Tips and Tricks for Validation of Quality Control Analytical Methods in Good Manufacturing Practice Mesenchymal Stromal Cell Production

Viganò, Mariele; Budelli, Silvia; Lavazza, Cristiana; Montemurro, Tiziana; Montelatici, Elisa; Cesare, Stefania de; Lazzari, Lorenza; Orlandi, Alessia; Lunghi, G.; Giordano, Rosaria

doi:10.1155/2018/3038565

Cited by 29 publications

(31 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As well as ensuring identity, purity is one of the minimal criteria for cell-based products [31]. In comparison to iPSCs, which can be clonally expanded indefinitely, iHEPs present a high proliferative activity and are usually expanded from a bulk population [32].…”

Section: Discussionmentioning

confidence: 99%

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

View full text Add to dashboard Cite

Background: Hepatocyte-like cells (iHEPs) generated by transcription factor-mediated direct reprogramming of somatic cells have been studied as potential cell sources for the development of novel therapies targeting liver diseases. The mechanisms involved in direct reprogramming, stability after long-term in vitro expansion, and safety profile of reprogrammed cells in different experimental models, however, still require further investigation.Methods: iHEPs were generated by forced expression of Foxa2/Hnf4a in mouse mesenchymal stromal cells and characterized their phenotype stability by in vitro and in vivo analyses.Results: The iHEPs expressed mixed hepatocyte and liver progenitor cell markers, were highly proliferative, and presented metabolic activities in functional assays. A progressive loss of hepatic phenotype, however, was observed after several passages, leading to an increase in alpha-SMA + fibroblast-like cells, which could be distinguished and sorted from iHEPs by differential mitochondrial content. The resulting purified iHEPs proliferated, maintained liver progenitor cell markers, and, upon stimulation with lineage maturation media, increased expression of either biliary or hepatocyte markers. In vivo functionality was assessed in independent pre-clinical mouse models. Minimal engraftment was observed following transplantation in mice with acute acetaminophen-induced liver injury. In contrast, upon transplantation in a transgenic mouse model presenting host hepatocyte senescence, widespread engraftment and uncontrolled proliferation of iHEPs was observed, forming islands of epithelial-like cells, adipocytelike cells, or cells presenting both morphologies. Conclusion: The results have significant implications for cell reprogramming, suggesting that iHEPs generated by Foxa2/Hnf4a expression have an unstable phenotype and depend on transgene expression for maintenance of hepatocyte-like characteristics, showing a tendency to return to the mesenchymal phenotype of origin and a compromised safety profile.

show abstract

Section: Discussionmentioning

confidence: 99%

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

View full text Add to dashboard Cite

show abstract

“…This involves the need for developing more advanced methods to detect adventitious viruses with improved efficiency, sensitivity and specificity of currently available methods such as retroviral reverse transcriptase, detection of viruses in cell culture and animal host systems or electron microscopy [127]. PCR-based methods and advanced nucleic acid-based technologies, namely high-throughput sequencing (HTS) or next-generation sequencing (NGS), afford a wide array of opportunities for patient safety and product efficacy [129,130,131]. These in vitro tests provide quantitative methods for evidencing infectious agents (adenovirus, enterovirus, cytomegalovirus, and Epstein–Barr virus) for the purpose of achieving a rapid release of hMSC-based products [129].…”

Section: Minimal Criteria For Hmsc Characterizationmentioning

confidence: 99%

“…PCR-based methods and advanced nucleic acid-based technologies, namely high-throughput sequencing (HTS) or next-generation sequencing (NGS), afford a wide array of opportunities for patient safety and product efficacy [129,130,131]. These in vitro tests provide quantitative methods for evidencing infectious agents (adenovirus, enterovirus, cytomegalovirus, and Epstein–Barr virus) for the purpose of achieving a rapid release of hMSC-based products [129].…”

Section: Minimal Criteria For Hmsc Characterizationmentioning

confidence: 99%

Principal Criteria for Evaluating the Quality, Safety and Efficacy of hMSC-Based Products in Clinical Practice: Current Approaches and Challenges

et al. 2019

View full text Add to dashboard Cite

Human Mesenchymal Stem Cells (hMSCs) play an important role as new therapeutic alternatives in advanced therapies and regenerative medicine thanks to their regenerative and immunomodulatory properties, and ability to migrate to the exact area of injury. These properties have made hMSCs one of the more promising cellular active substances at present, particularly in terms of the development of new and innovative hMSC-based products. Currently, numerous clinical trials are being conducted to evaluate the therapeutic activity of hMSC-based products on specific targets. Given the rapidly growing number of hMSC clinical trials in recent years and the complexity of these products due to their cellular component characteristics and medicinal product status, there is a greater need to define more stringent, specific, and harmonized requirements to characterize the quality of the hMSCs and enhance the analysis of their safety and efficacy in final products to be administered to patients. These requirements should be implemented throughout the manufacturing process to guarantee the function and integrity of hMSCs and to ensure that the hMSC-based final product consistently meets its specifications across batches. This paper describes the principal phases involved in the design of the manufacturing process and updates the specific technical requirements needed to address the appropriate clinical use of hMSC-based products. The challenges and limitations to evaluating the safety, efficacy, and quality of hMSCs have been also reviewed and discussed.

show abstract

“…12604013, Monza, Italy) for 5 min. Single-cell suspensions were counted directly with a GMP-validated Nucleocoun-ter® NC-100™ (ChemoMetec, Allerod, Denmark) as previously described [28]. Twenty thousand cells were embedded in a drop of 50 μl BME2, cultured with expansion medium, counted, and reseeded every 2 weeks.…”

Section: In Vitro Growth Curvementioning

confidence: 99%

Standardized GMP-compliant scalable production of human pancreas organoids

et al. 2020

Self Cite

View full text Add to dashboard Cite

Background: Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. Methods: Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis.Results: This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operatordepending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a welldefined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three-and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%). Conclusion: This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

show abstract

Tips and Tricks for Validation of Quality Control Analytical Methods in Good Manufacturing Practice Mesenchymal Stromal Cell Production

Cited by 29 publications

References 19 publications

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Principal Criteria for Evaluating the Quality, Safety and Efficacy of hMSC-Based Products in Clinical Practice: Current Approaches and Challenges

Standardized GMP-compliant scalable production of human pancreas organoids

Contact Info

Product

Resources

About