Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells

Ma, Zhiyao; Toledo, Marcelo Augusto Szymanski de; Wanek, Paul; Mabrouk, Mohamed H Elsafi; Smet, Francis; Pulak, Rock; Pieske, Simon; Piotrowski, Tobias; Herfs, Werner; Brecher, Christian; Schmitt, Robert; Wagner, Wolfgang; Zenke, Martin

doi:10.3389/fbioe.2022.755983

Cited by 8 publications

(4 citation statements)

References 42 publications

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“…Several robotic cell culture systems, ranging from small liquid handling devices [56,57] to large stand-alone multifunction platforms [58][59][60][61][62][63][64][65][66], have been reported. For instance, the CompacT SelecT (CTST) platform developed by Tristan et al, at the National Center for Advancing Translational Sciences, is a large stand-alone unit that incorporates an incubator, microscope, cell counter, several peristaltic pumps, chilled media storage area, and robotic plate handling device [58].…”

Section: Discussionmentioning

confidence: 99%

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

et al. 2023

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Background Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation. Methods In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed. Results Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory. Conclusion We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

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Section: Discussionmentioning

confidence: 99%

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

et al. 2023

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“…Recently, a method has been described to generate such aggregates by a self-detaching mechanism from microcontact printed substrates, [68] which facilitates upscaling for automated production. [69,70] Such automated platforms enable quality assurance in production which is necessary for clinical application. For the development of an advanced therapy medicinal product (ATMP), reproducible quality has to be assured to meet the quality-related regulation requirements.…”

Section: Automated and Standardized Cell Culturementioning

confidence: 99%

Transformative Materials to Create 3D Functional Human Tissue Models In Vitro in a Reproducible Manner

Gerardo‐Nava,

Jansen,

Günther

et al. 2023

Adv Healthcare Materials

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Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state‐of‐the‐art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large‐scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life‐like human tissue models that provide a platform to answer health‐based scientific questions.

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“…Unlike traditional single-cell-based flow cytometry and cell sorting, there are advanced sorting techniques and machines where clusters of cells can be sorted. Cell cluster sorting was employed for the automated differentiation of patient-specific iPSCs into blood cells [43]. For efficient differentiation of iPSCs into blood cells, the initial iPSC cluster and the size of EBs matter.…”

Section: Imaging Flow Cytometry and Pluripotent Stem Cell Researchmentioning

confidence: 99%

“…Based on the optical density and fluorescence intensity, cell clusters were characterized, and as they exited the flow cell, using an air sorting mechanism, it was sorted. The cell cluster size cut-off threshold was set based on the Minimum Time of Flight [43]. Thus, the cell clusters, iPSC clusters and EBs, of desired size and expression of respective markers can be sorted.…”

Section: Imaging Flow Cytometry and Pluripotent Stem Cell Researchmentioning

confidence: 99%

Pluripotent stem cells: Basic biology or else differentiations aimed at translational research and the role of flow cytometry

Bose

Nihad

2023

Cytometry Pt A

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Pluripotent stem cell research has revolutionized the modern era for the past 14 years with the advent of induced pluripotent stem cells. Before this time, scientists had access to human and mouse embryonic stem cells primarily for basic research and an attempt towards lineage-specific differentiations for cell therapy applications. Regarding pluripotent stem cells, expression of bonafide marker proteins such as Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin28 have been considered giving a perfect readout for pluripotent stem cells and assessed using an analytical flow cytometer. In addition to the intracellular markers, surface markers such as stagespecific embryonic antigen-1 for mouse cells and SSEA-4 for human cells are needed to sort pure populations of stem cells for further downstream applications for cell therapy. The surface marker SSEA-4 is the most appropriate for obtaining pure populations of human pluripotent stem cells. When differentiated in a controlled manner using growth factors or small molecules, it is mandatory to assess the downregulation of pluripotency markers (Oct4, Nanog, Sox2, and Klf4) with subsequent up-regulation of stage-specific differentiation markers. Such assessments are done using flow cytometry. Pluripotent stem cells have a high teratoma-forming potential in vivo. Small amounts of undifferentiated PSCs might lead to dangerous teratomas upon transplantation if leftover in the pool of differentiated cells. Hence, flow cytometry is essential for sorting out PSC populations with teratoma-forming potential. The pure populations of differentiated progenitors need to be flow-sorted before differentiating them further for cell therapy applications. For example, Glycoprotein 2 is a specific cell-surface marker for pancreatic progenitors that enables one to sort the pancreatic progenitors differentiated from human PSCs. Taken together, analytical flow cytometry, and cell sorting provide indispensable tools in PSC research and cell therapy.

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Cell Cluster Sorting in Automated Differentiation of Patient-specific Induced Pluripotent Stem Cells Towards Blood Cells

Abstract: Graphical AbstractClonal iPS cell lines and embryoid bodies (EB) of defined size are obtained by cell cluster sorting in an automated platform.

Cited by 8 publications

References 42 publications

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

Automating iPSC generation to enable autologous photoreceptor cell replacement therapy

Transformative Materials to Create 3D Functional Human Tissue Models In Vitro in a Reproducible Manner

Pluripotent stem cells: Basic biology or else differentiations aimed at translational research and the role of flow cytometry

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