A novel acoustic cell processing platform for cell concentration and washing

Acoustofluidics involves the interaction of ultrasonic standing waves with particle suspension flows. The field has seen considerable growth in the last decade, particularly for diagnostic MEMS scale applications but also in biological applications. Typically, an ultrasonic standing wave is generated across a fluid flow by a piezo-electric transducer and an opposite acoustic reflector. The scattering of the ultrasound field by the suspended particle results in an acoustic radiation force acting on the suspended particle. The strength of the acoustic radiation force is a function of fluid and particle density and compressibility and particle size. The dynamics of the particle are then controlled by a number of forces, such as the fluid drag force, gravity/buoyancy force, acoustic radiation force, and inter-particle forces. FloDesign Sonics has developed a novel acoustic cell processing platform based on multi-dimensional standing waves. The platform has broad applications in biopharmaceutical, e.g., cell clarification, continuous manufacturing, and cell processing within cellular therapy applications, e.g., cell concentration and wash, cell culturing, and microcarrier/cell separation. In fed batch cell clarification, e.g., CHO cells for mAb production, the multi-dimensional standing wave is designed to trap the cells in the acoustic field. The three-dimensional acoustic radiation forces cause the trapped cells to form tightly packed cylindrical shaped clusters of cells, which continuously settle out due to enhanced gravitational separation. This technology is single use, continuous, and scalable. A small scale clarification product operating at 4 L/hr was launched in April of 2016. Scaling of the technology has been successfully shown with larger units operating at flow rates of 10 and 50 L/hr, providing cell clarification efficiencies of 90% across a wide range of feed stream cell densities up to 80 M cells/ml. The same platform technology has been modified to enable a single use (gamma irradiated) continuous cell concentration and wash application for manufacturing of cell based therapies. The device has been designed to be able to process several liters of a suspended cell culture, e.g., T-cells, at concentrations of 1 to 10 M cells/ml. The cell suspension flows through the device. The acoustic radiation force field is used to trap and hold the cells in the acoustic field. After concentrating the cells, one or multiple washing steps are accomplished by flowing the washing fluid through the device, using the acoustic field to trap the cells while displacing the original cell culture fluid. The holdup volume of the device is about 30 ml. Depending on cell concentration and initial volume of the cell suspension, measured cell recoveries of 90% have been achieved with concentration factors of 20 to 50 for Jurkat T-cell suspensions. Scaling strategies used previously for cell clarification will be used to scale up the current cell concentration device to accommodate large volumes

Volume reduction, cell washing and affinity cell selection using multi-dimensional acoustic standing wave technology

Acoustic Cell Processing is a unique acousto-fluidics platform technology for shear-free manipulation of cells using ultrasonic standing waves. The platform has broad applications in the field of cell and gene therapy, e.g., cell concentration and washing, cell culturing, microcarrier/cell separation, acoustic affinity cell selection and label-free cell selection. The acoustic radiation force exerted by the ultrasonic standing wave on the suspended cells in combination with fluid drag forces and gravitational forces is used to manipulate the cells and achieve a certain cell processing unit operation, e.g., separate, concentrate, or wash. The technology is single-use, continuous, and can be scaled up, down or out. It therefore allows for a flexible and modular approach that can be customized to process a desired cell count, cell culture volume or cell concentration within a given required process time. Utilizing its proprietary multi-dimensional standing wave platform, FloDesign Sonics (FD Sonics) has been developing two applications for cell and gene therapy manufacturing, an Acoustic Concentrate-Wash (ACW) and Acoustic Affinity Cell Selection (AACS) system for closed and shear free Cell and Gene Therapy manufacturing, namely CAR-T immunocellular therapies. The ACW technology has been applied to Jurkat T-cells and primary cultures of T-cells of 1-2 Liters (L) with cell concentrations ranging from 1 million cells per milliliter (ml) to 40 million cells per ml. The process flow rate varies from 2-3 L/hour with average cell recoveries of more than 80% in 60 to 90 minutes. The efficiency of the cell washing process ranges from 95-99% depletion of a model protein (BSA), depending on the wash methodology. The AACS technology is a scalable acoustic affinity cell selection method using acoustic (non-paramagnetic) affinity beads for positive or negative cell selection. A multi-dimensional acoustic standing wave is then used to separate the affinity bead-cell complexes from the unbound cells, thereby completing the process of a negative or positive cell selection. A population of 1 billion CAR-T cells containing 30% T-Cell Receptor positive (TCR+) and 70% T-cell Receptor Negative (TCR-) cells has been depleted of 99% of its TCR+ population. The TCR- cell recovery for this process was above 70% and the full process took less than 2 hours. When used for positive selection of CD3+ cells, AACS allowed for an enrichment of 2.5-fold in CD3+ population. ACW and AACS are powerful acoustic-based cell processing technologies that lower cost and risk while enabling a modular, automation-friendly manufacturing process for cell and gene therapy manufacturing. Please click Additional Files below to see the full abstract

Comparability: manufacturing, characterization and controls, report of a UK Regenerative Medicine Platform Pluripotent Stem Cell Platform Workshop, Trinity Hall, Cambridge, 14–15 September 2015

This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this ‘may be difficult for cell-based medicinal products’. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates

Dimerization of CPAP Orchestrates Centrosome Cohesion Plasticity*

Centrosome cohesion and segregation are accurately regulated to prevent an aberrant separation of duplicated centrosomes and to ensure the correct formation of bipolar spindles by a tight coupling with cell cycle machinery. CPAP is a centrosome protein with five coiled-coil domains and plays an important role in the control of brain size in autosomal recessive primary microcephaly. Previous studies showed that CPAP interacts with tubulin and controls centriole length. Here, we reported that CPAP forms a homodimer during interphase, and the fifth coiled-coil domain of CPAP is required for its dimerization. Moreover, this self-interaction is required for maintaining centrosome cohesion and preventing the centrosome from splitting before the G2/M phase. Our biochemical studies show that CPAP forms homodimers in vivo. In addition, both monomeric and dimeric CPAP are required for accurate cell division, suggesting that the temporal dynamics of CPAP homodimerization is tightly regulated during the cell cycle. Significantly, our results provide evidence that CPAP is phosphorylated during mitosis, and this phosphorylation releases its intermolecular interaction. Taken together, these results suggest that cell cycle-regulated phosphorylation orchestrates the dynamics of CPAP molecular interaction and centrosome splitting to ensure genomic stability in cell division