Micro Theater

Strategies for harnessing the immune cells or stem cells to combat cancer and autoimmune disorders using cell therapy represent a promising approach in the field of personalized medicine.  In this context, many efforts are emerging for developing and engineering more potent T cell, Stem cell and NK cell–based cell and gene therapies. 
Commonly employed cell purification platforms for large-scale enrichment and isolation of immune cells or stem cells for cell therapy applications, separate cells based on a single surface antigen. Enrichment based on multiple cell surface markers or different expression levels of these markers often requires the use of a traditional cell sorter. 
Here we describe the use of the Sony CGX10 Cell Isolation System, a fully closed system suitable for GMP-compliant cell sorting. The CGX10 Cell Isolation System utilizes a novel microfluidics technology to achieve high purity cell isolation within a closed and sterile microfluidics chip.

PRESENTERS:
Aditi Singh, PhD, Sony Biotechnology Inc., USA

Optimal culture conditions increase cell expansion, so bioreactors are a logical step in upstream cell and gene therapy production. Media exchange and cell separation are two critical operations performed routinely on a repeated and regular schedule to maintain favorable culture conditions in small scale cultures. Through use of automation tools and purpose-built reactor components these two operations can be successfully automated in bioreactors.  These tools will directly improve product yields and quality and will also free personnel to perform other critical tasks. In this presentation we will introduce the concepts, techniques, and the components of highly automated and efficient systems to perform media exchange and cell separation in stirred tank bioreactors used for cell and gene therapy applications.

PRESENTERS:
George Barringer, PhD, Getinge Applikon Bioreactors, USA

The iPSC Neurodegenerative Disease Initiative (iNDI), led by the National Institutes of Health (NIH) and started in mid-2020, is the largest-ever iPSC genome engineering project attempted. The goal of the iNDI project is to generate a standardized set of disease models for over 100 single nucleotide variant (SNV) mutations associated with Alzheimer’s disease and related dementias (ADRD) in isogenic iPSC lines. The iNDI cell lines and related phenotypic data sets will be broadly shared by the NIH. Synthego, a genome engineering company, was selected as a partner to generate a significant number of the required iPSC lines. Utilizing state of the art CRISPR knock-in methods (editing design, optimization, zygosity control, analysis) and automation (cell transfection and clonal isolation), we generated clonal lines for 23 of the target mutations in the KOLF2.1 iPSC line. In less than 6 months spanning late 2020 to early 2021, at least 3 clonal homozygous and 6 clonal heterozygous mutation lines were generated for a total of 264 clonal lines (>8,000 vials of cells produced). Here we describe the use of our automated, high throughput CRISPR editing platform, ECLIPSE, for rapidly generating these isogenic iPSC lines as part of a valuable contribution to the iNDI project.

PRESENTERS:
Kevin Holden, PhD, Synthego, USA

The CellRaft technology developed by Cell Microsystems allows for streamlined and efficient cell line development by leveraging the combined power of our CytoSort Array tissue culture consumable with our automated isolation and imaging instrument, the CellRaft AIR® System.  The AIR System offers an automated solution to an otherwise labor-intensive workflow and supports cell health, time-course imaging for clonal verification, and automated isolation for downstream propagation, altogether providing an alternative that is more time, labor, and cost efficient for cell line development.  We have extensively demonstrated that the CellRaft AIR System can accelerate workflows for both 2D and 3D cell culture using a variety of cell types, including sensitive cells such as embryonic and induced pluripotent stem cells.  

PRESENTERS:
Jessica, Hartman, PhD, Cell Microsystems, USA

KEY LEARNINGS:
1. How Catalent is developing an HLA-homozygous (HLA-h) universal cell bank from cord blood units accounting for key regulatory aspects such as donor eligibility and consent, GMP manufacturing and commercial use consent.
2. Our characterization criteria of the banks to ensure high genomic integrity, effectiveness of reprogramming and unlimited self-renewal.
3. Our initiatives to develop advanced GMP-compliant protocols are suitable for differentiating iPSCs into various cell types of medical interest including retinal pigment epithelium, mesenchymal stromal cells, natural killer cells and more.

