Cryopreservation of Human Pluripotent Stem Cell-Derived Cardiomyocytes: Strategies, Challenges, and Future Directions

Preininger, Marcela K.; Singh, Monalisa; Xu, Chunhui

doi:10.1007/978-3-319-45457-3_10

Cited by 14 publications

(15 citation statements)

References 75 publications

(114 reference statements)

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“…Another key element in the large-scale manufacturing of cell-based therapies is maintaining the viability of cryopreserved cells, such that the therapeutic potential of these cells remains intact [25]. We extensively characterized the cryopreserved hiPSCs upon thaw, and confirmed their quality.…”

Section: Discussionmentioning

confidence: 74%

“…Cryopreservation of cell-based therapeutic products is a critical aspect of cell therapy [24][25][26]. Master and working cell banks of iPSCs could be readily used for subsequent rounds of expansion and differentiation into the desired cell therapy product.…”

Section: Quality Assessment Of Hpscs Cryopreserved Following Expansiomentioning

confidence: 99%

“…A centrifugation speed of 782× g was used. To optimize the formation of the fluidized bed, three flow rates (25,30,35 mL/min) were tested in increasing order. Prior to each run, the feed source was sampled in triplicate to determine cell density going into the kSep.…”

Section: Downstream Processing: Flow Rate Optimization For Fluidized mentioning

confidence: 99%

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End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

Pandey

Tomney

Woon

et al. 2019

IJMS

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Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.Int. J. Mol. Sci. 2020, 21, 89 2 of 29 recapitulate in vivo conditions. To replace the number of cells lost during a myocardial infarction, for example, approximately 1 × 10 9 cells are required per patient dose [7].Given that 2D-based cell culture platforms are nonscalable with minimal capacity for expansion, achieving high cell densities in a 2D system would involve costly arrangements including extensive manual effort, laboratory space and personnel. These platforms also often do not possess adequate systems to control or monitor parameters, such as the production of key metabolites by hiPSCs in culture. Moreover, iPSC-derived cardiomyocytes remain phenotypically immature [8], despite a number of studies demonstrating enhanced maturation through the modulation of existing methodologies [9][10][11][12][13].Recent innovations in suspension culture systems provide robust, controlled and scalable platforms beyond conventional 2D approaches, which can be translated to current Good Manufacturing Practice (cGMP) compliant processes [14][15][16]. A number of studies have demonstrated the feasibility of hPSC expansion in suspension cultures using aggregate [14,16,17] and microcarrier (MC)-based [18][19][20] three dimensional (3D) culture systems. Aggregate-based 3D culture provides a more physiologically relevant microenvironment, but has been shown not only to require the small molecule, Y27632, for the survival of hPSCs [15], but also sequential passaging to achieve high fold expansion [21]. Not without its own advantages, microcarrier-based culture systems facilitate a larger surface...

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Section: Discussionmentioning

confidence: 74%

Section: Quality Assessment Of Hpscs Cryopreserved Following Expansiomentioning

confidence: 99%

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End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

Pandey

Tomney

Woon

et al. 2019

IJMS

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“…While simple hPSC-CM differentiation protocols have become much more precise and efficient (Denning et al, 2016), there is a slight dearth of improvement and understanding with regards to cryopreservation. The review by Preininger and colleagues is a helpful navigator (Preininger et al, 2016). Among the publications that do openly report or specifically investigate cryopreservation of hPSC-CMs, as noted in the Introduction, there are widely varying rates of recovery (Burridge et al, 2015;Chen et al, 2015;van den Brink et al, 2020;Xu et al, 2011), and anecdotally recovery rates tend to be lower.…”

Section: Commentary Background Informationmentioning

confidence: 99%

“…The usability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) for applications within regenerative medicine, toxicology screening, and disease modelling, depends heavily on efficient reproducible bioprocesses. One of these bioprocesses is hPSC-CM cryopreservation, as it enables the generation, transportation, and application of large differentiation batches, which simplifies supply chains and should reduce variability and cost (Dunn & Palecek, 2018;Preininger, Singh, & Xu, 2016). Even on a small scale, reliable freezing and thawing of hPSC-CM batches greatly facilitates project Figure 1 Overview of general hPSC-CM differentiation and handling workflow.…”

Section: Introductionmentioning

confidence: 99%

Simple Workflow and Comparison of Media for hPSC‐Cardiomyocyte Cryopreservation and Recovery

Miller

Genehr

Telugu

et al. 2020

CP Stem Cell Biology

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Great progress has been made with protocols for the differentiation and functional application of hPSC-cardiomyocytes (hPSC-CMs) in recent years; however, the cryopreservation and recovery of hPSC-CMs still presents challenges and few reports describe in detail the protocols and general workflow. In order to facilitate cryopreservation and recovery of hPSC-CMs for a wide range of applications, we provide detailed information and step-by-step protocols. The protocols are simple and use common reagents. They are comprised of a fast dissociation, cryopreservation using standard equipment, and gentle recovery following thawing. We discuss various features of the protocols, as well as their utilization in the context of common hPSC-CM differentiation and application workflows. Finally, we compare two proprietary and two common in-house formulations of cryopreservation media used for hPSC-CMs, and despite differences in their price and composition find broadly similar recovery rates and cellular function after thawing.

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Advancing Cardiomyocyte Maturation: Current Strategies and Promising Conductive Polymer‐Based Approaches

Elkhoury,

Kodeih,

Enciso‐Martínez

et al. 2024

Adv Healthcare Materials

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Cardiovascular diseases are a leading cause of mortality and pose a significant burden on healthcare systems worldwide. Despite remarkable progress in medical research, the development of effective cardiovascular drugs has been hindered by high failure rates and escalating costs. One contributing factor is the limited availability of mature cardiomyocytes (CMs) for accurate disease modeling and drug screening. Human induced pluripotent stem cell‐derived CMs offer a promising source of CMs; however, their immature phenotype presents challenges in translational applications. This review focuses on the road to achieving mature CMs by summarizing the major differences between immature and mature CMs, discussing the importance of adult‐like CMs for drug discovery, highlighting the limitations of current strategies, and exploring potential solutions using electro‐mechano active polymer‐based scaffolds based on conductive polymers. However, critical considerations such as the trade‐off between three‐dimensional systems and nutrient exchange, biocompatibility, degradation, cell adhesion, longevity, and integration into wider systems must be carefully evaluated. Continued advancements in these areas will contribute to a better understanding of cardiac diseases, improved drug discovery, and the development of personalized treatment strategies for patients with cardiovascular disorders.This article is protected by copyright. All rights reserved

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Cryopreservation of Human Pluripotent Stem Cell-Derived Cardiomyocytes: Strategies, Challenges, and Future Directions

Cited by 14 publications

References 75 publications

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

Simple Workflow and Comparison of Media for hPSC‐Cardiomyocyte Cryopreservation and Recovery

Advancing Cardiomyocyte Maturation: Current Strategies and Promising Conductive Polymer‐Based Approaches

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