finally, based on the results from 12 independent donors, the possible predictive potential of this technique was assessed. Online monitoring of oxygen as a non-destructive method to quantify cells in engineered 3D tissue constructs. This project aimed at assessing a technique to monitor graft quality during production and/or at release. A quantitative method to monitor the cells number in a 3D construct, based on the on-line measurement of the oxygen consumption in a perfusion based bioreactor system was developed. Oxygen levels dissolved in the medium were monitored on line, by two chemo-optic flow-through micro-oxygen sensors connected at the inlet and the outlet of the bioreactor scaffold chamber. A destructive DNA assay served to quantify the number of cells at the end of the culture. Thus the oxygen consumption per cell could be calculated as the oxygen drop across the perfused constructs at the end of the culture period and the number of cells quantified by DNA. The method developed would allow to non-invasively monitoring in real time the number of chondrocytes on the scaffold. Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing. The aim of this project was to upscale the size of engineered human cartilage grafts. The main aim of this project consisted in the design and prototyping of a direct perfusion bioreactor system, based on fluidodynamic models (realized in collaboration with the Institute for Bioengineering of Catalonia, Spain), able to guarantee homogeneous seeding and culture conditions trough the entire scaffold surface. The system was then validated and the capability to reproducibly support the process of tissue development was tested by histological, biochemical and biomechanical assays. Within the same project the automation of the designed scaled up bioreactor system, thought as a stand alone system, was proposed. A prototype was realized in collaboration with Applikon Biotechnology BV, The Netherlands. The developed system allows to achieve within a closed environment both cell seeding and culture, controlling some important environmental parameters (e.g. temperature, CO2 and O2 tension), coordinating the medium flow and tracking culture development. The system represents a relevant step toward process automation in tissue engineering and, as previously discussed, enhancing the automation level is an important requirement in order to move towards standardized protocols of graft generation for the clinical practice. These techniques will be critical towards a controlled and standardized procedure for clinical implementation of tissue engineering products and will provide the basis for controlled in vitro studies on cartilage development. Indeed the resulting methods have already been integrated in a streamlined, controlled, bioreactor based protocol for the in vitro production of up scaled engineered cartilage drafts. Moreover the techniques described will serve as the foundation for a recently approved Collaborative Project funded by the European Union, having the goal to produce cartilage tissue grafts. In order to reach this goal the research based technologies and processes described in this dissertation will be adapted for GMP compliance and conformance to regulatory guidelines for the production of engineered tissues for clinical use, which will be tested in a clinical trial

Aerospace medicine and biology: A continuing bibliography with indexes (supplement 335)

This bibliography lists 143 reports, articles and other documents introduced into the NASA Scientific and Technical Information System during March, 1990. Subject coverage includes: aerospace medicine and psychology, life support systems and controlled environments, safety equipment, exobiology and extraterrestrial life, and flight crew behavior and performance

Expansion of human mesenchymal stem/stromal cells (hMSCs) in bioreactors using microcarriers: lessons learnt and what the future holds

Human mesenchymal stem/stromal cells (hMSCs) present a key therapeutic cellular intervention for use in cell and gene therapy (CGT) applications due to their immunomodulatory properties and multi-differentiation capability. Some of the indications where hMSCs have demonstrated pre-clinical or clinical efficacy to improve outcomes are cartilage repair, acute myocardial infarction, graft versus host disease, Crohn’s disease and arthritis. The current engineering challenge is to produce hMSCs at an affordable price and at a commercially-relevant scale whilst minimising process variability and manual, human operations. By employing bioreactors and microcarriers (due to the adherent nature of hMSCs), it is expected that production costs would decrease due to improved process monitoring and control leading to better consistency and process efficiency, and enabling economies of scale. This approach will result in off the shelf (allogeneic) hMSC-based products becoming more accessible and affordable. Importantly, cell quality, including potency, must be maintained during the bioreactor manufacturing process. This review aims to examine the various factors to be considered when developing a hMSC manufacturing process using microcarriers and bioreactors and their potential impact on the final product. As concluding remarks, gaps in the current literature and potential future areas of research are also discussed

Cells in Space

Discussions and presentations addressed three aspects of cell research in space: the suitability of the cell as a subject in microgravity experiments, the requirements for generic flight hardware to support cell research, and the potential for collaboration between academia, industry, and government to develop these studies in space. Synopses are given for the presentations and follow-on discussions at the conference and papers are presented from which the presentations were based. An Executive Summary outlines the recommendations and conclusions generated at the conference