PRESENTERS:
Boris Greber, Catalent Cell & Gene Therpay, USA

Human primary cells secrete extracellular matrices in vitro that retain properties of the tissue of origin. These tissue-specific ECMs promote maturation of iPSC-derived somatic cells in 2D culture. At StemBioSys we have developed processes for reproducibly manufacturing matrices capable of promoting rapid and spontaneous maturation of iPSC-derived cardiomyocytes, neurons, hepatocytes, and beta cells in high-throughput. Moreover, iPSCs from patients with inherited diseases exhibit the disease phenotype in 2D culture. Thus, this technology has the potential to dramatically improve the utility of iPSCs in preclinical drug testing applications for efficacy or toxicity screening.

PRESENTERS:
Travis Block, PhD, StemBioSys, Inc., USA

Physiologically relevant 3D cell models are essential for pre-clinical research and drug discovery. 3D cell cultures enable better representation of the in vivo conditions such as tumor microenvironment. 3D cell models offer the possibility to understand cancer progression better functionally as compared to 2D cultures. In this session, we present an advancement in biomarker staining and detection – the automated immunofluorescence staining of patient-derived 3D cell models using our proprietary microfluidic Pu·MA System combined with high resolution confocal imaging. The Pu·MA System can perform tedious protocols such as supernatant sampling, dose responses and IF staining with minimum perturbation to the samples. The Pu·MA System with proprietary microfluidic flowchips, performs “hands-off” automated fluid transfers at the touch of a screen. Using this system, we studied the expression of epithelial-to-mesenchymal transition and stem cell markers in patient derived breast cancer models. Here we used cells from primary TNBC tumors, that were grown into 3D tumoroids. Two patient-derived models with different metastatic potential were evaluated for the expression of different biomarkers, in response to different drug treatments, along with viability response. These methods using the Pu·MA System demonstrate applications such as tumor drug-sensitivities to an in-depth analysis of organoids and 3D cell models.

PRESENTERS:
Evan F. Cromwell, PhD, Protein Fluidics, Inc., USA
Katya Nikolov, MD, Protein Fluidics, Inc., USA

Using a novel DLP based bioprinting method, we developed a patient specific biomimetic model for spinal cord injury repair. The model effectively captured the spinal cord anatomy and mechanical property leading to effective neural stem cell delivery and maturation in vivo at the spinal cord lesion site. Given the microscale printing resolution of our bioprinter, linear microchannels were readily fabricated in the biomimetic implant to align and guide regenerating axons for functional recovery. The bioinks used for printing the model were highly biocompatible with tunable degradation rate which significantly reduced foreign body reaction to the implant and further improved functional outcomes. The bioprinting platform and methodology developed here can be readily extended to other stem cell delivery and therapeutic applications as well as disease modeling purposes.

PRESENTERS:
Wei Zhu, PhD, Allegro 3D, USA 

Routine cell therapies for solid organs such as the heart, pancreas, liver or brain will require large cell quantities estimated at 10^8 to 10^9 cells per patient. Robust, efficient, and economically viable systems are required to produce such amounts of cells in a reproducible manner. Stirred-tank bioreactors have emerged as promising cultivation systems, which facilitate close control of critical process parameters and have proven their value for efficient process scaling. In three-dimensional (3D) aggregate-based hPSC suspension cultivation, cells, nutrients, and gases are distributed homogenously. Controlling the aggregate size is a key factor specific to hPSC bioprocessing, because large aggregates might reach physical and physiological limits. Innovations in impeller designs, such as the 8-blade impeller, improve the cultivation of stem cells as aggregates or on microcarriers in the DASbox® Mini Bioreactor System. The use of this impeller supported the formation and growth of stem cell aggregates in stirred-tank bioreactors. 
The presentation demonstrates the advantages of process monitoring and control with the DASbox Mini Bioreactor System. By using the 8-blade impeller and precisely monitoring and controlling critical process parameters, the process strategy yielded a 70-fold expansion of the cells in seven days, achieving an unmatched cell density of 35 X 10^6 cells/mL.

PRESENTERS:
Philipp Nold, PhD, Eppendorf SE, Germany

Join us to learn about the importance of using genetically diverse stem cell models to promote the development of inclusive regenerative medicine technologies. Together, we will discuss how the stem cell community can increase the genetic diversity of cell lines and disease models so that the resulting scientific discoveries can be used to develop targeted cures and improve health among genetically diverse groups.